Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.

Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. IMPORTANCE Streptococcus pneumoniae is a human pathogen that causes pneumonia, bacteremia, and meningitis. Vaccination provides protection only against a quarter of the known S. pneumoniae serotypes, and the bacterium is rapidly becoming resistant to antibiotics. As such, new treatments are required. One strategy is to sensitize the bacteria to killing by the immune system. In this study, we performed a genome-wide screen to identify genes that help this bacterium resist oxidative stress exerted by the host at sites of colonization and infection. By identifying a number of critical pneumococcal defense mechanisms, our work provides novel targets for antimicrobial therapy.

Role of Bacteriophages in the Evolution of Pathogenic Vibrios and Lessons for Phage Therapy.

Viruses of bacteria, i.e., bacteriophages (or phages for short), were discovered over a century ago and have played a major role as a model system for the establishment of the fields of microbial genetics and molecular biology. Despite the relative simplicity of phages, microbiologists are continually discovering new aspects of their biology including mechanisms for battling host defenses. In turn, novel mechanisms of host defense against phages are being discovered at a rapid clip. A deeper understanding of the arms race between bacteria and phages will continue to reveal novel molecular mechanisms and will be important for the rational design of phage-based prophylaxis and therapies to prevent and treat bacterial infections, respectively. Here we delve into the molecular interactions of Vibrio species and phages.

Development of a Monoclonal Antibody to a Vibriophage as a Proxy for Vibrio cholerae Detection.

Cholera is an acute watery, diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage is present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by Enzyme linked immunosorbent assay (ELISA) and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 × 105 PFU by Western blotting analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

Emergence and Evolutionary Response of Vibrio cholerae to Novel Bacteriophage, Democratic Republic of the Congo1.

Cholera causes substantial illness and death in Africa. We analyzed 24 toxigenic Vibrio cholerae O1 strains isolated in 2015-2017 from patients in the Great Lakes region of the Democratic Republic of the Congo. Strains originating in southern Asia appeared to be part of the T10 introduction event in eastern Africa. We identified 2 main strain lineages, most recently a lineage corresponding to sequence type 515, a V. cholerae cluster previously reported in the Lake Kivu region. In 41% of fecal samples from cholera patients, we also identified a novel ICP1 (Bangladesh cholera phage 1) bacteriophage, genetically distinct from ICP1 isolates previously detected in Asia. Bacteriophage resistance occurred in distinct clades along both internal and external branches of the cholera phylogeny. This bacteriophage appears to have served as a major driver for cholera evolution and spread, and its appearance highlights the complex evolutionary dynamic that occurs between predatory phage and bacterial host.

Bdellovibrio reynosensis sp. nov., from a Mexico soil sample.

A novel predatory bacterium, strain LBG001T, has been isolated from Reynosa, Mexico. The 16S rRNA shares approximately 97 % sequence identity with many reported strains in the genus Bdellovibrio including the type strain Bdellovibrio bacteriovorus HD100T. Phylogenetic trees based on the 16S rRNA gene and on 30 concatenated housekeeping genes or core genes showed that LBG001T is on a separate branch from the B. bacteriovorus group. LBG0001T has a genome size of 3 582 323 bp with a G+C content of 43.1 mol %. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values with other members of the genus Bdellovibrio (<79, <72 and <17 %, respectively) qualifies the strain to represent a new species in the genus. Strain LBG001T formed visible plaques on all 10 tested Gram-negative bacterial species. The phenotypic characteristics, phylogenetic analysis and genomic taxonomic studies support the classification of the strain as representing a new species for which the name Bdellovibrio reynosensis sp. nov. is proposed. The type strain is LBG001T(=ATCC TSD-288T =CM-CNRG 0932T).

Niche adaptation limits bacteriophage predation of Vibrio cholerae in a nutrient-poor aquatic environment.

