There is much human disadvantage and unmet need in the world, including deficits in basic resources and services considered to be human rights, such as drinking water, sanitation and hygiene, healthy nutrition, access to basic healthcare, and a clean environment. Furthermore, there are substantive asymmetries in the distribution of key resources among peoples. These deficits and asymmetries can lead to local and regional crises among peoples competing for limited resources, which, in turn, can become sources of discontent and conflict. Such conflicts have the potential to escalate into regional wars and even lead to global instability. Ergo: in addition to moral and ethical imperatives to level up, to ensure that all peoples have basic resources and services essential for healthy living and to reduce inequalities, all nations have a self-interest to pursue with determination all available avenues to promote peace through reducing sources of conflicts in the world. Microorganisms and pertinent microbial technologies have unique and exceptional abilities to provide, or contribute to the provision of, basic resources and services that are lacking in many parts of the world, and thereby address key deficits that might constitute sources of conflict. However, the deployment of such technologies to this end is seriously underexploited. Here, we highlight some of the key available and emerging technologies that demand greater consideration and exploitation in endeavours to eliminate unnecessary deprivations, enable healthy lives of all and remove preventable grounds for competition over limited resources that can escalate into conflicts in the world. We exhort central actors: microbiologists, funding agencies and philanthropic organisations, politicians worldwide and international governmental and non-governmental organisations, to engage - in full partnership - with all relevant stakeholders, to 'weaponise' microbes and microbial technologies to fight resource deficits and asymmetries, in particular among the most vulnerable populations, and thereby create humanitarian conditions more conducive to harmony and peace.

Antimicrobial peptides play a crucial role in innate immunity, whose components are mainly peptide-based molecules with antibacterial properties. Indeed, the exploration of the immune system over the past 40 years has revealed a number of natural peptides playing a pivotal role in the defence mechanisms of vertebrates and invertebrates, including amphibians, insects, and mammalians. This review provides a discussion regarding the antibacterial mechanisms of peptide-based agents and their structure-activity relationships (SARs) with the aim of describing a topic that is not yet fully explored. Some growing evidence suggests that innate immunity should be strongly considered for the development of novel antibiotic peptide-based libraries. Also, due to the constantly rising concern of antibiotic resistance, the development of new antibiotic drugs is becoming a priority of global importance. Hence, the study and the understanding of defence phenomena occurring in the immune system may inspire the development of novel antibiotic compound libraries and set the stage to overcome drug-resistant pathogens. Here, we provide an overview of the importance of peptide-based antibacterial sources, focusing on accurately selected molecular structures, their SARs including recently introduced modifications, their latest biotechnology applications, and their potential against multi-drug resistant pathogens. Last, we provide cues to describe how antibacterial peptides show a better scope of action selectivity than several anti-infective agents, which are characterized by non-selective activities and non-targeted actions toward pathogens.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge and is often associated with other health consequences mainly in pregnant women. BV is described by an imbalance in the vaginal microbiota where strictly and facultative anaerobic bacteria outgrow the lactic acid- and hydrogen peroxide-producing Lactobacillus species. The species involved in BV are capable to grow and form a polymicrobial biofilm in the vaginal epithelium. The treatment of BV is usually performed using broad-spectrum antibiotics, including metronidazole and clindamycin. However, these conventional treatments are associated with high recurrence rates. The BV polymicrobial biofilm may have an important role on the treatment outcome and is accounted as one of the factors for treatment failure. Other possible reasons for treatment failure include the presence of species resistant to antibiotics or the chance of reinfection after treatment. Therefore, novel strategies to increase the rates of treatment have been studied namely the use of probiotics and prebiotics, acidifying agents, antiseptics, plant-based products, vaginal microbiota transplantation, and phage endolysins. Although some of them are still in an initial phase of development with very preliminary results, they show great perspectives for application. In this review, we aimed to study the role of the polymicrobial nature of BV in treatment failure and explore a few alternatives for treatment.

Synthetic biology uses molecular biology to implement genetic circuits that perform computations. These circuits can process inputs and deliver outputs according to predefined rules that are encoded, often entirely, into genetic parts. However, the field has recently begun to focus on using mechanisms beyond the realm of genetic parts for engineering biological circuits. We analyse the use of electrogenic processes for circuit design and present a model for a merged genetic and electrogenetic toggle switch operating in a biofilm attached to an electrode. Computational simulations explore conditions under which bistability emerges in order to identify the circuit design principles for best switch performance. The results provide a basis for the rational design and implementation of hybrid devices that can be measured and controlled both genetically and electronically.

Within the European-funded Coordination and Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), the Workshop 'Education in Food Systems Microbiome Related Sciences: Needs for Universities, Industry and Public Health Systems' brought together over 70 researchers, public health and industry partners from all over the world to work on elaborating microbiome-related educational needs in food systems. This publication provides a summary of discussions held during and after the workshop and the resulting recommendations.

Metallic nanoparticles (MeNPs) are widely used in many areas such as biomedicine, packaging, cosmetics, colourants, agriculture, antimicrobial agents, cleaning products, as components of electronic devices and nutritional supplements. In addition, some MeNPs exhibit quantum properties, making them suitable materials in the photonics, electronic and energy industries. Through the lens of technology, microbes can be considered nanofactories capable of producing enzymes, metabolites and capping materials involved in the synthesis, assembly and stabilization of MeNPs. This bioprocess is considered more ecofriendly and less energy intensive than the current chemical synthesis routes. However, microbial synthesis of MeNPs as an alternative method to the chemical synthesis of nanomaterials still faces some challenges that need to be solved. Some of these challenges are described in this Editorial.

The junction of bioelectrochemical systems and synthetic biology opens the door to many potentially groundbreaking technologies. When developing these possibilities, choosing the correct chassis organism can save a great deal of engineering effort and, indeed, can mean the difference between success and failure. Choosing the correct chassis for a specific application requires a knowledge of the metabolic potential of the candidate organisms, as well as a clear delineation of the traits, required in the application. In this review, we will explore the metabolic and electrochemical potential of a single genus, Marinobacter. We will cover its strengths, (salt tolerance, biofilm formation and electrochemical potential) and weaknesses (insufficient characterization of many strains and a less developed toolbox for genetic manipulation) in potential synthetic electromicrobiology applications. In doing so, we will provide a roadmap for choosing a chassis organism for bioelectrochemical systems.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

Biology leverages a range of electrical phenomena to extract and store energy, control molecular reactions and enable multicellular communication. Microbes, in particular, have evolved genetically encoded machinery enabling them to utilize the abundant redox-active molecules and minerals available on Earth, which in turn drive global-scale biogeochemical cycles. Recently, the microbial machinery enabling these redox reactions have been leveraged for interfacing cells and biomolecules with electrical circuits for biotechnological applications. Synthetic biology is allowing for the use of these machinery as components of engineered living materials with tuneable electrical properties. Herein, we review the state of such living electronic components including wires, capacitors, transistors, diodes, optoelectronic components, spin filters, sensors, logic processors, bioactuators, information storage media and methods for assembling these components into living electronic circuits.

Ergot alkaloids are a large family of fungal specialized metabolites that are important as toxins in agriculture and as the foundation of powerful pharmaceuticals. Fungi from several lineages and diverse ecological niches produce ergot alkaloids from at least one of several branches of the ergot alkaloid pathway. The biochemical and genetic bases for the different branches have been established and are summarized briefly herein. Several pathway branches overlap among fungal lineages and ecological niches, indicating activities of ergot alkaloids benefit fungi in different environments and conditions. Understanding the functions of the multiple genes in each branch of the pathway allows researchers to parse the abundant genomic sequence data available in public databases in order to assess the ergot alkaloid biosynthesis capacity of previously unexplored fungi. Moreover, the characterization of the genes involved in the various branches provides opportunities and resources for the biotechnological manipulation of ergot alkaloids for experimentation and pharmaceutical development.

Disruptive innovation is needed to raise the threshold of sustainable building performance, so that our buildings improve on net zero impacts and have a life-promoting impact on the natural world. This article outlines a new approach to next-generation sustainable architecture, which draws on the versatile metabolisms of microbes as a platform by incorporating microbial technologies and microbially produced materials into the practice of the built environment. The regenerative architecture arising from these interventions includes a broad range of advances from using new materials, to creating bioreceptive surfaces that promote life, and providing green, bio-remediating energy from waste. Such innovations are presently reaching the marketplace as novel materials like Biocement® with lower embodied carbon than conventional materials that adopt microbially facilitated processes, and as novel utilities like PeePower® that transforms urine into electrical energy and bioreactor-based building systems such as the pioneering BIQ building in Hamburg. While the field is still young, some of these products (e.g. mycelium biocomposites) are poised for uptake by the public-private economic axis to become mainstream within the building industry. Other developments are creating new economic opportunities for local maker communities that empower citizens and catalyse novel vernacular building practices. In particular, the activation of the microbial commons by the uptake of microbial technologies and materials through daily acts of living, 'democratises' resource harvesting (materials and energy) in ways that sustain life, and returns important decisions about how to run a home back to citizens. This disruptive move re-centres the domestic-commons economic axis to the heart of society, setting the stage for new vernacular architectures that support increasingly robust and resilient communities.

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.

