A global medical crisis is unfolding as antibiotics lose effectiveness against a growing number of bacterial pathogens. Horizontal gene transfer (HGT) contributes significantly to the rapid spread of ...resistance, yet the transmission dynamics of genes that confer antibiotic resistance are poorly understood. Multiple mechanisms of HGT liberate genes from normal vertical inheritance. Conjugation by plasmids, transduction by bacteriophages, and natural transformation by extracellular DNA each allow genetic material to jump between strains and species. Thus, HGT adds an important dimension to infectious disease whereby an antibiotic resistance gene (ARG) can be the agent of an outbreak by transferring resistance to multiple unrelated pathogens. Here, we review the small number of cases where HGT has been detected in clinical environments. We discuss differences and synergies between the spread of plasmid-borne and chromosomal ARGs, with a special consideration of the difficulties of detecting transduction and transformation by routine genetic diagnostics. We highlight how 11 of the top 12 priority antibiotic-resistant pathogens are known or predicted to be naturally transformable, raising the possibility that this mechanism of HGT makes significant contributions to the spread of ARGs. HGT drives the evolution of untreatable “superbugs” by concentrating ARGs together in the same cell, thus HGT must be included in strategies to prevent the emergence of resistant organisms in hospitals and other clinical settings.
Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate ...in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.
Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological ...analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.
DNA topology has fundamental control over the ability of transcription factors to access their target DNA sites at gene promoters. However, the influence of DNA topology on protein-DNA and ...protein-protein interactions is poorly understood. For example, relaxation of DNA supercoiling strongly induces the well-studied pathogenicity gene ssrA (also called spiR) in Salmonella enterica, but neither the mechanism nor the proteins involved are known. We have found that relaxation of DNA supercoiling induces expression of the Salmonella pathogenicity island (SPI)-2 regulator ssrA as well as the SPI-1 regulator hilC through a mechanism that requires the two-component regulator OmpR-EnvZ. Additionally, the ompR promoter is autoregulated in the same fashion. Conversely, the SPI-1 regulator hilD is induced by DNA relaxation but is repressed by OmpR. Relaxation of DNA supercoiling caused an increase in OmpR binding to DNA and a concomitant decrease in binding by the nucleoid-associated protein FIS. The reciprocal occupancy of DNA by OmpR and FIS was not due to antagonism between these transcription factors, but was instead a more intrinsic response to altered DNA topology. Surprisingly, DNA relaxation had no detectable effect on the binding of the global repressor H-NS. These results reveal the underlying molecular mechanism that primes SPI genes for rapid induction at the onset of host invasion. Additionally, our results reveal novel features of the archetypal two-component regulator OmpR. OmpR binding to relaxed DNA appears to generate a locally supercoiled state, which may assist promoter activation by relocating supercoiling stress-induced destabilization of DNA strands. Much has been made of the mechanisms that have evolved to regulate horizontally-acquired genes such as SPIs, but parallels among the ssrA, hilC, and ompR promoters illustrate that a fundamental form of regulation based on DNA topology coordinates the expression of these genes regardless of their origins.
More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our ...knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ70 (including phoP, slyA, and invF) from which we identified the –10 and –35 motifs of σ70-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.
The evolution of new gene networks is a primary source of genetic innovation that allows bacteria to explore and exploit new niches, including pathogenic interactions with host organisms. For ...example, the archetypal DNA binding protein, OmpR, is identical between Salmonella Typhimurium serovar Typhimurium and Escherichia coli, but regulatory specialization has resulted in different environmental triggers of OmpR expression and largely divergent OmpR regulons. Specifically, ompR mRNA and OmpR protein levels are elevated by acid pH in S. Typhimurium but not in E. coli. This differential expression pattern is due to differences in the promoter regions of the ompR genes and the E. coli ompR orthologue can be made acid-inducible by introduction of the appropriate sequences from S. Typhimurium. The OmpR regulon in S. Typhimurium overlaps that of E. coli at only 15 genes and includes many horizontally acquired genes (including virulence genes) that E. coli does not have. We found that OmpR binds to its genomic targets in higher abundance when the DNA is relaxed, something that occurs in S. Typhimurium as a result of acid stress and which is a requirement for optimal expression of its virulence genes. The genomic targets of OmpR do not share a strong nucleotide sequence consensus: we propose that the ability of OmpR to recruit additional genes to its regulon arises from its modest requirements for specificity in its DNA targets with its preference for relaxed DNA allowing it to cooperate with DNA-topology-based allostery to modulate transcription in response to acid stress.
