The white-rot fungus Pleurotus ostreatus is an agaricomycete that is frequently used in molecular genetics studies as many useful tools are applicable to the fungus. In particular, efficient gene targeting using homologous recombination and CRISPR/Cas9 enables the introduction of a mutation in the gene of interest for functional analysis. Multiple genes encoding various lignocellulose-degrading enzymes are predicted to be present in the genome; therefore, analyses of multiple-gene mutants are required to elucidate the mechanisms underlying lignocellulose degradation by P. ostreatus. Conventional tools for generating multiple-gene mutations in P. ostreatus are laborious and time-consuming. Therefore, more efficient and practical methods are needed. In this study, we introduced CRISPR/Cas9-assisted multiple-gene mutations using a polycistronic tRNA and CRISPR guide RNA approach. The frequency (triple-gene mutation in fcy1, vp2, and   62347) was only 3.3% when a tetracistronic tRNA-sgRNA containing four different sgRNAs targeting fcy1, vp2, vp3, or   62347 was expressed. It increased to 20% (triple-gene mutation in vp1, vp2, and vp3) after a tricistronic tRNA-sgRNA was expressed with replaced/modulated promoter and tRNA sequences. This study demonstrated, for the first time, the applicability of a strategy to induce multiple-gene mutations in P. ostreatus in a transformation experiment.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as an important global threat in recent years. The objective of the present study was to characterize the molecular characteristics, antibiotic resistance pattern and the distribution of virulence factors in CRKP isolated from different clinical specimens. A total of 60 clinical CRKP isolates were collected from clinical samples. Based on Clinical Laboratory Standards Institute guidelines, antimicrobial susceptibility testing was assessed by the disk diffusion method. Carbapenem and aminoglycoside resistance determinants in addition to virulence genes were inspected by PCR. Molecular characteristics of CRKP isolates were analyzed by random amplified polymorphic DNA (RAPD) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR. All isolates were resistant to imipenem, meropenem, cefoxitin, levofloxacin, cefotaxime, ceftazidime and ciprofloxacin. Resistance to tetracycline, gentamicin and kanamycin were detected in 53%, 75% and 72% of isolates, respectively. The most common carbapenem resistance genes were OXA-48 (28.5%) and NDM (22%). The most common aminoglycosides resistance genes were aac6´Ib (57%) and aac(3)-IVa (28%). The most prevalent virulence genes were mrkD (82%), entB (62%) and ybts (58%). ERIC and RAPD analyses revealed 55 and 53 different patterns of CRKP isolates, respectively. We conclude that CRKP infections have been associated with different genotypes and that the carbapenemase type (OXA-48) and AME gene (aac6´-Ib) were widely distributed in CRKP isolates in our hospital. Continued compliance with existing phenotypes and genotypes, and strict enforcement of infection control guidelines, are recommended approaches for the prevention and dissemination of these strains.

A soil-dwelling cyanobacterial strain (KLS-BP-3A_PS), has been isolated from the biodiversity rich Northeast region of India and characterized using a polyphasic approach. The strain was collected from a field covered with grass, near a stream from the Unakoti district of Tripura. Upon culturing in the laboratory, initial studies indicated the strain to be showing typical Nostoc or Nostoc-like morphology. Subsequently, 16S rRNA gene phylogenetic analyses using Neighbour joining, Maximum-likelihood, and Bayesian inference methods gave a distinct and stable positioning of the strain inside the genus Desikacharya. Upon recovery of the full-length operon of the 16S-23S ITS region with both tRNAs (tRNAIle and tRNAAla), the folded secondary structures revealed unique patterns of the D1-D1', V2, Box-B, and V3 regions of the strain KLS-BP-3A_PS as compared to phylogenetically related species of the genus Desikacharya. The total evidence approach indicated conclusively that the strain under investigation is a new species of the genus Desikacharya, which we describe as Desikacharya kailashaharensis in accordance with the International Code of Nomenclature for algae, fungi, and plants. Further, 16S rRNA gene phylogeny and evaluation of the 16S-23S ITS operons along with implying a re-examination of the family level affiliation of Desikacharya as well its generic limits may be in order. Notably, this study brings into focus the very less explored Northeast region of India which shares two global biodiversity hotspots in the world.

