A Vibrio cholerae Anti-Phage System Depletes Nicotinamide Adenine Dinucleotide to Restrict Virulent Bacteriophages.

Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with either protein and should be able to antagonize other anti-phage systems that deplete NAD+.

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.

CRISPR Gene Editing of Virulent Bacteriophage ICP1.

Tools for site-directed mutagenesis of virulent bacteriophages (phages; viruses of bacteria) have traditionally lagged those for bacteria, hindering their study. CRISPR gene editing represents a new and highly efficient method for editing virulent phage genomes. Here, I describe methods for using CRISPR gene editing for site-directed mutagenesis of ICP1, a virulent phage of Vibrio cholerae The first section outlines methods of constructing a plasmid for CRISPR editing of an ICP1 gene. The second section outlines methods of transferring the plasmid to an editing-competent strain of V. cholerae The third section outlines methods of selecting for and storing the edited phage.

CRISPR Gene Editing of a Virulent Bacteriophage.

Genome editing by site-directed mutagenesis is an important tool in biological research. CRISPR gene editing is the latest such tool developed, and one that is widely applicable to study organisms from all kingdoms of life. Here, I introduce a method for making site-directed, defined mutations in a virulent bacteriophage (a bacterial virus) using CRISPR gene editing. The ability to precisely edit the genomes of virulent phages will facilitate the study of their gene requirements for infection of host bacteria and advance our ability to engineer phages for use as therapeutic agents to combat bacterial infections. The protocol introduced here was developed as part of Cold Spring Harbor's Advanced Bacterial Genetics course.

Vibrio cholerae O1 associated with recent endemic cholera shows temporal changes in serotype, genotype, and drug-resistance patterns in Bangladesh.

BACKGROUND: Despite the advancement in our understanding of cholera and its etiological agent, Vibrio cholerae, the prevention and treatment of the disease are often hindered due to rapid changes in drug response pattern, serotype, and the major genomic islands namely, the CTX-prophage, and related genetic characteristics. In the present study, V. cholerae (n = 172) associated with endemic cholera in Dhaka during the years 2015-2021 were analyzed for major phenotypic and genetic characteristics, including drug resistance patterns. RESULTS: Results revealed that the V. cholerae strains belonged to serogroup O1 biotype El Tor carrying El Tor -specific genes rtxC, tcpA El Tor, and hlyA El Tor, but possessed classical-biotype cholera toxin. Serotypes of V. cholerae strains differed temporally in predominance with Inaba during 2015-2017, and again in 2020-2021, while Ogawa was the predominant serotype in 2018-2019. Also, ctxB1 was predominant in V. cholerae associated with cholera during 2015-2017, while ctxB7 was predominant in 2018, and in the subsequent years, as observed until 2021. V. cholerae strains differed in their antibiotic resistance pattern with a majority (97%) being multi-drug resistant (MDR) and belonging to six sub-groups. Notably, one of these MDR strains was resistant to eleven of the eighteen antibiotics tested, with resistance to fourth-generation cephalosporin (cefepime), and aztreonam. This extreme drug resistant (XDR) strain carried resistance-related genes namely, extended-spectrum ß-lactamases (ESBL), blaOXA-1 and blaPER-3. CONCLUSION: The observed temporal switching of serotypes, as well as the ctxB genotype, and the emergence of MDR/XDR V. cholerae and their association with endemic cholera in Dhaka underscore the need for routine monitoring of the pathogen for proper patient management.

High-Throughput Mutant Screening via Transposon Sequencing.

Transposon mutagenesis has been the method of choice for genetic screens and selections in bacteria by virtue of the transposon being linked to the disrupted gene, simplifying its identification. Transposon sequencing (Tn-seq) is a high-throughput version of transposon mutant screening, in which massively parallel sequencing is used to simultaneously follow the fitness of all mutants in a complex library. In a single experiment, one can use Tn-seq to interrogate the contribution of all genes of a bacterium to fitness under a condition of interest. Here, we introduce a method to construct a saturating transposon insertion library in Gram-negative bacteria, to capture the transposon junctions en masse, and to identify essential genes and conditional genes using massively parallel sequencing. The accompanying protocol was developed as part of Cold Spring Harbor's Advanced Bacterial Genetics course.

High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing.

Transposon mutagenesis greatly facilitates the study of gene function in microorganisms ranging from viruses to fungi. Traditionally, one would study individual transposon mutants with interesting phenotypes one mutant at a time. Here, we describe methods for the study of tens of thousands of transposon mutants in parallel in the bacterial pathogen Vibrio cholerae using transposon-sequencing. The first section outlines methods for making a saturated transposon mutant library. The second section outlines methods for massively parallel sequencing of the transposon junctions. The third section outlines methods for analyzing the sequence data to calculate the fitness contribution of genes.

