Significance: The ability of bacteria to survive antibiotic challenge without mutation (a phenomenon known as “adaptive resistance”) has been traditionally viewed as the consequence of bacterial responses to environmental cues. This study shows that adaptive resistance can also occur in the absence of known environmental stimuli. Cell-to-cell fluctuations in critical physiological traits preadapt certain cells within an isogenic culture to survive lethal selection, and growth of the “lucky” survivors generates bacterial subpopulations with increased antibiotic resistance. Phenotypic heterogeneity must therefore be added to the list of factors that limit the efficacy of antibiotic treatment.

The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvAB^OFF and opvAB^ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB^OFF subpopulation is killed and the opvAB^ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB^OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only. A tradeoff can increase the adaptive capacity of an organism at the expense of lowering the fitness conferred by specific traits. This study describes a tradeoff that confers bacteriophage resistance in Salmonella enterica at the expense of reducing its pathogenic capacity. Phase variation of the opvAB operon creates two subpopulations of bacterial cells, each with a distinct lipopolysaccharide structure. One subpopulation is large and virulent but sensitive to phages that use the lipopolysaccharide O-antigen as receptor, while the other is small and avirulent but phage resistant. In the presence of a phage that targets the O-antigen, only the avirulent subpopulation survives. However, phase variation permits resuscitation of the virulent opvAB^OFF subpopulation as soon as phage challenge ceases. This transient tradeoff may illustrate the adaptive value of epigenetic mechanisms that generate bacterial subpopulations in a reversible manner.

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression. Salmonella infection is one of the major causes of foodborne illness worldwide. During infection, Salmonella expresses a set of virulence genes encoded in discrete regions of the genome. The expression of these genes is tightly regulated, being specific for different stages of the Salmonella infection process. While many regulatory mechanisms that lead to the activation of infection-related gene expression have been described, little is known about silencing mechanisms under conditions when expression is not needed and may rather represent a burden than a benefit for the bacterial fitness. Here, we report a condition-specific silencing mechanism of bacterial virulence. That is, the global transcriptional regulator CRP-cAMP represses, indirectly through a post-transcriptional mechanism, the expression of the major Salmonella virulence regulator HilD. In bacteria, post-transcriptional regulation has so far been mainly focused on 5’ untranslated regions (5’UTR). Remarkably, here we describe a molecular mechanism targeting the 3’untranslated region (3’UTR) of the mRNA of the major regulator of Salmonella virulence by a small non-coding RNA under the transcriptional control of the global regulator CRP-cAMP. Our data highlight the importance of 3’UTR in the regulation of gene expression in bacteria.

Salmonella enterica pathogenicity island 1 (SPI-1) is a gene cluster that encodes a type III secretion system and effectors involved in epithelial cell invasion. SPI-1 undergoes bistable expression, with concomitant formation of SPI-1^ON and SPI-1^OFF lineages. This study describes single cell analysis of SP1-1 bistability and epithelial cell invasion, and reports the unsuspected observation that optimal invasion of epithelial cells requires the presence of both SPI-1^ON and SPI-1^OFF subpopulations. The contribution of SPI-1^OFF cells to optimal invasion may rely on their ability to invade epithelial cells if a SPI-1^ON subpopulation is present. In fact, Salmonella SPI-1 mutants are also able to invade epithelial cells in the presence of SPI-1^ONSalmonellae, a phenomenon described in the 1990’s by Galán and co-workers. Invasion by SPI-1^OFF cells does not seem to involve a diffusible factor. A small number of SPI-1^ON cells is sufficient to endow the bacterial population with invasion capacity, a feature that may permit host colonization regardless of the bottlenecks encountered by Salmonella populations inside animals.