Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knock-out by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene was may attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence several strategies are proposed to enhance gene knockout and plasmid curing.

Saccharomyces cerevisiae with its robustness and good acid tolerance, is an attractive candidate for use in various industries, including waste-based biorefineries where a high-value organic acid is produced, such as fumaric acid could be beneficial. However, this yeast is not a natural producer of dicarboxylic acids, and genetic engineering of S. cerevisiae strains is required to achieve this outcome. Disruption of the natural FUM1 gene and the recombinant expression of fumarase and malate transporter genes improved the malic acid-to-fumaric acid conversion by engineered S. cerevisiae strains. The efficacy of the strains was significantly influenced by the source of the fumarase gene (yeast versus bacterial), the presence of the XYNSEC signal secretion signal and the available oxygen in synthetic media cultivations. The ΔFUM1Ckr_fum + mae1 and ΔFUM1(ss)Ckr_fum + mae1 strains converted extracellular malic acid into 0.98 and 1.11 g/L fumaric acid under aerobic conditions.

A Gram-positive facultative anaerobe, nonspore forming, and nonmotile bacterial strain M31 was isolated from faecal contaminated soil. The strain is previously reported to produce a novel antimicrobial lipopeptide and displayed probiotic properties. The strain M31 is catalase negative and fermented d-galactose, d-glucose, esculin, d-maltose, d-lactose, d-melibiose, d-raffinose, d-saccharose (weak reaction), d-xylose (weak reaction), d-ribose (weak reaction), and l-arabinose (weak reaction). The majority of fatty acids were C16:0 (53.9%), C18:0 (26.9%), and C19:0 cyclo ω8c (19.1%). The genome is 2 234 040 bp long with 38.81% guanine-cytosine (GC) content. The pairwise ortho average nucleotide identity and digital DNA-DNA hybridization values of strain M31 with its closest relative species from Limosilactobacillus reuteri clade and Lm. rudii is below the recommended cut-off of 95% and 70%, respectively. Herein, we propose Lm. walteri sp. nov. as a novel species of the genus Limosilactobacillus with M31 = MTCC 12838 = JCM 32759 = KCTC 25569.

Thiobencarb is a highly effective thiocarbamate herbicide frequently used in rice fields globally. In this study, three bacterial strains (Dechloromonas sp. Th1, Thauera sp. Th2, and Azoarcus sp. Th3) isolated from immobilized biomass were analyzed for thiobencarb degradation under anaerobic conditions, with nitrate serving as an electron acceptor. The experimental results showed that thiobencarb was transformed by Dechloromonas sp. Th1 and Thauera sp. Th2 to produce high concentrations of metabolites in a mineral medium. Dechloromonas sp. Th1 dechlorinated the herbicide to benzyl mercaptan, which was then degraded by Thauera sp. Th2 and Azoarcus sp. Th3. Azoarcus sp. Th3 effectively degraded intermediates, i.e. 4-chlorobenzyl alcohol, 4-chlorobenzoic acid, and benzoic acid, produced from the degradation by Dechloromonas sp. Th1 and Thauera sp. Th2. The cross-feeding, nutrient sharing, and cooperation of all isolates in the degradation process decreased the concentrations of intermediate products. The determination of the degradation kinetics showed that the utilization in the exponential phase of the mixed bacteria was consistent with the Michaelis-Menten model, with a maximum degradation rate of 1.56 ± 0.16 µM day-1. This study showed the degradation mechanisms in bacteria and the synergistic process in the degradation of thiobencarb and its metabolites.

An activity-based labeling (ABL) approach was investigated for the phenol-oxidizing bacterium, Pseudomonas sp. CF600. Phenol-grown cells were exposed to several different terminal diynes, and following cell breakage, extracts of these cells were added to copper-catalyzed alkyne/azide cycloaddition reactions containing Alexa Fluor 647 azide. Analysis of total cell proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and near-infrared scanning demonstrated covalent fluorescent labeling of a 58- and a 34-kDa polypeptide in all diyne-treated cell types. Further studies using 1,4-diethynylbenzene (DEB) demonstrated that these labeled polypeptides were consistently detected in cells grown on substrates that exhibited phenol-dependent O2 uptake activity but not observed when cells were grown on substrates such as dextrose or catechol that did not support this activity. Fluorescent labeling of the two polypeptides in DEB-treated, phenol-grown cells was time dependent and was inhibited by several known substrates for phenol hydroxylase. These results suggest that diverse diynes act as mechanism-based inactivators of phenol hydroxylase in Pseudomonas sp. CF600 and that this effect can be exploited by ABL approaches to selectively label the major 58- and 34-kDa subunits of the hydroxylase component of this complex enzyme.