Pathogen life history theory assumes a positive relationship between pathogen load in host tissues and pathogen transmission. Empirical evidence for this relationship is surprisingly rare due to the ...difficulty of measuring transmission for many pathogens. The comparative method, where a common host is experimentally infected with a set of pathogen strains, is a powerful approach for investigating the relationships between pathogen load and transmission. The validity of such experimental estimates of strain-specific transmission is greatly enhanced if they can predict the pathogen population strain structure in nature. Borrelia burgdorferi is a multi-strain, tick-borne spirochete that causes Lyme disease in North America. This study used 11 field-collected strains of B. burgdorferi, a rodent host (Mus musculus, C3H/HeJ) and its tick vector (Ixodes scapularis) to determine the relationship between pathogen load in host tissues and lifetime host-to-tick transmission (HTT). Mice were experimentally infected via tick bite with 1 of 11 strains. Lifetime HTT was measured by infesting mice with I. scapularis larval ticks on 3 separate occasions. The prevalence and abundance of the strains in the mouse tissues and the ticks were determined by qPCR. We used published databases to obtain estimates of the frequencies of these strains in wild I. scapularis tick populations. Spirochete loads in ticks and lifetime HTT varied significantly among the 11 strains of B. burgdorferi. Strains with higher spirochete loads in the host tissues were more likely to infect feeding larval ticks, which molted into nymphal ticks that had a higher probability of B. burgdorferi infection (i.e., higher HTT). Our laboratory-based estimates of lifetime HTT were predictive of the frequencies of these strains in wild I. scapularis populations. For B. burgdorferi, the strains that establish high abundance in host tissues and that have high lifetime transmission are the strains that are most common in nature.
Summary
The conjugative IncHI1 plasmid pSfR27 from Shigella flexneri 2a strain 2457T encodes the Sfh protein, a paralogue of the global transcriptional repressor H‐NS. Sfh allows pSfR27 to be ...transmitted to new bacterial hosts with minimal impact on host fitness, providing a ‘stealth’ function whose molecular mechanism has yet to be determined. The impact of the Sfh protein on the Salmonella enterica serovar Typhimurium transcriptome was assessed and binding sites for Sfh in the Salmonella Typhimurium genome were identified by chromatin immunoprecipitation. Sfh did not bind uniquely to any sites. Instead, it bound to a subset of the larger H‐NS regulatory network. Analysis of Sfh binding in the absence of H‐NS revealed a greatly expanded population of Sfh binding sites that included the majority of H‐NS target genes. Furthermore, the presence of plasmid pSfR27 caused a decrease in H‐NS interactions with the S. Typhimurium chromosome, suggesting that the A + T‐rich DNA of this large plasmid acts to titrate H‐NS, removing it from chromosomal locations. It is proposed that Sfh acts as a molecular backup for H‐NS and that it provides its ‘stealth’ function by replacing H‐NS on the chromosome, thus minimizing disturbances to the H‐NS‐DNA binding pattern in cells that acquire pSfR27.
Summary
Although Escherichia coli and Salmonella enterica inhabit similar niches and employ similar genetic regulatory programmes, we find that they differ significantly in their DNA supercoiling ...responses to environmental and antibiotic challenges. Whereas E. coli demonstrates large dynamic transitions in supercoiling in response to growth phase, osmotic pressure and novobiocin treatment, supercoiling levels are much less variable in S. enterica. The FIS protein is a global regulator of supercoiling in E. coli, but it was found to have less influence over supercoiling control in S. enterica. These inter‐species differences fine‐tune gene promoters to endogenous supercoiling and FIS levels. Transferring a Salmonella virulence gene promoter (PssrA) into a new enteric host (E. coli) caused aberrant expression in response to stimulatory signals. Reciprocal horizontal transfer of topA promoters, which control expression of topoisomerase I, between E. coli and S. enterica revealed how these orthologous promoters have evolved to respond differentially to FIS and supercoiling levels in their cognate species. This also identified a previously unrecognized osmoregulation of topA expression that is independent of FIS and supercoiling in both E. coli and S. enterica. These findings suggest that E. coli and S. enterica may be unexpectedly divergent in their global regulation of cellular physiology.
Attention to environmental sources of Mycobacterium avium complex (MAC) infection is a vital component of disease prevention and control. We investigated MAC colonization of household plumbing in ...suburban Philadelphia, Pennsylvania, USA. We used variable-number tandem-repeat genotyping and whole-genome sequencing with core genome single-nucleotide variant analysis to compare M. avium from household plumbing biofilms with M. avium isolates from patient respiratory specimens. M. avium was recovered from 30 (81.1%) of 37 households, including 19 (90.5%) of 21 M. avium patient households. For 11 (52.4%) of 21 patients with M. avium disease, isolates recovered from their respiratory and household samples were of the same genotype. Within the same community, 18 (85.7%) of 21 M. avium respiratory isolates genotypically matched household plumbing isolates. Six predominant genotypes were recovered across multiple households and respiratory specimens. M. avium colonizing municipal water and household plumbing may be a substantial source of MAC pulmonary infection.
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