Zinc is an essential trace metal required for numerous cellular processes in all forms of life. In order to maintain zinc homeostasis, bacteria have developed several transport systems to regulate its uptake. In this study, we investigated zinc transport systems in the enteric pathogen Vibrio cholerae , the causative agent of cholera. Bioinformatic analysis predicts that two gene clusters, VC2081 to VC2083 (annotated as zinc utilization genes znuABC ) and VC2551 to VC2555 (annotated as z inc- r egulated g enes zrgABCDE ), are regulated by the putative zinc uptake regulator Zur. Using promoter reporter and biochemical assays, we confirmed that Zur represses znuABC and zrgABCDE promoters in a Zn ²⁺ -dependent manner. Under Zn ²⁺ -limiting conditions, we found that mutations in either the znuABC or zrgABCDE gene cluster affect bacterial growth, with znuABC mutants displaying a more severe growth defect, suggesting that both ZnuABC and ZrgABCDE are involved in Zn ²⁺ uptake and that ZnuABC plays the predominant role. Furthermore, we reveal that ZnuABC and ZrgABCDE are important for V. cholerae colonization in both infant and adult mouse models, particularly in the presence of other intestinal microbiota. Collectively, our studies indicate that these two zinc transporter systems play vital roles in maintaining zinc homeostasis during V. cholerae growth and pathogenesis.

Colistin is a drug of last resort for the treatment of many multidrug-resistant Gram-negative bacteria, including carbapenem-resistant Klebsiella pneumoniae (CRKP). However, bacteria readily acquire resistance to this antibiotic via lipopolysaccharide modifications caused by spontaneous mutations or from enzymes acquired by lateral gene transfer. The fitness cost associated with these modifications remains poorly understood.

Vibrio cholerae is the causative agent of cholera, a dehydrating diarrheal disease. This Gram-negative pathogen is able to modulate its gene expression in order to combat stresses encountered in both aquatic and host environments, including stress posed by reactive oxygen species (ROS). In order to further the understanding of V. cholerae’s transcriptional response to ROS, we performed an RNA sequencing analysis to determine the transcriptional profile of V. cholerae when exposed to hydrogen hydroperoxide. Of 135 differentially expressed genes, VC0139 was amongst the genes with the largest induction. VC0139 encodes a protein homologous to the DPS (DNA-binding protein from starved cells) protein family, which are widely conserved and are implicated in ROS resistance in other bacteria. Using a promoter reporter assay, we show that during exponential growth, dps is induced by H₂O₂ in a manner dependent on the ROS-sensing transcriptional regulator, OxyR. Upon entry into stationary phase, the major stationary phase regulator RpoS is required to transcribe dps. Deletion of dps impaired V. cholerae resistance to both inorganic and organic hydroperoxides. Furthermore, we show that Dps is involved in resistance to multiple environmental stresses. Finally, we found that Dps is important for V. cholerae adult mouse colonization, but becomes dispensable in the presence of antioxidants. Taken together, our results suggest that Dps plays vital roles in both V. cholerae stress resistance and pathogenesis.

Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species (ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis (Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair (MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC^- mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC^- mice was significantly higher than that in NAC⁺ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms. Cholera is a devastating diarrheal disease that is still endemic to many developing nations, with the worst outbreak in history having occurred recently in Yemen. Vibrio cholerae, the causative agent of cholera, transitions from aquatic reservoirs to the human gastrointestinal tract, where it expresses virulence factors to facilitate colonization of the small intestines and to combat host innate immune effectors, such as reactive oxygen species (ROS). We applied a genome-wide transposon screen (Tn-seq) and identified that deletion of mutS, which is part of DNA mismatch repair system, drastically increased colonization in ROS-rich mice. The deletion of mutS led to the accumulation of catalase-overproducing mutants and a high frequency rugose phenotype when exposed to ROS selective pressures in vivo. Additionally, ROS elevated mutation frequency in wildtype, both in vitro and in vivo. Our data imply that V. cholerae may modulate mutation frequency as a temporal adaptive strategy to overcome oxidative stress and to enhance infectivity.

The estimation of a postmortem interval (PMI) is particularly important for forensic investigations. The aim of this study was to assess the succession of bacterial communities associated with the decomposition of mouse cadavers and determine the most important biomarker taxa for estimating PMIs. High-throughput sequencing was used to investigate the bacterial communities of gravesoil samples with different PMIs, and a random forest model was used to identify biomarker taxa. Redundancy analysis was used to determine the significance of environmental factors that were related to bacterial communities. Our data showed that the relative abundance of Proteobacteria, Bacteroidetes and Firmicutes showed an increasing trend during decomposition, but that of Acidobacteria, Actinobacteria and Chloroflexi decreased. At the genus level, Pseudomonas was the most abundant bacterial group, showing a trend similar to that of Proteobacteria. Soil temperature, total nitrogen, NH₄⁺-N and NO₃⁻-N levels were significantly related to the relative abundance of bacterial communities. Random forest models could predict PMIs with a mean absolute error of 1.27 days within 36 days of decomposition and identified 18 important biomarker taxa, such as Sphingobacterium, Solirubrobacter and Pseudomonas. Our results highlighted that microbiome data combined with machine learning algorithms could provide accurate models for predicting PMIs in forensic science and provide a better understanding of decomposition processes.

Citrate is a ubiquitous compound and can be utilized by many bacterial species, including enteric pathogens, as a carbon and energy source. Genes involved in citrate utilization have been extensively studied in some enteric bacteria, such as Klebsiella pneumoniae, however their role in pathogenesis is still not clear. In this study, we investigated citrate utilization and regulation in Vibrio cholerae, the causative agent of cholera. The putative anaerobic citrate fermentation genes in V. cholerae, consisting of citCDEFXG, citS-oadGAB, and two-component system (TCS) genes citAB, are highly homologous to those in K. pneumoniae. Deletion analysis shows that these cit genes are essential for V. cholerae growth when citrate is the sole carbon source. The expression of citC and citS operons was dependent on citrate and CitAB, whose transcription was autorepressed and regulated by another TCS regulator ArcA. In addition, citrate fermentation was under the control of catabolite repression. Mouse colonization experiments showed that V. cholerae can utilize citrate in vivo using the citrate fermentation pathway and that V. cholerae likely needs to compete with other members of the gut microbiota to access citrate in the gut.