Elemental selenium (Se0 ) nanomaterials undergo allotropic transition from thermodynamically-unstable to more stable phases. This process is significantly different when Se0 nanoparticles (NPs) are produced via physico-chemical and biological pathways. While the allotropic transition of physico-chemically synthesized Se0 is fast (minutes to hours), the biogenic Se0 takes months to complete. The biopolymer layer covering biogenic Se0 NPs might be the main factor controlling this retardation, but this still remains an open question. Phylogenetically-diverse bacteria reduce selenium oxyanions to red amorphous Se0 allotrope, which has low market value. Then, red Se0 undergoes allotropic transition to trigonal (metallic grey) allotrope, the end product having important industrial applications (e.g. semiconductors, alloys). Is it not yet clear whether biogenic Se0 presents any biological function, or it is mainly a detoxification and respiratory by-product. The better understanding of this transition would benefit the recovery of Se0 NPs from secondary resources and its targeted utilization with respect to each allotropic stage. This review article presents and critically discusses the main physico-chemical methods and biosynthetic pathways of Se0 (bio)mineralization. In addition, the article proposes a conceptual model for the resource recovery potential of trigonal selenium nanomaterials in the context of circular economy.

Clostridium spp. are suitable for the bioconversion of C1 -gases (e.g., CO2 , CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C4 or C6 fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.

Lactococcus is one of the earliest identified fermentative bacterial genera and among its member species, the dairy-associated Lactococcus lactis and Lactococcus cremoris are undoubtedly the best studied. These two species are believed to have evolved from plant-associated lactococci and through genome decay and acquisition of plasmids, have adapted to the dairy niche. The past decade has witnessed a surge of activity in novel lactococcal species identification from insect, plant and animal sources. Currently, 22 Lactococcus species are described and in this review, we summarise the genome characteristics of and phylogenetic relationships among these species. Furthermore, we explore the role of mobile elements including plasmids and bacteriophages in the diversification of lactococcal species. The pace of identification of novel lactococcal species suggests that the number of lactococcal species is likely to continue to grow. With additional sequence data for the emerging species, it will be possible to perform pathogenicity/virulence risk evaluations and generate extensive insights into the niche adaptation strategies through which they have evolved.

In addition to being an important intermediate in the TCA cycle, L-malate is also widely used in the chemical and beverage industries. Due to the resulting high demand, numerous studies investigated chemical methods to synthesize L-malate from petrochemical resources, but such approaches are hampered by complex downstream processing and environmental pollution. Accordingly, there is an urgent need to develop microbial methods for environmentally-friendly and economical L-malate biosynthesis. The rapid progress and understanding of DNA manipulation, cell physiology, and cell metabolism can improve industrial L-malate biosynthesis by applying intelligent biochemical strategies and advanced synthetic biology tools. In this paper, we mainly focused on biotechnological approaches for enhancing L-malate synthesis, encompassing the microbial chassis, substrate utilization, synthesis pathway, fermentation regulation, and industrial application. This review emphasizes the application of novel metabolic engineering strategies and synthetic biology tools combined with a deep understanding of microbial physiology to improve industrial L-malate biosynthesis in the future.

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public. Since the last such effort 5 years ago, 1168 genetic functions have been updated, allowing the construction of a new metabolic model of this organism of environmental and industrial interest. The emphasis in this review is on new metabolic insights, the role of metals in metabolism and macromolecule biosynthesis, functions involved in biofilm formation, features controlling cell growth, and finally, protein agents that allow class discrimination, thus allowing maintenance management, and accuracy of all cell processes. New 'genomic objects' and an extensive updated literature review have been included for the sequence, now available at the International Nucleotide Sequence Database Collaboration (INSDC: AccNum AL009126.4).

Climate change has rapidly altered many ecosystems, with detrimental effects for biodiversity across the globe. In recent years, it has become increasingly apparent that the microorganisms that live in and on animals can substantially affect host health and physiology, and the structure and function of these microbial communities can be highly sensitive to environmental variables. To date, most studies have focused on the effects of increasing mean temperature on gut microbiota, yet other aspects of climate are also shifting, including temperature variation, seasonal dynamics, precipitation and the frequency of severe weather events. This array of environmental pressures might interact in complex and non-intuitive ways to impact gut microbiota and consequently alter animal fitness. Therefore, understanding the impacts of climate change on animals requires a consideration of multiple types of environmental stressors and their interactive effects on gut microbiota. Here, we present an overview of some of the major findings in research on climatic effects on microbial communities in the animal gut. Although ample evidence has now accumulated that shifts in mean temperature can have important effects on gut microbiota and their hosts, much less work has been conducted on the effects of other climatic variables and their interactions. We provide recommendations for additional research needed to mechanistically link climate change with shifts in animal gut microbiota and host fitness.

Solute binding proteins (SBPs) are of central physiological relevance for prokaryotes. These proteins present substrates to transporters, but they also stimulate different signal transduction receptors. SBPs form a superfamily of at least 33 protein Pfam families. To assess possible links between SBP sequence and the ligand recognized, we have inspected manually all SBP three-dimensional structures deposited in the protein data bank and retrieved 748 prokaryotic structures that have been solved in complex with bound ligand. These structures were classified into 26 SBP Pfam families. The analysis of the ligands recognized revealed that most families possess a preference for a compound class. There were three families each that bind preferentially saccharides and amino acids. In addition, we identified families that bind preferentially purines, quaternary amines, iron and iron-chelating compounds, oxoanions, bivalent metal ions or phosphates. Phylogenetic analyses suggest convergent evolutionary events that lead to families that bind the same ligand. The functional link between chemotaxis and compound uptake is reflected in similarities in the ligands recognized by SBPs and chemoreceptors. Associating Pfam families with ligand profiles will be of help to design experimental strategies aimed at the identification of ligands for uncharacterized SBPs.

Fungi and antifungal compounds are relevant to the United Nation's Sustainable Development Goals. However, the modes-of-action of antifungals-whether they are naturally occurring substances or anthropogenic fungicides-are often unknown or are misallocated in terms of their mechanistic category. Here, we consider the most effective approaches to identifying whether antifungal substances are cellular stressors, toxins/toxicants (that are target-site-specific), or have a hybrid mode-of-action as toxin-stressors (that induce cellular stress yet are target-site-specific). This newly described 'toxin-stressor' category includes some photosensitisers that target the cell membrane and, once activated by light or ultraviolet radiation, cause oxidative damage. We provide a glossary of terms and a diagrammatic representation of diverse types of stressors, toxic substances, and toxin-stressors, a classification that is pertinent to inhibitory substances not only for fungi but for all types of cellular life. A decision-tree approach can also be used to help differentiate toxic substances from cellular stressors (Curr Opin Biotechnol 2015 33: 228-259). For compounds that target specific sites in the cell, we evaluate the relative merits of using metabolite analyses, chemical genetics, chemoproteomics, transcriptomics, and the target-based drug-discovery approach (based on that used in pharmaceutical research), focusing on both ascomycete models and the less-studied basidiomycete fungi. Chemical genetic methods to elucidate modes-of-action currently have limited application for fungi where molecular tools are not yet available; we discuss ways to circumvent this bottleneck. We also discuss ecologically commonplace scenarios in which multiple substances act to limit the functionality of the fungal cell and a number of as-yet-unresolved questions about the modes-of-action of antifungal compounds pertaining to the Sustainable Development Goals.

The overall challenge of establishing a sustainable anthropogenic phosphate cycle did not change in the last decade, rather the need to act becomes ever more urgent. Here, I briefly highlight some developments in (poly)phosphate research within the last 10 years and dare a crystal ball look for topics that might help us towards a sustainable P society.

Biomineralization, the capacity to form minerals, has evolved in a great diversity of bacterial lineages as an adaptation to different environmental conditions and biological functions. Microbial biominerals often display original properties (morphology, composition, structure, association with organics) that significantly differ from those of abiotically formed counterparts, altogether defining the 'mineral phenotype'. In principle, it should be possible to take advantage of microbial biomineralization processes to design and biomanufacture advanced mineral materials for a range of technological applications. In practice, this has rarely been done so far and only for a very limited number of biomineral types. This is mainly due to our poor understanding of the underlying molecular mechanisms controlling microbial biomineralization pathways, preventing us from developing bioengineering strategies aiming at improving biomineral properties for different applications. Another important challenge is the difficulty to upscale microbial biomineralization from the lab to industrial production. Addressing these challenges will require combining expertise from environmental microbiologists and geomicrobiologists, who have historically been working at the forefront of research on microbe-mineral interactions, alongside bioengineers and material scientists. Such interdisciplinary efforts may in the future allow the emergence of a mineral biomanufacturing industry, a critical tool towards the development more sustainable and circular bioeconomies.

Microbial electrochemical systems (MESs) are a highly versatile platform technology with a particular focus on power or energy production. Often, they are used in combination with substrate conversion (e.g., wastewater treatment) and production of value-added compounds via electrode-assisted fermentation. This rapidly evolving field has seen great improvements both technically and biologically, but this interdisciplinarity sometimes hampers overseeing strategies to increase process efficiency. In this review, we first briefly summarize the terminology of the technology and outline the biological background that is essential for understanding and thus improving MES technology. Thereafter, recent research on improvements at the biofilm-electrode interface will be summarized and discussed, distinguishing between biotic and abiotic approaches. The two approaches are then compared, and resulting future directions are discussed. This mini-review therefore provides basic knowledge of MES technology and the underlying microbiology in general and reviews recent improvements at the bacteria-electrode interface.