Vibrio cholerae, the causative agent of cholera, has reservoirs in fresh and brackish water where it interacts with virulent bacteriophages. Phages are the most abundant biological entity on earth and coevolve with bacteria. It was reported that concentrations of phage and V. cholerae inversely correlate in aquatic reservoirs and in the human small intestine, and therefore that phages may quench cholera outbreaks. Although there is strong evidence for phage predation in cholera patients, evidence is lacking for phage predation of V. cholerae in aquatic environments. Here, we used three virulent phages, ICP1, ICP2, and ICP3, commonly shed by cholera patients in Bangladesh, as models to understand the predation dynamics in microcosms simulating aquatic environments. None of the phages were capable of predation in fresh water, and only ICP1 was able to prey on V. cholerae in estuarine water due to a requirement for salt. We conclude that ICP2 and ICP3 are better adapted for predation in a nutrient rich environment. Our results point to the evolution of niche-specific predation by V. cholerae-specific virulent phages, which complicates their use in predicting or monitoring cholera outbreaks as well as their potential use in reducing aquatic reservoirs of V. cholerae in endemic areas.

NusG prevents transcriptional invasion of H-NS-silenced genes.

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho's primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU.

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

A Tn-seq Screen of Streptococcus pneumoniae Uncovers DNA Repair as the Major Pathway for Desiccation Tolerance and Transmission.

Streptococcus pneumoniae is an opportunistic pathogen that is a common cause of serious invasive diseases such as pneumonia, bacteremia, meningitis, and otitis media. Transmission of this bacterium has classically been thought to occur through inhalation of respiratory droplets and direct contact with nasal secretions. However, the demonstration that S. pneumoniae is desiccation tolerant and, therefore, environmentally stable for extended periods of time opens up the possibility that this pathogen is also transmitted via contaminated surfaces (fomites). To better understand the molecular mechanisms that enable S. pneumoniae to survive periods of desiccation, we performed a high-throughput transposon sequencing (Tn-seq) screen in search of genetic determinants of desiccation tolerance. We identified 42 genes whose disruption reduced desiccation tolerance and 45 genes that enhanced desiccation tolerance. The nucleotide excision repair pathway was the most enriched category in our Tn-seq results, and we found that additional DNA repair pathways are required for desiccation tolerance, demonstrating the importance of maintaining genome integrity after desiccation. Deletion of the nucleotide excision repair gene uvrA resulted in a delay in transmission between infant mice, indicating a correlation between desiccation tolerance and pneumococcal transmssion. Understanding the molecular mechanisms that enable pneumococcal persistence in the environment may enable targeting of these pathways to prevent fomite transmission, thereby preventing the establishment of new colonization and any resulting invasive disease.

Genome-wide detection of conservative site-specific recombination in bacteria.

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon.

Nasopharyngeal Exposure to Streptococcus pneumoniae Induces Extended Age-Dependent Protection against Pulmonary Infection Mediated by Antibodies and CD138+ Cells.

Streptococcus pneumoniae commonly resides asymptomatically in the nasopharyngeal (NP) cavity of healthy individuals but can cause life-threatening pulmonary and systemic infections, particularly in the elderly. NP colonization results in a robust immune response that protects against invasive infections. However, the duration, mechanism, and cellular component of such responses are poorly understood. In this study, we found that repeated NP exposure of mice to S. pneumoniae TIGR4 strain results in pneumococcal-specific Ab responses that protect against lethal lung challenge. Abs were necessary and sufficient for protection because Ab-deficient µMT mice did not develop postexposure protection, only becoming resistant to lung infection after transfer of immune sera from NP-exposed mice. T cells contributed to immunity at the time of NP exposure, but neither CD4+ nor CD8+ T cells were required. The protective activity was detectable 20 wk after exposure and was maintained in irradiated mice, suggesting involvement of long-lived Ab-secreting cells (ASC), which are radioresistant and secrete Abs for extended periods of time in the absence of T cells or persistent Ag. CD138+ bone marrow cells, likely corresponding to long-lived ASC, were sufficient to confer protection. NP exposure of aged mice failed to protect against subsequent lung infection despite eliciting a robust Ab response. Furthermore, transfer of CD138+ bone marrow cells or sera from NP-exposed old mice failed to protect naive young mice. These findings suggest that NP exposure elicits extended protection against pneumococcal lung infection by generating long-lived CD138+ ASC and that the protective efficacy of these responses declines with age.

Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.

A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity.

Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.

Expanding the repertoire of conservative site-specific recombination in Clostridioides difficile.

Recent genomic analysis of an epidemic ribotype 027 (RT027) Clostridioides difficile strain revealed the presence of several chromosomal site-specific invertible sites hypothesized to control the expression of adjacent genes in a bimodal on-off mode. This process, named phase variation, is thought to enhance phenotypic variability under homogeneous conditions ultimately increasing population fitness in unpredictable environmental fluctuations. The full extent of phase variation mediated by DNA-inversions in C. difficile is currently unknown. Here, we sought to expand our previous analysis by screening for site-specific inversions in isolates that belong to the rapidly emerging ribotypes RT017 and RT078. We report the finding of one novel inversion site for which we demonstrate the inversion potential and quantify inversion proportions during exponential and stationary growth in both historic and modern isolates of the same ribotype. We then employ a computational approach to assess the prevalence of all sites identified so far in a large collection of sequenced C. difficile isolates. We show that phase-variable loci are widespread with some sites being present in virtually all analyzed strains. Furthermore, in our small subset of RT017 and RT078 strains, we detect no evidence of gain or loss of invertible sites in historic versus modern isolates demonstrating the relative stability of those genomic elements. Overall, our results support the idea that C. difficile has adopted phase variation mediated by DNA inversions as its major generator of diversity which could be beneficial during the pathogenesis process.

Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

A globally distributed mobile genetic element inhibits natural transformation of Vibrio cholerae.

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA(+) ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to ∼40% from 2006 to 2011. Thus, IdeA(+) ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.

Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility.

An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.

ManLMN is a glucose transporter and central metabolic regulator in Streptococcus pneumoniae.

Streptococcus pneumoniae is a common colonizer of the human nasopharynx and a leading cause of bacterial pneumonia and otitis media, among other invasive diseases. During both colonization and invasive disease S. pneumoniae ferments host-derived carbohydrates as its primary means of generating energy. This pathogen is adept at transporting and metabolizing a wide variety of carbohydrates. We found the highly conserved PTS ManLMN contributes to growth on glucose and is also essential for growth on a variety of nonpreferred carbohydrates, suggesting it is a multisubstrate transporter. Exploration of this phenotype revealed ManLMN is required for inducing expression of downstream metabolic genes in response to carbohydrate stimuli. We further demonstrate that ManLMN's role as a constitutively expressed transporter is likely unique and integral to pneumococcus's strategy of carbon catabolite repression (CCR). Using a selection for suppressors, we explored how ManLMN is integrated into the CCR regulatory framework in S. pneumoniae. We identified two hypothetical small proteins and the virulence regulator SmrC as potential mediators of CCR in connection with ManLMN. Characterization of these two hypothetical proteins revealed they influence transcriptional regulation of carbohydrate transporters. We propose a model unifying these observations in which ManLMN is a versatile surveyor of available carbohydrates in S. pneumoniae.

Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources.

Phosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, Vibrio cholerae, encounters phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo- and exonucleases. However, no transporter of nucleotides has been identified in V. cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V. cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.

Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi.

Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection. We performed genetic fitness analyses to identify genes encoding factors contributing to growth on glucose, maltose, mannose, trehalose and N-acetyl-glucosamine. We obtained insight into the potential functions of proteins predicted to be involved in carbohydrate utilization and identified additional factors previously unrecognized as contributing to the metabolism of the tested carbohydrates. Strong phenotypes were observed for the putative carbohydrate phosphotransferase transporters BB0408 and BBB29 as well as the response regulator Rrp1. We further validated Tn-seq for use in mouse studies and were able to correctly identify known infectivity factors as well as additional transporters and genes on lp54 that may contribute to optimal mouse infection. As such, this study establishes Tn-seq as a powerful method for both in vitro and in vivo studies of B. burgdorferi.