Background: The human intestine, in which Vibrio cholerae exerts its virulence, is an anaerobic environment. Results: When grown anaerobically with trimethylamine N-oxide (TMAO), V. cholerae exhibited enhanced growth and cholera toxin (CT) production was remarkably induced. Conclusion: Anaerobic TMAO respiration may serve as a signal to increase V. cholerae virulence. Significance: A novel growth condition that induces CT production is uncovered.

ABSTRACTPseudomonas aeruginosaundergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3−) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments againstP. aeruginosabiofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B12to be a molecule that can reinstate defective anaerobic growth ofP. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth ofP. aeruginosawith normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B12, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth ofP. aeruginosaand illustrates the ability of vitamin B12to modulate the biofilm-forming capacity ofP. aeruginosaunder such condition.

The pathogen is the causative agent of cholera. Emergence of antibiotic-resistant strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in We found that N16961, a pandemic strain, and its isogenic (p)ppGpp-overexpressing mutant ΔΔ are both more antibiotic-resistant than (p)ppGpp (ΔΔΔ) and Δ mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene in the (p)ppGpp mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including , was increased in (p)ppGpp, but not in the antibiotic-resistant ΔΔ mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp, and gene deletion restored viability of tetracycline-exposed (p)ppGpp cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in .

Background: Cholera toxin (CT) production is induced during anaerobic respiration with trimethylamine N-oxide (TMAO) in Vibrio cholerae. Results: A bacterial stringent response to nutrient starvation was activated during anaerobic TMAO respiration and influenced CT production. Conclusion: CT production during anaerobic TMAO respiration is mediated by stringent response in V. cholerae. Significance: A mechanism of TMAO-stimulated CT production is uncovered.

e Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel. Metagenomics aims to characterize a collection of genetic materials as they exist in a microbial ecosystem (1). This method stands in contrast to characterization by isolation of individual colonies. Because metagenomics offers a unique opportunity to study organisms that are not cultured in a laboratory, it opens access to a reservoir of novel microbial genes.The large bowels of mammalian species are colonized by microbial communities that are referred to as the gut microbiota. The communities, mostly bacterial in composition, have considerable biodiversity and gain much of their energy and carbon requirements from the hydrolysis of plant glycans and fermentation of the hydrolysis products. Additionally, some members of the community utilize mucins from mucus and the components of enterocytes sloughed from the intestinal mucosal surface (2, 3). Both the metabolic activities and antigenicity of the microbiota have important physiological and immunological repercussions for the host (4-7).Although many of the bacterial commensals of the human intestine have now been cultured (8), most information with regard to the bacterial community has been derived from high-throughput sequencing studies (3, 9). This strategy has revealed the complexity and functional potential of the communities but relies on gene annotations in public databases. However, these annotations, confounded by the detection of numerous hypothetical proteins of unknown function, may not reveal the full potential of proteins encoded by genes detected in as-yet uncultivated bacteria.Functional screens of metagenomic libraries of microbiota DNA can uncover important funct...