Globally, soils and sediments are affected by the bioturbation activities of benthic species. The consequences of these activities are particularly impactful in intertidal sediment, which is generally anoxic and nutrient-poor. Mangrove intertidal sediments are of particular interest because, as the most productive forests and one of the most important stores of blue carbon, they provide global-scale ecosystem services. The mangrove sediment microbiome is fundamental for ecosystem functioning, influencing the efficiency of nutrient cycling and the abundance and distribution of key biological elements. Redox reactions in bioturbated sediment can be extremely complex, with one reaction creating a cascade effect on the succession of respiration pathways. This facilitates the overlap of different respiratory metabolisms important in the element cycles of the mangrove sediment, including carbon, nitrogen, sulphur and iron cycles, among others. Considering that all ecological functions and services provided by mangrove environments involve microorganisms, this work reviews the microbial roles in nutrient cycling in relation to bioturbation by animals and plants, the main mangrove ecosystem engineers. We highlight the diversity of bioturbating organisms and explore the diversity, dynamics and functions of the sediment microbiome, considering both the impacts of bioturbation. Finally, we review the growing evidence that bioturbation, through altering the sediment microbiome and environment, determining a 'halo effect', can ameliorate conditions for plant growth, highlighting the potential of the mangrove microbiome as a nature-based solution to sustain mangrove development and support the role of this ecosystem to deliver essential ecological services.

Pseudomonas aeruginosa is an opportunistic pathogen able to infect any human tissue. One of the reasons for its high adaptability and colonization of host tissues is its capacity of maintaining iron homeostasis through a wide array of iron acquisition and removal mechanisms. Due to their ability to cause life-threatening acute and chronic infections, especially among cystic fibrosis and immunocompromised patients, and their propensity to acquire resistance to many antibiotics, the World Health Organization (WHO) has encouraged the scientific community to find new strategies to eradicate this pathogen. Several recent strategies to battle P. aeruginosa focus on targeting iron homeostasis mechanisms, turning its greatest advantage into an exploitable weak point. In this review, we discuss the different mechanisms used by P. aeruginosa to maintain iron homeostasis and the strategies being developed to fight this pathogen by blocking these mechanisms. Among others, the use of iron chelators and mimics, as well as disruption of siderophore production and uptake, have shown promising results in reducing viability and/or virulence of this pathogen. The so-called 'Trojan-horse' strategy taking advantage of the siderophore uptake systems is emerging as an efficient method to improve delivery of antibiotics into the bacterial cells. Moreover, siderophore transporters are considered promising targets for the developing of P. aeruginosa vaccines.

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea-host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.

Plasmid pUC11B is a 49.3-kb plasmid harboured by the fermented meat isolate Lactococcus lactis subsp. lactis UC11. Among other features, pUC11B encodes a pMRC01-like conjugation system and tetracycline-resistance. In this study, we demonstrate that this plasmid can be conjugated at high frequencies to recipient strains. Mutational analysis of the 22 genes encompassing the presumed pUC11B conjugation cluster revealed the presence of several genes with essential conjugation functions, as well as a gene, trsR, encoding a putative transcriptional repressor of this conjugation cluster. Furthermore, plasmid pUC11B encodes an anti-restriction protein, TrsAR, which facilitates higher conjugation frequencies when pUC11B is transferred into recipient strains containing Type II or Type III RM systems. These findings demonstrate how RM mechanisms can be circumvented when they act as a biological barrier for conjugation events.

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5-5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.

Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA , from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.

Purple phototrophic bacteria are one of the main actors in chemolithotrophic carbon fixation and, therefore, fundamental in the biogeochemical cycle. These microbes are capable of using insoluble electron donors such as ferrous minerals or even carbon-based electrodes. Carbon fixation through extracellular electron uptake places purple phototrophic bacteria in the field of microbial electrosynthesis as key carbon capturing microorganisms. In this work we demonstrate biomass production dominated by purple phototrophic bacteria with a cathode (-0.6 V vs. Ag/AgCl) as electron donor. In addition, we compared the growth and microbial population structure with ferrous iron as the electron donor. We detect interaction between the cathode and the consortium showing a midpoint potential of 0.05 V (vs. Ag/AgCl). Microbial community analyses revealed different microbial communities depending on the electron donor, indicating different metabolic interactions. Electrochemical measurements together with population analyses point to Rhodopseudomonas genus as the key genus in the extracellular electron uptake. Furthermore, the genera Azospira and Azospirillum could play a role in the photoelectrotrophic consortium.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Bacillus subtilis is a soil-dwelling bacterium that can interact with a plethora of other microorganisms in its natural habitat. Due to the versatile interactions and its ability to form nanotubes, i.e., recently described membrane structures that trade cytoplasmic content between neighbouring cells, we investigated the potential of HGT from B. subtilis to industrially-relevant members of lactic acid bacteria (LAB). To explore the interspecies HGT events, we developed a co-culturing protocol and provided proof of transfer of a small high copy non-conjugative plasmid from B. subtilis to LABs. Interestingly, the plasmid transfer did not involve conjugation nor activation of the competent state by B. subtilis. Moreover, our study shows for the first time non-conjugative cell-to-cell intraspecies plasmid transfer for non-competent Lactococcus lactis sp. cremoris strains. Our study indicates that cell-to-cell transformation is a ubiquitous form of HGT and can be potentially utilized as an alternative tool for natural (non-GMO) strain improvement.

The S-layer or surface layer protein (SLP) is the most ancient biological envelope, highly conserved in several Bacteria and Archaea. In lactic acid bacteria (LAB), SLP is only found in species belonging to the Lactobacillaceae family, many of them considered probiotic microorganisms. New reclassification of members within the Lactobacillaceae family (International Journal of Systematic and Evolutionary Microbiology, 2020, 70, 2782) and newly sequenced genomes demands an updated revision on SLP genes and domain organization. There is growing information concerning SLP occurrence, molecular biology, biophysical properties, and applications. Here, we focus on the prediction of slp genes within the Lactobacillaceae family, and specifically, on the neat interconnection between the two different modular SLP domain organizations and the new reclassified genera. We summarize the results in a concise tabulated manner to review the present knowledge on SLPs and discuss the most relevant and updated concepts regarding SLP sequence clustering. Our assessment is based on sequence alignments considering the new genera classification and protein domain definition with post-translational modifications. We analyse the difficulties encountered to resolve the SLPs 3D structure, describing the need for structure prediction approaches and the relation between protein structure and its anchorage mechanism to the cell wall. Finally, we enumerate new SLP applications regarding heterologous display, pathogen exclusion, immunostimulation, and metal binding.

Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the -10 and -35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus -10 and -35 sequence boxes strongly targeted by σ70 , incorporation of a strong ribosome binding site recognized broadly by Gram-negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which significantly improves the signal-to-background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.

Microbial attachment and biofilm formation is a ubiquitous behaviour of microorganisms and is the most crucial prerequisite of contact bioleaching. Monazite and xenotime are two commercially exploitable minerals containing rare earth elements (REEs). Bioleaching using phosphate solubilizing microorganisms is a green biotechnological approach for the extraction of REEs. In this study, microbial attachment and biofilm formation of Klebsiella aerogenes ATCC 13048 on the surface of these minerals were investigated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In a batch culture system, K. aerogenes was able to attach and form biofilms on the surface of three phosphate minerals. The microscopy records showed three distinctive stages of biofilm development for K. aerogenes commencing with initial attachment to the surface occurring in the first minutes of microbial inoculation. This was followed by colonization of the surface and formation of a mature biofilm as the second distinguishable stage, with progression to dispersion as the final stage. The biofilm had a thin-layer structure. The colonization and biofilm formation were localized toward physical surface imperfections such as cracks, pits, grooves and dents. In comparison to monazite and xenotime crystals, a higher proportion of the surface of the high-grade monazite ore was covered by biofilm which could be due to its higher surface roughness. No selective attachment or colonization toward specific mineralogy or chemical composition of the minerals was detected. Finally, in contrast to abiotic leaching of control samples, microbial activity resulted in extensive microbial erosion on the high-grade monazite ore.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

Serine proteases are among the most important biological additives in various industries such as detergents, leather, animal feed and food. A serine protease gene, Fgapt4, from Fusarium graminearum 2697 was identified, cloned and expressed in Pichia pastoris. The optimal pH and temperature of FgAPT4 were 8.5 and 40°C, respectively. The relative activity was >30% even at 10°C. It had a wide range of pH stability (4.0-12.0) and detergent compatibility. To improve the catalytic activity, a strategy combining molecular docking and evolutionary analysis was adopted. Twelve amino acid residue sites and three loops (A, B and C) were selected as potential hot spots that might play critical roles in the enzyme's functional properties. Twenty-eight mutants targeting changes in individual sites or loops were designed, and mutations with good performance were combined. The best mutant was FgAPT4-M3 (Q70N/D142S/A143S/loop C). The specific activity and catalytic efficiency of FgAPT4-M3 increased by 1.6 (1008.5 vs. 385.9 U/mg) and 2.2-fold (3565.1 vs. 1106.3/s/mM), respectively. Computational analyses showed that the greater flexibility of the substrate pocket may be responsible for the increased catalytic activity. In addition, its application in detergents indicated that FgAPT4-M3 has great potential in washing.

3D printing has emerged as a powerful way to produce complex materials on-demand. These printing technologies are now being applied in microbiology, with many recent examples where microbes and matrices are co-printed to create bespoke living materials. Here, we propose a new paradigm for microbial printing. In addition to its importance for materials, we argue that printing can be used to understand and engineer microbiome communities, analogous to its use in human tissue engineering. Many microbes naturally live in diverse, spatially structured communities that are challenging to study and manipulate. 3D printing offers an exciting new solution to these challenges, as it can precisely arrange microbes in 3D space, allowing one to build custom microbial communities for a wide range of purposes in research, medicine, and industry.