A polyphasic taxonomic approach was used to characterize a novel bacterium, designated strain CC-YST710T, isolated from poultry manure sampled in Taiwan. Cells of strain CC-YST710T were aerobic, Gram-stain-negative, nonmotile, nonspore-forming rods, displaying positive reactions for catalase, and oxidase activities. Strain CC-YST710T was found to grow optimally at 30°C, pH 7.0, and in the presence of 2% (w/v) NaCl. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, four unidentified aminolipids, one unidentified aminophospholipid, and five unidentified lipids. The major polyamine was spermidine. The dominating cellular fatty acids (> 5%) included C16:0, C18:0, and C18:1ω7c/C18:1ω6c. Based on 16S rRNA gene analysis, this isolate showed the closest phylogenetic relationship with 'Pseudogemmobacter humicola' (97.6%), followed by Pseudogemmobacter bohemicus (97.2%) and 'Pseudogemmobacter hezensis' (97.5%). The draft genome (4.3 Mb) had 62.9 mol% G + C content. CC-YST710T can be distinguished from other Pseudogemmobacter species due to the exclusive presence of key genes encoding p-hydroxybenzoate hydroxylase, protocatechuate 3, 4-dioxygenase (α and β chain), and homogentisate 1, 2-dioxygenase involved in the degradation of phenolic compounds such as p-hydroxybenzoic acid, protocatechuate, and homogentisate, respectively. Orthologous average nucleotide identity (OrthoANI) of the isolate with the type strains of the genera Pseudogemmobacter were 77.6%‒78.0% (n = 3), followed by Tabrizicola (72.3%‒73.7%, n = 5), and Gemmobacter(72.3%‒73.5%, n = 7). Based on its distinct phylogenetic, phenotypic, and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence, OrthoANI, digital DDH, and the phylogenomic placement, strain CC-YST710T is considered to represent a novel Pseudogemmobacter species, for which the name Pseudogemmobacter faecipullorum sp. nov. (type strain CC-YST710T = BCRC 81286T = JCM 34182T).

Stress response and checkpoint activation are the main determinants of cellular survival in adverse conditions. In Schizosaccharomyces pombe, these are controlled by the Mitogen Activated Protein Kinase Spc1 and the Cyclin dependent Kinase Cdc2 respectively. Cdc2 is regulated positively by Cdc25 and negatively by Wee1. Changes in Cdc2 activity can be sensed by Spc1 resulting in the modulation of mitotic timing by Spc1. Functional cross talks between cell cycle regulation and MAPK machinery during regulation of mitotic timing are well characterised but the presence of similar communication during stress response remains unexplored. In this study we report how the checkpoint activator kinase Wee1 can also influence the transcriptional response to oxidative stress. We show that deletion of Wee1 results in changes in gene expression of the cells, especially with respect to genes whose expression is known to be regulated by Spc1. These differences are seen in unperturbed cells as well as during oxidative stress. Moreover, such variations extend beyond what could be expected to occur due to the known enhanced Spc1 activity of these cells. This is the first depiction of the influence of Wee1 and consequently Cdc2 activity on transcriptional response to oxidative stress.

Foodborne and zoonotic viral pathogens are responsible for substantial morbidity and mortality worldwide. These viruses can be transmitted through foods such as dairy products to humans and cause several acute and chronic diseases. This study aimed to investigate the prevalence and profile of different foodborne and zoonotic viruses in raw cow milk samples. We collected 492 raw cow milk samples from local dairy markets in Qazvin, Iran. Then we evaluated the presence of hepatitis A virus, noroviruses, rotavirus, astrovirus, bovine leukaemia virus (BLV) and tick-borne encephalitis virus (TBEV) in samples using conventional and nested reverse transcription-polymerase chain reaction methods. We found that 34.95, 7.72, 25.81, 14.63, 66.86, 12.80 and 21.34% of raw milk samples were contaminated with norovirus GI, norovirus GII, hepatitis A virus, rotavirus, astrovirus, BLV and TBEV viruses, respectively. Interestingly, the samples collected from the city's south area revealed a higher prevalence of foodborne and zoonotic viruses. Astrovirus and its combination with norovirus GI were the most prevalent virus profiles. Also, the highest correlations were observed among the presence of rotavirus and hepatitis A viruses (0.36) and TBEV and norovirus GII (0.31). Considering the prevalence rate and virus profiles of different foodborne and zoonotic viruses in raw milk samples, hygiene practices and the pasteurization process are strongly suggested to be conducted throughout the cow milk production chain and in dairy industries to prevent infections with these pathogens.