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.

Isolation and Sequencing of Novel Vibrio Species.

Whole-genome sequencing of bacteria facilitates their genotypic analysis and expands our understanding of the tree of life on Earth. The Vibrio genus comprises many halophilic species of bacteria, including some that are pathogenic to humans and other animals. Here I describe methods for isolating and sequencing both known and novel species of Vibrio from saltwater environments. The first section outlines methods of isolating and phenotypically characterizing strains, followed by purification of their total DNA. The second and third sections outline methods of whole-genome sequencing and assembly, annotation, and phylogenetic analysis.

De Novo Genome Sequencing, Annotation, and Taxonomy of Unknown Bacteria.

Whole-genome sequencing of viruses and bacteria has become routine thanks to advances in DNA-sequencing technologies. Parallel advances in computing power and software design allow for billions of base pairs of sequence information to be analyzed in hours to minutes. Here, I describe methods to isolate known as well as new species of bacteria from the environment; to purify, sequence, assemble, and bioinformatically annotate their genomes; and to determine their place in the tree of life by phylogenetic analysis. The protocol introduced here was developed as part of Cold Spring Harbor's Advanced Bacterial Genetics course.

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.

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.

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).

An Intranasal Vaccine Based on Outer Membrane Vesicles Against SARS-CoV-2.

The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.

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.

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.

Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System.

The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5'-TT-3' PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5' position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5'-RTT-3' PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5'-RTT-3' PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5' spacer pyrimidine.IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.

Prophage-Dependent Neighbor Predation Fosters Horizontal Gene Transfer by Natural Transformation.

Natural transformation is a broadly conserved mechanism of horizontal gene transfer (HGT) in bacteria that can shape their evolution through the acquisition of genes that promote virulence, antibiotic resistance, and other traits. Recent work has established that neighbor predation via type VI secretion systems, bacteriocins, and virulent phages plays an important role in promoting HGT. Here, we demonstrate that in chitin estuary microcosms, Vibrio cholerae K139 lysogens exhibit prophage-dependent neighbor predation of nonlysogens to enhance HGT. Through predation of nonlysogens, K139 lysogens also have a fitness advantage under these microcosm conditions. The ecological strategy revealed by our work provides a better understanding of the evolutionary mechanisms used by bacteria to adapt in their natural setting and contributes to our understanding of the selective pressures that may drive prophage maintenance in bacterial genomes.IMPORTANCE Prophages are nearly ubiquitous in bacterial species. These integrated phage elements have previously been implicated in horizontal gene transfer (HGT) largely through their ability to carry out transduction (generalized or specialized). Here, we show that prophage-encoded viral particles promote neighbor predation leading to enhanced HGT by natural transformation in the waterborne pathogen Vibrio cholerae Our findings contribute to a comprehensive understanding of the dynamic forces involved in prophage maintenance which ultimately drive the evolution of naturally competent bacteria in their natural environment.

Colistin-resistant Escherichia coli carrying mcr-1 in food, water, hand rinse, and healthy human gut in Bangladesh.

BACKGROUND: One of the most significant public health concerns in today's world is the persistent upsurge of infections caused by multidrug resistant bacteria. As a result, clinicians are being forced to intervene with either less effective backup drugs or ones with substantial side-effects. Colistin is a last resort antimicrobial agent for the treatment of infections caused by multi-drug resistant gram-negative bacteria. METHODS: Escherichia coli (n = 65) isolated from street food (n = 20), hand rinse (n = 15), surface water (n = 10), and healthy human stool (n = 20) were tested for colistin resistance gene mcr-1 and response to antimicrobial agents. Antimicrobial resistance genes and virulence genes were detected by employing polymerase chain reaction. DNA fingerprinting of the strains were determined by pulsed-field gel electrophoresis. RESULTS: Screening of E. coli allowed us to confirm colistin resistance marker gene mcr-1 in 13 strains (street food, n = 4; hand rinse, n = 2; surface water, n = 4; and stool, n = 3); and two of these E. coli strains carrying mcr-1 harbored bla TEM gene encoding extended spectrum beta lactamase. Antibiotic assay results revealed all 13 E. coli strains carrying mcr-1 to be multi-drug resistant (MDR), including to colistin. The minimum inhibitory concentration (MIC) for colistin ranged from 2 to 6 µg/ml. DNA sequencing confirmed homogeneity of the nucleotide sequence for mcr-1, but the E. coli strains were heterogenous, as confirmed by pulsed-field gel electrophoresis suggesting horizontal transmission of colistin resistance in Bangladesh. CONCLUSION: Widespread dissemination of E. coli strains carrying mcr-1 encoding resistance to colistin in the present study is alarming as this is the last resort drug for the treatment of infections caused by MDR gram-negative bacteria resistant to almost all drugs used commonly.

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.