Microorganisms are exposed in their natural niches to a wide diversity of signal molecules. Specific detection of these signals results in alterations in microbial metabolism and physiology. Auxins like indole-3-acetic acid are key phytohormones that regulate plant growth and development. Nonetheless, auxin biosynthesis is not restricted to plants but is ubiquitous in all kingdoms of life. This wide phylogenetic distribution of auxins production, together with the diversity of regulated cellular processes, have made auxins key intra- and inter-kingdom signal molecules in life modulating, for example microbial physiology, metabolism and virulence. Despite their increasing importance as global signal molecules, the mechanisms by which auxins perform their regulatory functions in microorganisms are largely unknown. In this article, we outline recent research that has advanced our knowledge of the mechanisms of bacterial auxin perception. We also highlight the potential applications of this research in aspects such as antibiotic production, biosensor design, plant microbiome engineering and antivirulence therapies.

Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD+ regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities.

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 μg/ml mediating an anodic current of 15.1 μA/cm2 . On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 μA/cm2 . Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.

Sanghuangporus baumii is a medicinal fungi with anti-inflammatory, liver protection and antitumour effects. Terpenoids are one of the main medicinal ingredients of S. baumii. However, terpenoid production by wild-type S. baumii cannot meet the market demand, which affects its application in medical care. Therefore, exploring how to increase terpenoid content in S. baumii is a promising path in this research field. Salicylic acid (SA) is a secondary metabolite. In this study, a concentration of 350 μmol/L SA was added into fungal cultivations for 2 and 4 days, and then the transcriptome and metabolome of untreated mycelia and treated with SA were analysed. The expression of some genes in the terpenoids biosynthesis pathway increased in SA-induced cultivations, and the content of isopentenyl pyrophosphate (IPP) and geranylgeranyl-PP (GGPP) increased significantly as well as the contents of triterpenoids, diterpenoids, sesquiterpenoids and carotenoids. The gene FPS was considered to be a key gene regulating terpenoid biosynthesis. Therefore, FPS was overexpressed in S. baumii by Agrobacterium tumefaciens-mediated genetic transformation. The gene FPS and its downstream gene (LS) expression levels were confirmed to be increased in the FPS overexpressing transformant, and terpenoid content was 36.98% higher than that of the wild-type strain in the evaluated cultivation conditions.

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5'-NGG-3' PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5'-NGG-3' PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

Soft rot pectobacteria (SRP) are phytopathogens of the genera Pectobacterium and Dickeya that cause soft rots on a wide range of crops and ornamental plants. SRP produce plant cell wall degrading enzymes (PCWDEs), including pectinases. Bdellovibrio and like organisms are bacterial predators that can prey on a variety of Gram-negative species, including SRP. In this research, a low methoxyl pectin (LMP)-based immobilization system for B. bacteriovorus is established. It takes advantage that pectin residues induce PCWDE secretion by the pathogens, bringing upon the release of the encapsulated predators. Three commercial LMPs differing in the degree of esterification (DE) and amidation (DA) were tested as potential carriers, by examining their effect on SRP growth, enzymes secretion and substrate breakdown. A clear advantage was observed for pectin 5 CS with the lowest DE and DA content. The degradation of 5 CS pectin-based carriers was further optimized by reducing cross-linker and pectin concentration, by adding gelatin and by dehydration. This resulted in SRP-induced disintegration of the carrier within 72 h. The released encapsulated predator caused a large decrease in SRP population while its own significantly increased, demonstrating the efficiency of this system in which the pathogen brings about its own demise.

Amino acids are important nutrients and also serve as signals for diverse signal transduction pathways. Bacteria use chemoreceptors to recognize amino acid attractants and to navigate their gradients. In Escherichia coli two likely paralogous chemoreceptors Tsr and Tar detect 9 amino acids, whereas in Pseudomonas aeruginosa the paralogous chemoreceptors PctA, PctB and PctC detect 18 amino acids. Here, we show that the phytobacterium Pectobacterium atrosepticum uses the three non-homologous chemoreceptors PacA, PacB and PacC to detect 19 proteinogenic and several non-proteinogenic amino acids. PacB recognizes 18 proteinogenic amino acids as well as 8 non-proteinogenic amino acids. PacB has a ligand preference for the three branched chain amino acids L-leucine, L-valine and L-isoleucine. PacA detects L-proline next to several quaternary amines. The third chemoreceptor, PacC, is an ortholog of E. coli Tsr and the only one of the 36 P. atrosepticum chemoreceptors that is encoded in the cluster of chemosensory pathway genes. Surprisingly, in contrast to Tsr, which primarily senses serine, PacC recognizes aspartate as the major chemoeffector but not serine. Our results demonstrate that bacteria use various strategies to sense a wide range of amino acids and that it takes more than one chemoreceptor to achieve this goal.

The worldwide demand for reduced and restricted use of pesticides in agriculture due to serious environmental effects, health risks and the development of pathogen resistance calls for the discovery of new bioactive compounds. In the medical field, antibiotic-resistant microorganisms have become a major threat to man, increasing mortality. Endophytes are endosymbiotic microorganisms that inhabit plant tissues without causing any visible damage to their host. Many endophytes secrete secondary metabolites with biological activity against a broad range of pathogens, making them potential candidates for novel drugs and alternative pesticides of natural origin. We isolated endophytes from wild plants in Israel, focusing on endophytes that secrete secondary metabolites with biological activity. We isolated 302 different endophytes from 30 different wild plants; 70 of them exhibited biological activity against phytopathogens. One biologically active fungal endophyte from the genus Penicillium, isolated from a squill (Urginea maritima) leaf, was further examined. Chloroform-based extraction of its growth medium was similarly active against phytopathogens. High-performance liquid chromatography separation followed by gas chromatography/mass spectrometry analysis revealed a single compound-mycophenolic acid-as the main contributor to the biological activity of the organic extract.

Hydrogen-oxidising bacteria play a key role in maintaining the composition of gases within the atmosphere and are ubiquitous in agricultural soils. While studies have shown that hydrogen accumulates in soil surrounding legume nodules and the soil surface, soils as a whole act as a net sink for hydrogen, raising questions about how hydrogen is internally recycled by soils. Can the energy derived from hydrogen oxidation be directly funnelled into plants to promote their growth or does it only act as a booster for other plant-growth promoting bacteria? Moreover, while the fertilisation effect of hydrogen on plants has previously been shown to be beneficial, questions remain about the upper limit of hydrogen uptake by plants before it becomes detrimental. Agricultural practices such as fertilisation may impact the balance of hydrogen-oxidisers and hydrogen-producers in these ecosystems, potentially having detrimental effects on not only agricultural land but also global biogeochemical cycles. In this perspectives piece, we highlight the importance of understanding the contribution of hydrogen to agricultural soils and the effects of agricultural practices on the ability for bacteria to cycle hydrogen in agricultural soils. We propose a framework to gain better insights into microbial hydrogen cycling within agroecosystems, which could contribute to the development of new agricultural biotechnologies.

Protein nanowires are critical electroactive components for electron transfer of Geobacter sulfurreducens biofilm. To determine the applicability of the nanowire proteins in improving bioelectricity production, their genes including pilA, omcZ, omcS and omcT were overexpressed in G. sulfurreducens. The voltage outputs of the constructed strains were higher than that of the control strain with the empty vector (0.470-0.578 vs. 0.355 V) in microbial fuel cells (MFCs). As a result, the power density of the constructed strains (i.e. 1.39-1.58 W m-2 ) also increased by 2.62- to 2.97-fold as compared to that of the control strain. Overexpression of nanowire proteins also improved biofilm formation on electrodes with increased protein amount and thickness of biofilms. The normalized power outputs of the constructed strains were 0.18-0.20 W g-1 that increased by 74% to 93% from that of the control strain. Bioelectrochemical analyses further revealed that the biofilms and MFCs with the constructed strains had stronger electroactivity and smaller internal resistance, respectively. Collectively, these results demonstrate for the first time that overexpression of nanowire proteins increases the biomass and electroactivity of anode-attached microbial biofilms. Moreover, this study provides a new way for enhancing the electrical outputs of MFCs.

Environmental water is considered one of the main vehicles for the transmission of antimicrobial resistance (AMR), posing an increasing threat to humans and animals health. Continuous efforts are being made to eliminate AMR; however, the detection of AMR pathogens from water samples often requires at least one culture step, which is time-consuming and can limit sensitivity. In this study, we employed comparative genomics to identify the prevalence of AMR genes within among: Escherichia coli, Klebsiella, Salmonella enterica and Acinetobacter, using publicly available genomes. The mcr-1, blaKPC (KPC-1 to KPC-4 alleles), blaOXA-48, blaOXA-23 and blaVIM (VIM-1 and VIM-2 alleles) genes are of great medical and veterinary significance, thus were selected as targets for the development of isothermal loop-mediated amplification (LAMP) detection assays. We also developed a rapid and sensitive sample preparation method for an integrated culture-independent LAMP-based detection from water samples. The developed assays successfully detected the five AMR gene markers from pond water within 1 h and were 100% sensitive and specific with a detection limit of 0.0625 μg/mL and 10 cfu/mL for genomic DNA and spiked bacterial cells, respectively. The integrated detection can be easily implemented in resource-limited areas to enhance One Health AMR surveillances and improve diagnostics.