Escherichia coli is able to ferment mixed carbon sources and produce various fermentation end-products. In this study, the function of FhlA protein in the specific growth rate (µ), metabolism, regulation of ΔpH and proton ATPase activity was investigated. Reduced µ in fhlA mutant of ∼25% was shown, suggesting the role of FhlA in the growth process. The utilization rate of glycerol is decreased in fhlA ∼ 2 fold, depending on the oxidation-reduction potential values. Bacteria regulate the activity of hydrogenase enzymes during growth depending on the external pH, which manifests as a lack of hydrogen gas generation during glycerol utilization at pH values below 5.9. It is suggested that cells maintain ΔpH during the fermentative growth via formate-lactate-succinate exchange. The decrement of the value of pHin, but not of pHex in mutant cells, is regulating ΔpH and consequently proton motive force generation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a serious contagious disease. MTB-encoded small regulatory RNA (sRNA) MTS2823 was reported to be upregulated in the plasma of TB patients. Nevertheless, whether MTS2823 is implicated in MTB drug resistance is unclear. Human macrophage cell line THP-1 was infected with the drug-susceptible strain H37Rv or the multidrug-resistant (MDR) strain 8462. Colony-forming unit assay was implemented for evaluating intracellular growth of the MTB strains. Enzyme-linked immunosorbent assay was used for measurement of inflammatory cytokines. Real-time quantitative polymerase chain reaction was utilized to assess MTS2823 and recombinase A (recA) expression in strains 8462 and H37Rv. Nitric oxide (NO) production in the MDR strain-infected THP-1 cells was measured. In this study, MTS2823 was found to display a low level in the MDR strain. Overexpressing MTS2823 promoted intracellular growth of the MDR strain and inhibited inflammatory cytokine and NO production in infected THP-1 cells. RecA might be a target of MTS2823 in the MDR strain. Overall, MTB-encoded sRNA MTS2823 displays a low level and regulates the growth of the MDR strain in THP-1 cells by modulating recA.

Erythritol is a 4-carbon polyol produced with the aid of microbes in presence of hyper-osmotic stress. It is the most effective sugar alcohol that is produced predominantly by fermentation. In comparison to various polyols, it has many precise functions and is used as a flavor enhancer, sequestrant, humectant, nutritive sweetener, stabilizer, formulation aid, thickener, and a texturizer. Erythritol production is a common trait in a number of the yeast genera viz., Trigonopsis, Candida, Pichia, Moniliella, Yarrowia, Pseudozyma, Trichosporonoides, Aureobasidium, and Trichoderma. Extensive work has been carried out on the biological production of erythritol through Yarrowia, Moniliella, Candida, and other yeast strains, and numerous strategies used to improve erythritol productivity through mutagenesis and genetic engineering are discussed in this review.

Noncanonical D-amino acids are involved in peptidoglycan and biofilm metabolism in bacteria. Previously, we identified amino acid racemases with broad substrate specificity, including YgeA from Escherichia coli, which strongly prefers homoserine as a substrate. In this study, we investigated the functions of this enzyme in vivo. When wild-type and ygeA-deficient E. coli strains were cultured in minimal medium containing D-homoserine, the D-homoserine level was significantly higher in the ygeA-deficient strain than in the wild-type strain, in which it was almost undetectable. Additionally, D-homoserine was detected in YgeA-expressed E. coli cells cultured in minimal medium containing L-homoserine. The growth of the ygeA-deficient strain was significantly impaired in minimal medium with or without supplemental D-homoserine, while L-methionine, L-threonine or L-isoleucine, which are produced via L-homoserine, restored the growth impairment. Furthermore, the wild-type strain formed biofilms significantly more efficiently than the ygeA-deficient strain. Addition of L- or D-homoserine significantly suppressed biofilm formation in the wild-type strain, whereas this addition had no significant effect in the ygeA-deficient strain. Together, these data suggest that YgeA acts as an amino acid racemase and plays a role in L- and D-homoserine metabolism in E. coli.

Acute kidney injury (AKI) is a global public health hazard with high morbidity and mortality. Sepsis accounts for nearly half of all causes of AKI. Scientists have made a great effort to explore effective therapeutic agents with limited side effects in the treatment of AKI, but have had little success. With the development of gut flora study, Akkermansia muciniphila (A. muciniphila) has been proven to prevent different organs by regulating the inflammatory response. However, the reno-protective function is still unknown. Here, the AKI model was induced using lipopolysaccharide (LPS) in mice with or without pretreatment of A. muciniphila. Renal function and histological change were measured. Inflammatory factors were detected by ELISA and rt-PCR. TLR4/NF-κB signaling factors and NLRP3 inflammasome were tested by western blot and immunohistochemistry. Pretreatment of A. muciniphila markedly inhibited inflammatory response and ameliorated kidney histopathological changes. Furthermore, the TLR4, p-NF-κB p65, and downstream IκBα were notably activated in the model group and inhibited by A. muciniphila. A similar effect was found in the regulation of NLRP3 inflammasome. In conclusion, pretreatment with A. muciniphila could protect against LPS-induced AKI by inhibition of the TLR4/NF-κB pathway and NLRP3 inflammasome activation. It may be a new therapeutic strategy for AKI prevention and treatment in the future.

