Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in Nature

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD50) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-β; IL-1β; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses

Microbial Fuel Cells and Microbial Ecology: Applications in Ruminant Health and Production Research

Microbial fuel cell (MFC) systems employ the catalytic activity of microbes to produce electricity from the oxidation of organic, and in some cases inorganic, substrates. MFC systems have been primarily explored for their use in bioremediation and bioenergy applications; however, these systems also offer a unique strategy for the cultivation of synergistic microbial communities. It has been hypothesized that the mechanism(s) of microbial electron transfer that enable electricity production in MFCs may be a cooperative strategy within mixed microbial consortia that is associated with, or is an alternative to, interspecies hydrogen (H2) transfer. Microbial fermentation processes and methanogenesis in ruminant animals are highly dependent on the consumption and production of H2in the rumen. Given the crucial role that H2 plays in ruminant digestion, it is desirable to understand the microbial relationships that control H2 partial pressures within the rumen; MFCs may serve as unique tools for studying this complex ecological system. Further, MFC systems offer a novel approach to studying biofilms that form under different redox conditions and may be applied to achieve a greater understanding of how microbial biofilms impact animal health. Here, we present a brief summary of the efforts made towards understanding rumen microbial ecology, microbial biofilms related to animal health, and how MFCs may be further applied in ruminant research

β-Lysine discrimination by lysyl-tRNA synthetase

AbstractElongation factor P is modified with (R)-β-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α- and β-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α-lysine, while the G469A and A233S/G469A variants decreased stable α-lysyl-adenylate formation. A233S LysRS recognized β-lysine better than wildtype, suggesting a role for this residue in discriminating α- and β-amino acids. Both enantiomers of β-lysine were substrates for tRNA aminoacylation by LysRS, which, together with the relaxed specificity of the A233S variant, suggest a possible means to develop systems for in vivo co-translational insertion of β-amino acids

H-NS promotes looped domain formation in the bacterial chromosome

SummaryThe bacterial chromosome is organized into loops, which constitute topologically isolated domains. It is unclear which proteins are responsible for the formation of the topological barriers between domains. The abundant DNA-binding histone-like nucleoid structuring protein (H-NS) is a key player in the organization and compaction of bacterial chromosomes [1,2]. The protein acts by bridging DNA duplexes [3], thus allowing for the formation of DNA loops. Here, genome-wide studies of H-NS binding suggest that this protein is directly involved in the formation or maintenance of topological domain barriers

Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium

The PhoPQ two-component system acts a transcriptional regulator that responds to Mg2+ starvation both in Escherichia coli and Salmonella typhimurium (Garcia et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular Mg2+, this two-component system allows S. typhimurium to sense the transition from an extracellular environment to a subcellular location. Concomitantly with this transition, a set of virulence factors essential for survival in the intracellular environment is activated by the PhoPQ system (Groisman et al. 1989; Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the PhoPQ regulon in pathogens must contain target genes specifically contributing, to the virulence phenotype. To verify this hypothesis, we compared the composition of the PhoPQ regulon between E. coli and S. typhimurium using a combination of expression experiments and motif data. PhoPQ-dependent genes in both organisms were identified from PhoPQ-related microarray experiments. To distinguish between direct and indirect targets, we searched for the presence of the regulatory motif in the promoter region of the identified PhoPQ-dependent genes. This allowed us to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S. typhimurium LT2. Comparison of both regulons revealed a very limited overlap of PhoPQ-dependent genes between both organisms. These results suggest that the PhoPQ system has acquired a specialized function during evolution in each of these closely related species that allows adaptation to the specificities of their lifestyles (e.g., pathogenesis in S. typhimurium)

Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium

The PhoPQ two-component system acts as a transcriptional regulator that responds to Mg(2+) starvation both in Escherichia coli and Salmonella typhimurium (Garcia et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular Mg(2+), this two-component system allows S. typhimurium to sense the transition from an extracellular environment to a subcellular location. Concomitantly with this transition, a set of virulence factors essential for survival in the intracellular environment is activated by the PhoPQ system (Groisman et al. 1989; Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the PhoPQ regulon in pathogens must contain target genes specifically contributing to the virulence phenotype. To verify this hypothesis, we compared the composition of the PhoPQ regulon between E. coli and S. typhimurium using a combination of expression experiments and motif data. PhoPQ-dependent genes in both organisms were identified from PhoPQ-related microarray experiments. To distinguish between direct and indirect targets, we searched for the presence of the regulatory motif in the promoter region of the identified PhoPQ-dependent genes. This allowed us to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S. typhimurium LT2. Comparison of both regulons revealed a very limited overlap of PhoPQ-dependent genes between both organisms. These results suggest that the PhoPQ system has acquired a specialized function during evolution in each of these closely related species that allows adaptation to the specificities of their lifestyles (e.g., pathogenesis in S. typhimurium).status: publishe

Silencing by H-NS Potentiated the Evolution of <i>Salmonella</i>

<div><p>The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in <i>Salmonella</i>, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in <i>Salmonella</i>. Six independently derived <i>S.</i> Typhimurium <i>hns</i> mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the <i>hns</i> mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the <i>Salmonella</i> Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in <i>Salmonella hns</i> mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type <i>Salmonella</i> lineages did not acquire these mutations. The <i>stpA</i> missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.</p></div