A major challenge facing bacterial intestinal pathogens is competition for nutrient sources with the host microbiota. Vibrio cholerae is an intestinal pathogen that causes cholera, which affects millions each year; however, our knowledge of its nutritional requirements in the intestinal milieu is limited. In this study, we demonstrated that V. cholerae can grow efficiently on intestinal mucus and its component sialic acids and that a tripartite ATP-independent periplasmic SiaPQM strain, transporter-deficient mutant NC1777, was attenuated for colonization using a streptomycin-pretreated adult mouse model. In in vivo competition assays, NC1777 was significantly outcompeted for up to 3 days postinfection. NC1777 was also significantly outcompeted in in vitro competition assays in M9 minimal medium supplemented with intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria (members of Actinobacillus, Corynebacterium, Mycoplasma, and Streptomyces), Bacteroidetes (mainly Bacteroides, Capnocytophaga, and Prevotella), Firmicutes (members of Streptococcus, Staphylococcus, Clostridium, and Lactobacillus), and Proteobacteria (including Escherichia, Shigella, Salmonella, Citrobacter, Haemophilus, Klebsiella, Pasteurella, Photobacterium, Vibrio, and Yersinia species), mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others.

BackgroundBacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.ResultsHerein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.ConclusionsOur data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5439-1) contains supplementary material, which is available to authorized users.

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Vibrio parahaemolyticus is a halophile that is the predominant cause of bacterial seafood-related gastroenteritis worldwide. To survive in the marine environment, V. parahaemolyticus must have adaptive strategies to cope with salinity changes. Six putative compatible solute (CS) transport systems were previously predicted from the genome sequence of V. parahaemolyticus RIMD2210633. In this study, we determined the role of the four putative betaine-carnitine-choline transporter (BCCT) homologues VP1456, VP1723, VP1905, and VPA0356 in the NaCl stress response. Expression analysis of the four BCCTs subjected to NaCl upshock showed that VP1456, VP1905, and VPA0356, but not VP1723, were induced. We constructed in-frame single-deletion mutant strains for all four BCCTs, all of which behaved similarly to the wild-type strain, demonstrating a redundancy of the systems. Growth analysis of a quadruple mutant and four BCCT triple mutants demonstrated the requirement for at least one BCCT for efficient CS uptake. We complemented Escherichia coli MHK13, a CS synthesis-and transporter-negative strain, with each BCCT and examined CS uptake by growth analysis and 1 H nuclear magnetic resonance (NMR) spectroscopy analyses. These data demonstrated that VP1456 had the most diverse substrate transport ability, taking up glycine betaine (GB), proline, choline, and ectoine. VP1456 was the sole ectoine transporter. In addition, the data demonstrated that VP1723 can transport GB, proline, and choline, whereas VP1905 and VPA0356 transported only GB. Overall, the data showed that the BCCTs are functional and that there is redundancy among them. Vibrio parahaemolyticus is a Gram-negative halophile inhabiting a wide range of aquatic ecosystems and causes bacteriumrelated seafood gastroenteritis in humans. Recent years have seen an increase in the incidence of infections worldwide, mostly during the warmer summer months (1-3). Vibrio parahaemolyticus is found in estuarine and marine environments as free-living organisms or in associations with fish and other marine species (4-7). Human infections occur usually through consumption of contaminated shellfish such as oysters. Vibrio parahaemolyticus is generally faced with salt concentrations of 3.5% salinity (35 ppt), but in estuarine systems and in shallow oyster beds during the summer months, this concentration can be much lower or higher. Salinity shifts in the environment pose tremendous osmotic challenges to bacteria that must respond swiftly by equating their intracellular osmotic potential with that of the external environment in order to maintain the positive turgor pressure required for normal growth (8-10). This is typically achieved via two strategies: the accumulation of inorganic ions, such as potassium ions (K ϩ ), and the accumulation of low-molecular-weight organic compounds, termed compatible solutes (CSs), which can be amassed in high concentrations without disturbing vital cellular function (8-10). The accumulation of CSs such as trehalose; free amino acids such as glutamate...

Edited by Chris WhitfieldNonulosonic acids (NulOs) are a diverse family of ␣-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryoticspecific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-D-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-Dalanyl modification. In silico analysis suggested that Leg5Ac7 AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning required and high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants, or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, labs, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument, and material batch. Though the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability.

L-ascorbic acid, commonly known as vitamin C, is a ubiquitous 6-carbon carbohydrate characterized by its ability to scavenge free radicals. In enteric bacteria, L-ascorbate can be utilized as a nutrient using the UlaABCDEF and UlaG-UlaRpathway under anaerobic conditions. In this study, we identified homologs of the Ula system within Vibrio cholerae and showed that V. cholerae is able to utilize L-ascorbate as an energy source. Growth pattern assays of a ulaG in-frame deletion mutant demonstrated that ulaG is essential for L-ascorbate fermentation. Expression analysis showed that ula catabolism and transport genes were significantly induced in cells grown in the presence of L-ascorbate compared to glucose and these genes were also highly induced during growth on intestinal mucus. In in vitro growth competition assays, the ulaG mutant was outcompeted by wild type when grown in intestinal mucus suggesting the Ula system could be important for fitness. Within the ula operon in V. cholerae and all Vibrio species a homology of ORF VCA0243 is present that encodes a pyridoxal phosphate (PLP) phosphatase. This enzyme in E. coli, converts the active form of vitamin B6 PLP to its inactive form pyridoxal (PL). In V. splendidus and related species, the aerobic and anaerobic L-ascorbate pathway genes cluster together and both systems contain a PLP phosphatase. An in-frame deletion mutant of vca0243 resulted in a growth defect in L-ascorbate fermentation as well as additional carbon and amino acid sources indicating a role in cellular metabolism. Phylogenetic analysis of UlaG and UlaD suggested the region was acquired by horizontal gene transfer.ImportanceL-ascorbate is a carbohydrate present in the human intestine, available for microbial consumption and several enteric species have been shown to utilize this compound as an energy source. We demonstrated that L-ascorbate fermentation genes are also present among marine bacteria from the family Vibrionaceae and that the human pathogen V. cholerae can ferment L-ascorbate as an energy source. Within the Ula operon in all Vibrionaceae, a putative pyridoxal phosphate phosphatase was present that was required for L-ascorbate fermentation and cellular metabolism in general. The Ula system was present among a limited number of genera within Vibrionaceae; Vibrio, Aliivibrio and Photobacterium and showed an evolutionary history consistent with horizontal transfer between genera and species.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92 − are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.