The excessive consumption of alcohol results in a dysbiosis of the gut microbiota, which subsequently impairs the gut microbiota-brain/liver axes and induces cognitive dysfunction and hepatic injury. This study aimed to investigate the potential effect of Acetobacter pasteurianus BP2201 in reducing the negative effects of alcohol consumption on cognitive function and liver health by modulating the gut microbiota-brain/liver axes. Treatment with A. pasteurianus BP2201 improved alcohol-induced hippocampal damage, suppressed neuroinflammation, promoted neuroprotein expression in the hippocampus and enhanced cognitive function. At the same time, A. pasteurianus BP2201 can also reduce serum lipid levels, relieve oxidative stress, inhibit TLR4/MyD88/NF-κB pathway, reduce the secretion of TNF-α and IL-1β, so as to improve alcoholic liver injury. Concomitantly, the treatment with A. pasteurianus BP2201 leads to a shift in the intestinal microbiota structure towards that of healthy individuals, inhibiting the proliferation of harmful bacteria and promoting the recovery of beneficial bacteria. In addition, it also improves brain cognitive dysfunction and liver health by affecting the gut microbiota-brain/liver axes by promoting the synthesis of relevant amino acids and the metabolism of nucleotide base components. These findings demonstrate the potential of regulating the gut microbiome and gut microbiota-brain/liver axes to mitigate alcohol-induced disease.

Treatment of wastewater contaminated with high sulfate concentrations is an environmental imperative lacking a sustainable and environmental friendly technological solution. Microbial electrochemical technology (MET) represents a promising approach for sulfate reduction. In MET, a cathode is introduced as inexhaustible electron source for promoting sulfate reduction via direct or mediated electron transfer. So far, this is mainly studied in batch mode representing straightforward and easy-to-use systems, but their practical implementation seems unlikely, as treatment capacities are limited. Here, we investigated bioelectrochemical sulfate reduction in flow mode and achieved removal efficiencies (Esulfate , 89.2 ± 0.4%) being comparable to batch experiments, while sulfate removal rates (Rsulfate , 3.1 ± 0.2 mmol L-1 ) and Coulombic efficiencies (CE, 85.2 ± 17.7%) were significantly increased. Different temperatures and hydraulic retention times (HRT) were applied and the best performance was achieved at HRT 3.5 days and 30°C. Microbial community analysis based on amplicon sequencing demonstrated that sulfate reduction was mainly performed by prokaryotes belonging to the genera Desulfomicrobium, Desulfovibrio, and Desulfococcus, indicating that hydrogenotrophic and heterotrophic sulfate reduction occurred by utilizing cathodically produced H2 or acetate produced by homoacetogens (Acetobacterium). The advantage of flow operation for bioelectrochemical sulfate reduction is likely based on higher absolute biomass, stable pH, and selection of sulfate reducers with a higher sulfide tolerance, and improved ratio between sulfate-reducing prokaryotes and homoacetogens.

This Lilliput explores the current epidemiological and virological arguments for a zoonotic origin of the COVID-19 pandemic. While the role of bats, pangolins and racoon dogs as viral reservoirs has not yet been proven, a spill-over of a coronavirus infection from animals into humans at the Huanan food market in Wuhan has a much greater plausibility than alternative hypotheses such as a laboratory virus escape, deliberate genetic engineering or introduction by cold chain food products. This Lilliput highlights the dynamic nature of the animal-human interface for viral cross-infections from humans into feral white tail deer or farmed minks (reverse zoonosis). Surveillance of viral infections at the animal-human interface is an urgent task since live animal markets are not the only risks for future viral spill-overs. Climate change will induce animal migration which leads to viral exchanges between animal species that have not met in the past. Environmental change and deforestation will also increase contact between animals and humans. Developing an early warning system for emerging viral infections becomes thus a societal necessity not only for human but also for animal and environmental health (One Health concept). Microbiologists have developed tools ranging from virome analysis in key suspects such as viral reservoirs (bats, wild game animals, bushmeat) and in humans exposed to wild animals, to wastewater analysis to detect known and unknown viruses circulating in the human population and sentinel studies in animal-exposed patients with fever. Criteria need to be developed to assess the virulence and transmissibility of zoonotic viruses. An early virus warning system is costly and will need political lobbying. The accelerating number of viral infections with pandemic potential over the last decades should provide the public pressure to extend pandemic preparedness for the inclusion of early viral alert systems.

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from -17.0 kJ/mol to -29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation-reduction cascade at larger scales.

Corynebacterium glutamicum experiences a transient iron limitation during growth in minimal medium, which can be compensated by the external supplementation of protocatechuic acid (PCA). Although C. glutamicum is genetically equipped to form PCA from the intermediate 3-dehydroshikimate catalysed by 3-dehydroshikimate dehydratase (encoded by qsuB), PCA synthesis is not part of the native iron-responsive regulon. To obtain a strain with improved iron availability even in the absence of the expensive supplement PCA, we re-wired the transcriptional regulation of the qsuB gene and modified PCA biosynthesis and degradation. Therefore, we ushered qsuB expression into the iron-responsive DtxR regulon by replacing the native promoter of the qsuB gene by the promoter PripA and introduced a second copy of the PripA -qsuB cassette into the genome of C. glutamicum. Reduction of the degradation was achieved by mitigating expression of the pcaG and pcaH genes through a start codon exchange. The final strain C. glutamicum IRON+ showed in the absence of PCA a significantly increased intracellular Fe2+ availability, exhibited improved growth properties on glucose and acetate, retained a wild type-like biomass yield but did not accumulate PCA in the supernatant. For the cultivation in minimal medium C. glutamicum IRON+ represents a useful platform strain that reveals beneficial growth properties on different carbon sources without affecting the biomass yield and overcomes the need of PCA supplementation.

The elimination of the expression of cellular functions that are not needed in a certain well-defined artificial environment, such as those used in industrial production facilities, has been the goal of many cellular minimization projects. The generation of a minimal cell with reduced burden and less host-function interactions has been pursued as a tool to improve microbial production strains. In this work, we analysed two cellular complexity reduction strategies: genome and proteome reduction. With the aid of an absolute proteomics data set and a genome-scale model of metabolism and protein expression (ME-model), we quantitatively assessed the difference of reducing genome to the correspondence of reducing proteome. We compare the approaches in terms of energy consumption, defined in ATP equivalents. We aim to show what is the best strategy for improving resource allocation in minimized cells. Our results show that genome reduction by length is not proportional to reducing resource use. When we normalize calculated energy savings, we show that strains with the larger calculated proteome reduction show the largest resource use reduction. Furthermore, we propose that reducing highly expressed proteins should be the target as the translation of a gene uses most of the energy. The strategies proposed here should guide cell design when the aim of a project is to reduce the maximum amount or cellular resources.

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.

The recent introduction of non-conventional yeast species as companion wine starters has prompted a growing interest in microbial interactions during wine fermentation. There is evidence of interactions through interference and exploitation competition, as well as interactions depending on physical contact. Furthermore, the results of some transcriptomic analyses suggest interspecific communication, but the molecules or biological structures involved in recognition are not well understood. In this work, we explored extracellular vesicles (EVs) as possible mediators of interspecific communication between wine yeasts. The transcriptomic response of Saccharomyces cerevisiae after 3 h of contact with a fraction enriched in EVs of Metschnikowia pulcherrima was compared with that induced by active M. pulcherrima cells. Interestingly, there is a high level of overlap between the transcriptomic profiles of yeast cells challenged by either M. pulcherrima whole cells or the EV-enriched fraction. The results indicate an upregulation of yeast metabolism in response to competing species (in line with previous results). This finding points to the presence of a signal, in the EV-enriched fraction, that can be perceived by the yeast cells as a cue for the presence of competitors, even in the absence of metabolically active cells of the other species.

Polymyxin resistance is conferred by MCR-1 (mobile colistin resistance 1)-induced lipopolysaccharide (LPS) modification of G- bacteria. However, the peptide MSI-1 exerts potent antimicrobial activity against mcr-1-carrying bacteria. To further investigate the potential role of MCR-1 in improving bacterial virulence and facilitating immune evasion, and the immunomodulatory effect of peptide MSI-1, we first explored outer membrane vesicle (OMV) alterations of mcr-1-carrying bacteria in the presence and absence of sub-MIC MSI-1, and host immune activation during bacterial infection and OMV stimulation. Our results demonstrated that LPS remodelling induced by MCR-1 negatively affected OMV formation and protein cargo by E. coli. In addition, MCR-1 diminished LPS-stimulated pyroptosis but facilitated mitochondrial dysfunction, further aggravating apoptosis in macrophages induced by OMVs of E. coli. Similarly, TLR4-mediated NF-κB activation was markedly alleviated once LPS was modified by MCR-1. However, peptide MSI-1 at the sub-MIC level inhibited the expression of MCR-1, further partly rescuing OMV alteration and attenuation of immune responses in the presence of MCR-1 during both infection and OMV stimulation, which can be exploited for anti-infective therapy.

Our planet teeters on the brink of massive ecosystem collapses, and arid regions experience manifold environmental and climatic challenges that increase the magnitude of selective pressures on already stressed ecosystems. Ultimately, this leads to their aridification and desertification, that is, to simplified and barren ecosystems (with proportionally less microbial load and diversity) with altered functions and food webs and modification of microbial community network. Thus, preserving and restoring soil health in such a fragile biome could help buffer climate change's effects. We argue that microorganisms and the protection of their functional properties and networks are key to fight desertification. Specifically, we claim that it is rational, possible and certainly practical to rely on native dryland edaphic microorganisms and microbial communities as well as dryland plants and their associated microbiota to conserve and restore soil health and mitigate soil depletion in newly aridified lands. Furthermore, this will meet the objective of protecting/stabilizing (and even enhancing) soil biodiversity globally. Without urgent conservation and restoration actions that take into account microbial diversity, we will ultimately, and simply, not have anything to protect anymore.