Bacteria have a mechanism to rescue stalled ribosomes known as trans-translation consisting of SsrA, a transfer-messenger RNA (tmRNA), and the small protein SmpB. Other alternative rescue mechanisms mediated by ArfA and ArfB proteins are present only in some species. Ribosome rescue mechanisms also play a role in tolerance to antibiotics and various stresses such as heat. This study shows that the genome of the soil bacterium A. vinelandii harbours genes encoding for tmRNA, SmpB, two paralogs of ArfA (arfA1 and arfA2), and ArfB. A number of mutant strains carrying mutations in the ssrA, arfA1, arfA2, and arfB genes were constructed and tested for their growth and susceptibility to heat and the antibiotic tetracycline. We found that the inactivation of both ssrA and one or the two arfA genes was detrimental to growth and caused a higher susceptibility to heat and to the antibiotic tetracycline. Interestingly, the arfB mutant strain was unable to grow after 2 h of incubation at 45°C. Inactivation of arfB in the ssrA-arfA1-arfA2 strain caused a lethal phenotype since the quadruple mutant could not be isolated. Taken together, our data suggest that both arfA1 and arfA2, as well as arfB, are functional as back up mechanisms, and that the ArfB pathway has an essential role that confers A. vinelandii resistance to high temperatures.

Plant-based dairy alternatives are gaining increasing interest, e.g. alternatives to yoghurt, cheese, and butter. In all these products butter flavor (diacetyl + acetoin) plays an important role. We previously have reported efficient butter flavor formation from low value dairy side streams using a dairy isolate of Lactococcus lactis deficient in lactate dehydrogenase. Here, we have tested the ability of this strain, RD1M5, to form butter flavor in plant milks based on oat and soy. We found that oat milk, with its high sugar content, supported more efficient production of butter aroma, when compared to soy milk. When supplemented with glucose, efficient butter aroma production was achieved in soy milk as well. We also carried out an extended adaptive laboratory evolution of the dairy strain in oat milk. After two months of adaptation, we obtained a strain with enhanced capacity for producing butter aroma. Despite of its high sugar content, RD1M5 and its adapted version only metabolized approximately 10% of the fermentable sugars available in the oat milk, which we found was due to amino acid starvation and partly starvation for vitamins. The study demonstrates that dairy cultures have great potential for use in plant-based fermentations.

Vibrio cholerae is an etiological cause of cholera and has been implicated in several epidemics. Exploration of the antimicrobial signatures of culinary spices has become an important industrial tool to suppress the growth of foodborne bacterial pathogens including Vibrio spp. The antibiofilm and antimotility activities of some selected natural antimicrobial agents were then evaluated. All the extracts showed vibriostatic activities with minimum inhibitory concentration (MIC) ranging from 0.1% to 0.4%. Cinnamon and black pepper demonstrated significant biofilm inhibition activity from 94.77% to 99.77% when administered at 100% MIC. Black pepper extract also demonstrated the highest biofilm inhibition activity against the established biofilms of V. cholerae O1 and O139. Cinnamon, calabash nutmeg, and black pepper significantly inhibited swimming and swarming motility by 85.51% to 94.87%. Sub-MICs (50% and 75%) of some extracts were also effective as an antibiofilm and antimotility agent against the tested strains. The findings of our study suggest the potential application of natural antimicrobial agents such as spices in food to inhibit biofilm formation and motility, which consequently mitigate the virulence and persistence of the pathogen in the food supply chain.

Streptomyces coelicolor A3(2) is considered as the model strain among the Streptomyces and has the capacity to produce several natural molecules. Our hypothesis was that cultivation of the strain onto a complex carbon source such as wheat bran (WB) would induce the production of various secondary metabolites due to the presence of complex polysaccharides. A multiapproach has been performed in order to investigate: (1) whether that strain could degrade lignocellulose; (2) which enzymatic and metabolic pathways secondary were over-expressed when grown on WB. The transcriptomic approach showed the expression of several CAZymes significantly expressed when grown on WB such as endoglucanases (encoding for GH74, GH5_8, and GH12) and xylanases (GH11 and CE4 encoding for respectively endo-1,4-beta-xylanase and an acetyl-xylan esterase). Enzymatic activities showed an expression of xylanase (115.3 ± 32.2 mUI/ml) and laccase-peroxidase (101.5 ± 10.9 mUI/ml) during WB degradation by S. coelicolor A3(2). Metabolomics showed that the production of secondary metabolites differed between growth on either glucose or WB as carbon source, which may be correlated to the complexity of carbon compounds within WB, which are similar to the ones encountered in soils and should represent more the in situ carbon conditions which Streptomyces might face off. This opens opportunities for the bioproduction of molecules of interest from WB.