The recent surge in beta-lactamase resistance has created superbugs, which pose a current and significant threat to public healthcare. This has created an urgent need to keep pace with the discovery of inhibitors that can inactivate these beta-lactamase producers. In this study, the in vitro and in vivo activity of 1,4,7-triazacyclononane-1,4,7 triacetic acid (NOTA)-a potential metallo-beta-lactamase (MBL) inhibitor was evaluated in combination with meropenem against MBL producing bacteria. Time-kill studies showed that NOTA restored the efficacy of meropenem against all bacterial strains tested. A murine infection model was then used to study the in vivo pharmacokinetics and efficacy of this metal chelator. The coadministration of NOTA and meropenem (100 mg/kg.bw each) resulted in a significant decrease in the colony-forming units of Klebsiella pneumoniae NDM-1 over an 8-h treatment period (>3 log10 units). The findings suggest that chelators, such as NOTA, hold strong potential for use as a MBL inhibitor in treating carbapenem-resistant Enterobacterale infections.

Azospirillum baldaniorum Sp 245 is a model plant growth-promoting rhizobacterium. The first cross-talk with plants takes place within the roots. Roots cells growth is constrained by the primary cell wall (CW). Also, neighboring CW form the apoplast that should affect cells signaling and biochemical messages. Studies on CW phenolic composition ferulate (FA), diferulates (DFA) and p-coumarate and polyamines (PA) metabolisms of A. baldaniorum Sp 245- inoculated roots and on bacterial PA production in culture media should help to understand more about the mechanisms involved in Azospirillum-root association. For this purpose, CW-bound FA, DFA and p-coumarate contents, putrescine (put) and spermidine contents, diamine and polyamine oxidases activities, and H2O2 content of Cucumis sativus roots from dark grown seedlings inoculated with A. baldaniorum Sp 245 were determined. Also, bacterial PA production under constant agitation or static conditions was evaluated. Results showed lesser contents of all phenolics, and higher FA/DFA ratio in CW of inoculated roots that should be responsible for roots growth promotion. Also, the increased put content, DAO activity, and H2O2 production in the roots should be associated to A. baldaniorum Sp 245 growth promotion in early stages. Finally, the participation of both PA in A. baldaniorum Sp 245 biofilm formation was demonstrated.

Strain GKT was isolated from the Kumbet plateu of Giresun in Turkey. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GKT belonged to genus Janthinobacterium and 16S rRNA gene sequence similarities with all type strains of the genus Janthinobacterium were 98.89%-99.78%. The calculated pairwise average nucleotide identity (ANI) values between strain GKT and all type strains of Janthinobacterium species were in the range of 79.8%-93.2%. In addition, digital DNA-DNA hybridization (dDDH) values were in the range of 23.0%-51.7%. Major fatty acids are C10:03OH, C12:0, C16:1ω7c, C16:0, and C18:1ω7c, and polar lipids included phosphatidylethanolamine, phosphatidylglycerol, also one unidentified phospholipid and one unidentified aminophospholipid. The respiratory quinone of strain GKT was determinated to be Q-8. The genome sizes of strain GKT was 6 197 538 bp with 63.16% G + C ratio. Strain GKT is Gram-stain-negative, aerobic, rod-shaped, and motile. A violet pigment was produced by strain GKT. The crude violacein pigments were separated into three diferent bands on a TLC sheet. Then violacein and deoxyviolacein were purifed by vacuum liquid column chromatography and identifed by NMR spectroscopy. The antimicrobial activities of purifed violacein and deoxyviolacein were screened for seven microorganisms. Based on the results of the morphological, biochemical, physiological, phylogenetic, and genomic characteristics, we propose classifying the strain GKT as representative of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium kumbetense sp. nov. is proposed (GKT = LMG 32662T = DSM 11423T).

Parabens are substances with antifungal and antibacterial properties, suspected to be endocrine disruptors and widely used as preservatives in cosmetics. In this case, exposure to these compounds is mainly dermal and interactions may occur with skin components including cutaneous mycobiota. In this work, we have explored the in vitro reciprocal interactions between three parabens (methylparaben, ethylparaben and propylparaben) and yeasts from the human cutaneous mycobiota (Candida parapsilosis, Cryptococcus uniguttulatus and Rhodotorula mucilaginosa) by studying the effect of these parabens on fungal growth and the fungal ability to metabolize the tested compounds. Our results showed that, at the tested concentrations, the growth of three strains of C. parapsilosis was not influenced by the presence of parabens. Whereas, using the same parabens concentrations, growth of C. uniguttulatus and R. mucilaginosa was completely inhibited by ethylparaben since the first day of contact, whereas these same fungi were not sensitive to the two other parabens, even after seven days of incubation. The presence of a lamellar wall in these basidiomycete fungi as well as the physico-chemical properties of ethylparaben could explain this selective inhibition. Additionally, C. parapsilosis and R. mucilaginosa degraded 90 to 100% of propylparaben after seven days of incubation but had no effect on the other tested parabens. Thus, their enzymes seem to only degrade long chain parabens. In the same conditions, C. uniguttulatus did not degrade any paraben. This inability may be due to the absence of fungal enzymes able to degrade parabens or to the possible inaccessibility of intracellular enzymes due to the polysaccharide capsule. Our work has shown that parabens can act differently from one fungus to another within the cutaneous mycobiota. These preliminary results have evidenced that in vitro parabens, contained in cosmetic products, could be involved in the occurrence of a state of dysbiosis. The tested yeasts from the cutaneous mycobiota can also be involved in the degradation of parabens and thereby reduce, according to the produced metabolites and their activities, the risk of endocrine disruption they can induce.