Pollution by metalloids, e.g., tellurite and selenite, is of serious environmental concern and, therefore, there is an increasing interest in searching for ecologically friendly solutions for their elimination. Some microorganisms are able to reduce toxic tellurite/selenite into less toxic elemental tellurium (Te) and selenium (Se). Here, we describe the use of the environmentally relevant β-proteobacterium Aromatoleum sp. CIB as a platform for tellurite elimination. Aromatoleum sp. CIB was shown to tolerate 0.2 and 0.5 mM tellurite at aerobic and anaerobic conditions, respectively. Furthermore, the CIB strain was able to reduce tellurite into elemental Te producing rod-shaped Te nanoparticles (TeNPs) of around 200 nm length. A search in the genome of Aromatoleum sp. CIB revealed the presence of a gene, AzCIB_0135, which encodes a new methyltransferase that methylates tellurite and also selenite. AzCIB_0135 orthologs are widely distributed in bacterial genomes. The overexpression of the AzCIB_0135 gene both in Escherichia coli and Aromatoleum sp. CIB speeds up tellurite and selenite removal, and it enhances the production of rod-shaped TeNPs and spherical Se nanoparticles (SeNPs), respectively. Thus, the overexpression of a methylase becomes a new genetic strategy to optimize bacterial catalysts for tellurite/selenite bioremediation and for the programmed biosynthesis of metallic nanoparticles of biotechnological interest.

Established vaccine hesitancy measurement instruments, such as the Vaccine Hesitancy Determinants Matrix, are not sufficiently equipped to adequately and consistently measure political and ideological attitudes. Focusing on Germany, which is a particularly interesting case since it witnessed the establishment of the by far most well-organized and sustained 'anti-Covid' movement in Europe, this quantitative study explores the impact of political ideology and partisanship on the degree of vaccine hesitancy based on four surveys (February-October 2021) among more than 30,000 individuals. We demonstrate that party affiliation, political ideology and region of residence all impact vaccine hesitancy. In fact, they turn out to have a greater impact than two factors often analysed with respect to vaccine hesitancy: gender and educational background. Further interaction models show that the effect of political ideology on vaccine hesitancy is moderated by age, gender and region of residency. For instance, while the more rightwing a young individual is, the more hesitant they are towards SARS-CoV-2 vaccination-for older individuals, this is not the case. Our findings are relevant for future investigators measuring vaccine hesitancy and policy makers contemplating the differential impact of complex public health interventions: as the impact of political and ideological attitudes on vaccine hesitancy are not adequately captured by established vaccine hesitancy measurement instruments, we recommend its modification to include a clear and harmonised definition of the political-ideological dimension of vaccine hesitancy together with pre-validated measurement items that improve future studies. In addition, we reason that vaccine hesitancy, while being an outcome of complex socio-political factors, is in itself an indicator for societal cohesion and anomie, the degree of which is associated with trust in (health) policy makers, (public) health authorities, health service providers, etc. Therefore, we further recommend that vaccine hesitancy questions should be integrated in pertinent national surveys.

A double-stranded RNA (dsRNA) phage phiYY is able to kill a pyomelanin-producing Pseudomonas aeruginosa strain, which was isolated from a 40-year-old man with interstitial lung disease (ILD) and chronic lung infection. Phage therapy was used as a last resort for this patient. The three-course nebulized phiYY treatment was used to reduce the bacterial burden and clinical symptoms of the patient. Recurrences of P. aeruginosa infections were observed 1-3 days post phage therapy. The recurrent isolates exhibited distinct antibiotic-susceptibility profiles compared with the original strain yet were still susceptible to phiYY. This assay represents the application of dsRNA phage in the treatment of chronic lung infection, albeit the safety and efficacy of the dsRNA phage require further assessment.

Indole-3-acetic acid (IAA) is emerging as a key intra- and inter-kingdom signal molecule that modulates a wide range of processes of importance during plant-microorganism interaction. However, the mechanisms by which IAA carries out its functions in bacteria as well as the regulatory processes by which bacteria modulate auxin production are largely unknown. Here, we found that IAA synthesis deficiency results in important global transcriptional changes in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. Most pronounced transcriptional changes were observed in various gene clusters for aromatic acid metabolism, including auxin catabolism. To delve into the corresponding molecular mechanisms, different regulatory proteins were biochemically characterized. Among them, a TyrR orthologue was essential for IAA production through the activation of the ipdc gene encoding a key enzyme for IAA biosynthesis. We showed that TyrR specifically recognizes different aromatic amino acids which, in turn, alters the interactions of TyrR with the ipdc promoter. Screening of mutants defective in various transcriptional and post-transcriptional regulators allowed the identification of additional regulators of IAA production, including PigP and quorum sensing-related genes. Advancing our knowledge on the mechanisms that control the IAA biosynthesis in beneficial phytobacteria is of biotechnological interest for improving agricultural productivity and sustainable agricultural development.

Smallpox caused by the variola virus (VARV) was one of the greatest infectious killers of mankind. Historical records trace back smallpox for at least a millennium while phylogenetic analysis dated the ancestor of VARV circulating in the 20th century into the 19th century. The discrepancy was solved by the detection of distinct VARV sequences first in 17th-century mummies and then in human skeletons dated to the 7th century. The historical records noted marked variability in VARV virulence which scientists tentatively associated with gene losses occurring when broad-host poxviruses narrow their host range to a single host. VARV split from camel and gerbil poxviruses and had no animal reservoir, a prerequisite for its eradication led by WHO. The search for residual pockets of VARV led to the discovery of the monkeypox virus (MPXV); followed by the detection of endemic smallpox-like monkeypox (mpox) disease in Africa. Mpox is caused by less virulent clade 2 MPXV in West Africa and more virulent clade 1 MPXV in Central Africa. Exported clade 2 mpox cases associated with the pet animal trade were observed in 2003 in the USA. In 2022 a world-wide mpox epidemic infecting more than 80,000 people was noted, peaking in August 2022 although waning rapidly. The cases displayed particular epidemiological characteristics affecting nearly exclusively young men having sex with men (MSM). In contrast, mpox in Africa mostly affects children by non-sexual transmission routes possibly from uncharacterized animal reservoirs. While African children show a classical smallpox picture, MSM mpox cases show few mostly anogenital lesions, low-hospitalization rates and 140 fatal cases worldwide. MPXV strains from North America and Europe are closely related, derived from clade 2 African MPXV. Distinct transmission mechanisms are more likely causes for the epidemiological and clinical differences between endemic African cases and the 2022 epidemic cases than viral traits.

Global population growth makes it necessary to increase agricultural production yields. However, climate change impacts and diseases caused by plant pathogens are challenging modern agriculture. Therefore, it is necessary to look for alternatives to the excessive use of chemical fertilizers and pesticides. The plant microbiota plays an essential role in plant nutrition and health, and offers enormous potential to meet future challenges of agriculture. In this context, here we characterized the antifungal properties of the rhizosphere bacterium Pantoea agglomerans 9Rz4, which is active against a broad spectrum of plant pathogenic fungi. Chemical analyses revealed that strain 9Rz4 produces the antifungal herbicolin A and its biosynthetic gene cluster was identified and characterized. We found that the only acyl-homoserine lactone-based quorum sensing system of 9Rz4 modulates herbicolin A gene cluster expression. No role of plasmid carriage in the production of herbicolin A was observed. Plant assays revealed that herbicolin A biosynthesis does not affect the root colonization ability of P. agglomerans 9Rz4. Current legislative restrictions are aimed at reducing the use of chemical pesticides in agriculture, and the results derived from this study may lay the foundations for the development of novel biopesticides from rhizosphere microorganisms.

In vitro models of the human colon have been used extensively in understanding the human gut microbiome (GM) and evaluating how internal and external factors affect the residing bacterial populations. Such models have been shown to be highly predictive of in vivo outcomes and have a number of advantages over animal models. The complexity required by in vitro models to closely mimic the physiology of the colon poses practical limits on their scalability. The scalable Mini Gut (MiGut) platform presented in this paper allows considerable expansion of model replicates and enables complex study design, without compromising on in vivo reflectiveness as is often the case with other model systems. MiGut has been benchmarked against a validated gut model in a demanding 9-week study. MiGut showed excellent repeatability between model replicates and results were consistent with those of the benchmark system. The novel technology presented in this paper makes it conceivable that tens of models could be run simultaneously, allowing complex microbiome-xenobiotic interactions to be explored in far greater detail, with minimal added resources or complexity. This platform expands the capacity to generate clinically relevant data to support our understanding of the cause-effect relationships that govern the GM.

Saccharomyces non-cerevisiae yeasts are gaining momentum in wine fermentation due to their potential to reduce ethanol content and achieve attractive aroma profiles. However, the design of the fermentation process for new species requires intensive experimentation. The use of mechanistic models could automate process design, yet to date, most fermentation models have focused on primary metabolism. Therefore, these models do not provide insight into the production of secondary metabolites essential for wine quality, such as aromas. In this work, we formulate a continuous model that accounts for the physiological status of yeast, that is, exponential growth, growth under nitrogen starvation and transition to stationary or decay phases. To do so, we assumed that nitrogen starvation is associated with carbohydrate accumulation and the induction of a set of transcriptional changes associated with the stationary phase. The model accurately described the dynamics of time series data for biomass and primary and secondary metabolites obtained for various yeast species in single culture fermentations. We also used the proposed model to explore different process designs, showing how the addition of nitrogen could affect the aromatic profile of wine. This study underlines the potential of incorporating yeast physiology into batch fermentation modelling and provides a new means of automating process design.

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

The production of biogenic palladium nanoparticles (bio-Pd NPs) is widely studied due to their high catalytic activity, which depends on the size of nanoparticles (NPs). Smaller NPs (here defined as <100 nm) are more efficient due to their higher surface/volume ratio. In this work, inductively coupled plasma-mass spectrometry (ICP-MS), flow cytometry (FCM) and transmission electron microscopy (TEM) were combined to obtain insight into the formation of these bio-Pd NPs. The precipitation of bio-Pd NPs was evaluated on a cell-per-cell basis using single-cell ICP-MS (SC-ICP-MS) combined with TEM images to assess how homogenously the particles were distributed over the cells. The results provided by SC-ICP-MS were consistent with those provided by "bulk" ICP-MS analysis and FCM. It was observed that heterogeneity in the distribution of palladium over an entire cell population is strongly dependent on the Pd2+ concentration, biomass and partial H2 pressure. The latter three parameters affected the particle size, ranging from 15.6 to 560 nm, and exerted a significant impact on the production of the bio-Pd NPs. The TEM combined with SC-ICP-MS revealed that the mass distribution for bacteria with high Pd content (144 fg Pd cell-1 ) indicated the presence of a large number of very small NPs (D50 = 15.6 nm). These results were obtained at high cell density (1 × 105  ± 3 × 104 cells μl-1 ) and H2 partial pressure (180 ml H2 ). In contrast, very large particles (D50 = 560 nm) were observed at low cell density (3 × 104  ± 10 × 102 cells μl-1 ) and H2 partial pressure (10-100 ml H2 ). The influence of the H2 partial pressure on the nanoparticle size and the possibility of size-tuned nanoparticles are presented.

Huangkui capsule (HKC), a traditional Chinese medicine, has been used for medication of kidney diseases, including diabetic nephropathy (DN). The current study aimed to evaluate the effects of HKC in the modulation of gut microbiota and the amelioration of metabolite levels by using non-obese diabetes (NOD) mice with DN. The microbiota from three parts of intestines (duodenum, ileum and colon) in NOD mice with and without HKC treatment were analysed using 16S rDNA sequencing techniques. Untargeted metabolomics in plasma of NOD mice were analysed with liquid mass spectrometry. Results showed that HKC administration ameliorated DN in NOD mice and the flora in duodenum were more sensitive to HKC intervention, while the flora in colon had more effects on metabolism. The bacterial genera such as Faecalitalea and Muribaculum significantly increased and negatively correlated with most of the altered metabolites after HKC treatment, while Phyllobacterium, Weissella and Akkermansia showed an opposite trend. The plasma metabolites, mainly including amino acids and fatty acids such as methionine sulfoxide, BCAAs and cis-7-Hexadecenoic acid, exhibited a distinct return to normal after HKC treatment. The current study thereby provides experimental evidence suggesting that HKC may modulate gut microbiota and subsequently ameliorate the metabolite levels in DN.

Identification of Nocardia and Mycobacterium species by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is still a challenging task that requires both suitable protein extraction procedures and extensive databases. This study aimed to evaluate the VITEK MS Plus system coupled with updated RUO (v4.17) and IVD (v3.2) databases for the identification of Nocardia spp. and Mycobacterium spp. clinical isolates. Sample preparation was carried out using the VITEK MS Mycobacterium/Nocardia kit for protein extraction. From 90 Nocardia spp. isolates analysed, 86 (95.6%) were correctly identified at species or complex level using IVD and 78 (86.7%) using RUO. Only two strains were misidentified as other species pertaining to the same complex. Among the 106 non-tuberculous Mycobacterium clinical isolates tested from a liquid culture medium, VITEK MS identified correctly at species or complex level 96 (90.6%) isolates in the IVD mode and 89 (84.0%) isolates in the RUO mode. No misidentifications were detected. Although the IVD mode was unable to differentiate members of the M. fortuitum complex, the RUO mode correctly discriminated M. peregrinum and M. septicum. The robustness and accuracy showed by this system allow its implementation for routine identification of these microorganisms in clinical laboratories.

The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health-promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein-encoding genes shared across a human-derived L. crispatus strain selection, which includes 200 currently available human-derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra-species micro-diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi-strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.

Bloodstream infections are serious and complex infectious diseases that often require a rapid diagnosis. Polymerase chain reaction coupled with quantum dot fluorescence analysis (PCR-QDFA) is a novel diagnostic technique. This study aimed to evaluate the diagnostic performance of PCR-QDFA for pathogen detection in patients with suspected bloodstream infections (BSIs). It evaluates 29 kinds of common pathogens (24 bacteria and 5 yeasts) from blood culture bottles. The results of PCR-QDFA identification and traditional microbial laboratory identification were compared, and the latter was used as the 'gold standard' to analyse the diagnostic performance of the PCR-QDFA. In total, 517 blood culture bottles were included in this study. The PCR-QDFA identified microorganisms in 368/422 (87.2%) samples with monomicrobial growth. For the pathogens on the PCR-QDFA list, the assay showed a higher sensitivity of 97.4% (368/378). When polymicrobial growth was analysed, the PCR-QDFA successfully detected 19/25 (76%) microorganisms on the PCR-QDFA list. In addition, 82/82 negative blood culture bottles also showed no pathogens by PCR-QDFA with a specificity of 100%. In conclusion, the PCR-QDFA assay could identify a majority of the common pathogens encountered in clinical practice, showing excellent diagnostic performance for pathogen detection in patients with suspected BSIs.

Patents and the systematic analysis thereof provide important decision information for a range of stakeholders pursuing diverse goals. However, in particular academia and small-scale private sector innovators underappreciate the value of patent information for identifying research gaps, ensuring originality of their work and, in turn, maximisation of limited (public) funds. By the same token, pertinent public sector organisations, such as regulators, require overviews of potentially upcoming technologies to adequately adapt regulatory protocols. The latter, in particular if contemporary scientific evidence is not sufficient, can require a substantial amount of time and lead to a delay in the marketing of important innovations. Patent landscape analysis (PLA) is a very useful method to create overviews of technology fields and thereby indicate if, inter alia, a specific line of scientific enquiry and its application have already been pursued or potential regulatory gaps will exist in the near- to mid-term future. The objective of this communication is to increase awareness of the utility of patent information and provide support in retrieval and analysis of pertinent information for those involved in biotech R&D. Based on Espacenet, a patent search engine, we provide basic guidance on search strategy development, piloting and execution, and data preparation and analysis. To highlight the value of PLA, we also summarise selected results of a PLA we performed recently for microbiome targeting interventions, also referred to as live biotherapeutic products.

Erythritol is produced in yeasts via the reduction of erythrose into erythritol by erythrose reductases (ERs). However, the genes codifying for the ERs involved in this reaction have not been described in any Saccharomyces species yet. In our laboratory, we recently showed that, during alcoholic fermentation, erythritol is differentially produced by Saccharomyces cerevisiae and S. uvarum species, the latter being the largest producer. In this study, by using BLAST analysis and phylogenetic approaches the genes GRE3, GCY1, YPR1, ARA1 and YJR096W were identified as putative ERs in Saccharomyces cerevisiae Then, these genes were knocked out in our S. uvarum strain (BMV58) with higher erythritol biosynthesis compared to control S. cerevisiae wine strain, to evaluate their impact on erythritol synthesis and global metabolism. Among the mutants, the single deletion of GRE3 markedly impacts erythritol production, although ΔYPR1ΔGCY1ΔGRE3 was the combination that most decreased erythritol synthesis. Consistent with the increased production of fermentative by-products involved in redox balance in the Saccharomyces uvarum strain BMV58, erythritol synthesis increases at higher sugar concentrations, hinting it might be a response to osmotic stress. However, the expression of GRE3 in the S. uvarum strain was found to peak just before the start of the stationary phase, being consistent with the observation that erythritol increases at the start of the stationary phase, when there is low sugar in the medium and nitrogen sources are depleted. This suggests that GRE3 plays its primary function to help the yeast cells to maintain the redox balance during the last phases of fermentation.

Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the -1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure-function relationship of GH43 multifunctional enzymes.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed-batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine-tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Muts ) growth during the induction phase for three fed-batch strategies, conducted at constant specific growth rates (μ-stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed-batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl2 ) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (Rmeoh/Δx ), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of Rmeoh/Δx compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L-1 to 928 mg L-1 ), while production yield improved by 85% (24.8 mg gDCW -1 to 46 mg gDCW -1 ).

In order to address the global crisis in pear productivity, there has been increased attention given to advocating for the use of organic fertilizers. As part of this effort, researchers have been investigating the microbial properties of organic fertilizers to better understand their potential impact on fruit productivity. Our research focused specifically on the impact of four different ratios of sheep manure (SM) and chemical fertilizers (CF) on pear productivity. We found that replacing CF with SM resulted in a proliferation of gammaproteobacteria, Chlamydiae, Bastocatellia and Clostridia in the soil rhizosphere, which is the region around the roots of plants where most nutrient uptake occurs. Using redundancy analysis, we were able to determine that SM was particularly effective at promoting the growth of gammaproteobacteria and clostridia, which were associated with C:N ratios around 14:1 as well as the availability of K, Fe, Zn and Cu. This combination of factors was conducive to a transition from vegetative growth to reproductive growth, resulting in an increase in pear production from 43 to 56 tons per hectare. We also discovered that Blastociella acts as a buffering system in regulating soil acidity. Taken together, our findings indicate that a combination of SM and CF can improve the abundance of beneficial bacteria in the rhizosphere, leading to an increase in pear productivity.

The characterization of bacterial strains with efficient root colonization ability and the mechanisms responsible for their efficient colonization is critical for the identification and application of beneficial bacteria. In this study, we found that Burkholderia strain B23 exhibited a strong niche differentiation between the rhizosphere and rhizoplane (a niche with more abundant easy-to-use nutrients but stronger selective pressures compared with the tightly adjacent rhizosphere) when inoculated into the field-grown citrus trees. Full-length 16S rDNA amplicon analysis demonstrated that the relative abundance of B23 in the rhizoplane microbiome at 3, 5, and 9 days post-inoculation (dpi) was always higher than that at 1 dpi, whereas its relative abundance in the rhizosphere microbiome was decreased continuously, as demonstrated by a 3.18-fold decrease at 9 dpi compared to 1 dpi. Time-series comparative expression profiling of B23 between the rhizoplane and rhizosphere was performed at representative time points (1, 3, and 9 dpi) through metatranscriptomic analysis, and the results demonstrated that multiple genes involved in the uptake and utilization of easy-to-use carbohydrates and amino acids and those involved in metabolism, energy production, replication, and translation were upregulated in the rhizoplane compared with the rhizosphere at 1 dpi and 3 dpi. Several genes involved in resistance to plant- and microbial competitor-derived stresses exhibited higher expression activities in the rhizoplane compared with the rhizosphere. Furthermore, gene loci responsible for the biosynthesis of the key antifungal and antibacterial metabolites occidiofungin and ornibactin were induced, and their expression levels remained relatively stable from 3 dpi to 9 dpi in the rhizoplane but not in the rhizosphere. Collectively, our findings provide novel lights into the mechanisms underlying the root colonization of the inoculated bacterial strains and serve as a basis for the identification of strains with efficient colonization ability, thus contributing to the development of beneficial bacteria applications.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

Gut microbiota plays important roles in host metabolism. Whether and how much the gut microbiota in different gut locations contributes to the variations of host serum metabolites are largely unknown, because it is difficult to obtain microbial samples from different gut locations on a large population scale. Here, we quantified the gut microbial compositions using 16S rRNA gene sequencing for 1070 samples collected from the ileum, cecum and faeces of 544 F6 pigs from a mosaic pig population. Untargeted metabolome measurements determined serum metabolome profiles. We found 1671, 12,985 and 103,250 significant correlations between circulating serum metabolites and bacterial ASVs in the ileum, cecum, and faeces samples. We detected nine serum metabolites showing significant correlations with gut bacteria in more than one gut location. However, most metabolite-microbiota pairwise associations were gut location-specific. Targeted metabolome analysis revealed that CDCA, taurine, L-leucine and N-acetyl-L-alanine can be used as biomarkers to predict porcine fatness. Enriched taxa in fat pigs, for example Prevotella and Lawsonia intracellularis were positively associated with L-leucine, while enriched taxa in lean pigs, such as Clostridium butyricum, were negatively associated with L-leucine and CDCA, but positively associated with taurine and N-acetyl-L-alanine. These results suggested that the contributions of gut microbiota in each gut location to the variations of serum metabolites showed spatial heterogeneity.

Hypertension is a significant risk factor of cardiovascular diseases (CVDs) with high prevalence worldwide, the current treatment has multiple adverse effects and requires continuous administration. The glucagon-like peptide-1 receptor (GLP-1R) agonists have shown great potential in treating diabetes mellitus, neurodegenerative diseases, obesity and hypertension. Butyric acid is a potential target in treating hypertension. Yet, the application of GLP-1 analogue and butyric acid in reducing blood pressure and reversing ventricular hypertrophy remains untapped. In this study, we combined the therapeutic capability of GLP-1 and butyric acid by transforming Clostridium butyricum (CB) with recombinant plasmid pMTL007 encoded with hGLP gene to construct the engineered probiotics Clostridium butyricum-pMTL007-GLP-1 (CB-GLP-1). We used spontaneous hypertensive rat (SHR) models to evaluate the positive effect of this strain in treating hypertension. The results revealed that the intragastric administration of CB-GLP-1 had markedly reduced blood pressure and improved cardiac marker ACE2, AT2R, AT1R, ANP, BNP, β-MHC, α-SMA and activating AMPK/mTOR/p70S6K/4EBP1 signalling pathway. The high-throughput sequencing further demonstrated that CB-GLP-1 treatments significantly improved the dysbiosis in the SHR rats via downregulating the relative abundance of Porphyromonadaceae at the family level and upregulating Lactobacillus at the genus level. Hence, we concluded that the CB-GLP-1 greatly improves blood pressure and cardiomegaly by restoring the gut microbiome and reducing ventricular hypertrophy in rat models. This is the first time using engineered CB in treating hypertension, which provides a new idea for the clinical treatment of hypertension.

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.

The soil bacterium Pseudomonas putida KT2440 has been shown to produce selenium nanoparticles aerobically from selenite; however, the molecular actors involved in this process are unknown. Here, through a combination of genetic and analytical techniques, we report the first insights into selenite metabolism in this bacterium. Our results suggest that the reduction of selenite occurs through an interconnected metabolic network involving central metabolic reactions, sulphur metabolism, and the response to oxidative stress. Genes such as sucA, D2HGDH and PP_3148 revealed that the 2-ketoglutarate and glutamate metabolism is important to convert selenite into selenium. On the other hand, mutations affecting the activity of the sulphite reductase decreased the bacteria's ability to transform selenite. Other genes related to sulphur metabolism (ssuEF, sfnCE, sqrR, sqr and pdo2) and stress response (gqr, lsfA, ahpCF and sadI) were also identified as involved in selenite transformation. Interestingly, suppression of genes sqrR, sqr and pdo2 resulted in the production of selenium nanoparticles at a higher rate than the wild-type strain, which is of biotechnological interest. The data provided in this study brings us closer to understanding the metabolism of selenium in bacteria and offers new targets for the development of biotechnological tools for the production of selenium nanoparticles.

Glycolytic flux is a fundamental index in microbial cell factories. A glycolytic flux biosensor that can monitor glucose metabolism efficiency is a promising strategy in rewiring metabolic flux to balance growth and biosynthesis. A key design feature of the glycolytic flux biosensors is the interaction between the global transcriptional factor Cra and its regulated promoters. However, overexpression and mutation of Cra has unpredictable effects on global metabolism in Escherichia coli. Therefore, new orthogonal biosensor design strategies should be developed to circumvent metabolic issues. In this report, the promoters in glycolytic flux biosensor were replaced with synthetic promoters of varying strengths or phage-derived promoters, and the Cra DNA-binding sites were deployed into promoters at different positions and distances to yield biosensors. The de nova biosensors that depended on Cra could sense Fructose-1,6-diphosphate (FBP) with broad dynamic ranges and low basal leakage. Then the negative-response biosensors were applied to fine-tune the target ATP synthesis gene, leading to the desired increase in pyruvate production (the highest 9.66 g/L) and cell growth. Moreover, the membrane synthesis gene plsC was also dynamically activated by the positive-response biosensor, leading to effective accumulation of lycopene in the cell membrane and a 50-fold increase in lycopene titre (100.3 mg/L) when compared with the control strain, demonstrating the effective and broader usages of our biosensors.

Currently, malaria is still one of the major public health problems commonly caused by the four Plasmodium species. The similar symptoms of malaria and the COVID-19 epidemic of fever or fatigue lead to frequent misdiagnosis. The disadvantages of existing detection methods, such as time-consuming, costly, complicated operation, need for experienced technicians, and indistinguishable typing, lead to difficulties in meeting the clinical requirements of rapid, easy, and accurate typing of common Plasmodium species. In this study, we developed and optimized a universal two-dimensional labelled probe-mediated melting curve analysis (UP-MCA) assay based on multiplex and asymmetric PCR for rapid and accurate typing of five Plasmodium species, including novel human Plasmodium, Plasmodium knowlesi (Pk), in a single closed tube following genome extraction. The assay showed a limit of detection (LOD) of 10 copies per reaction and could accurately distinguish Plasmodium species from intra-plasmodium and other pathogens. Additionally, we proposed and validated different methods of fluorescence quenching and tag design for probes that are suitable for UP-MCA assays. Moreover, the clinical performance of the Plasmodium UP-MCA assay using a base-quenched universal probe was evaluated using 226 samples and showed a sensitivity of 100% (164/164) and specificity of 100% (62/62) at a 99% confidence interval, with the microscopy method as the gold standard. In summary, the UP-MCA assay showed excellent sensitivity, specificity, and accuracy for genotyping Plasmodium species spp. Additionally, it facilitates convenient and rapid Plasmodium detection in routine clinical practice and has great potential for clinical translation.

The maintenance of the beneficial plant microbiome to control plant pathogens is an emerging concept of disease management, and necessitates a clear understanding of these microbial communities and the environmental factors that affect their diversity and compositional structure. As such, studies investigating the microbiome of economically significant cultivars within each growing region are necessary to develop adequate disease management strategies. Here, we assessed the relative impacts of growing season, management strategy, and geographical location on the fungal microbiome of 'Honeycrisp' apples from seven different orchard locations in the Atlantic Maritime Ecozone for two consecutive growing years. Though apple fruit tissue was dominated by relatively few fungal genera, significant changes in their fungal communities were observed as a result of environmental factors, including shifts in genera with plant-associated lifestyles (symbionts and pathogens), such as Aureobasidium, Alternaria, Penicillium, Diplodia, and Mycosphaerella. Variation in fungal composition between different tissues of fruit was also observed. We demonstrate that growing season is the most significant factor affecting fungal community structure and diversity of apple fruit, suggesting that future microbiome studies should take place for multiple growing seasons to better represent the host-microbiome of perennial crops under different environmental conditions.