Pathogenic Adaptation of Intracellular Bacteria by Rewiring a Cis-Regulatory Input Function

The acquisition of DNA by horizontal gene transfer enables bacteria to adapt to previously unexploited ecological niches. Although horizontal gene transfer and mutation of protein-coding sequences are well-recognized forms of pathogen evolution, the evolutionary significance of cis-regulatory mutations in creating phenotypic diversity through altered transcriptional outputs is not known. We show the significance of regulatory mutation for pathogen evolution by mapping and then rewiring a cis-regulatory module controlling a gene required for murine typhoid. Acquisition of a binding site for the Salmonella pathogenicity island-2 regulator, SsrB, enabled the srfN gene, ancestral to the Salmonella genus, to play a role in pathoadaptation of S. typhimurium to a host animal. We identified the evolved cis-regulatory module and quantified the fitness gain that this regulatory output accrues for the bacterium using competitive infections of host animals. Our findings highlight a mechanism of pathogen evolution involving regulatory mutation that is selected because of the fitness advantage the new regulatory output provides the incipient clones

Interdomain Linkers of Homologous Response Regulators Determine Their Mechanism of Action

OmpR and PhoB are response regulators that contain an N-terminal phosphorylation domain and a C-terminal DNA binding effector domain connected by a flexible interdomain linker. Phosphorylation of the N terminus results in an increase in affinity for specific DNA and the subsequent regulation of gene expression. Despite their sequence and structural similarity, OmpR and PhoB employ different mechanisms to regulate their effector domains. Phosphorylation of OmpR in the N terminus stimulates the DNA binding affinity of the C terminus, whereas phosphorylation of the PhoB N terminus relieves inhibition of the C terminus, enabling it to bind to DNA. Chimeras between OmpR and PhoB containing either interdomain linker were constructed to explore the basis of the differences in their activation mechanisms. Our results indicate that effector domain regulation by either N terminus requires its cognate interdomain linker. In addition, our findings suggest that the isolated C terminus of OmpR is not sufficient for a productive interaction with RNA polymerase

The Escherichia coli CpxA-CpxR Envelope Stress Response System Regulates Expression of the Porins OmpF and OmpC

We performed transposon mutagenesis of a two-color fluorescent reporter strain to identify new regulators of the porin genes ompF and ompC in Escherichia coli. Screening of colonies by fluorescence microscopy revealed numerous mutants that exhibited interesting patterns of porin expression. One mutant harbored an insertion in the gene encoding the histidine kinase CpxA, the sensor for a two-component signaling system that responds to envelope stress. The cpxA mutant exhibited increased transcription of ompC and a very strong decrease in transcription of ompF under conditions in which acetyl phosphate levels were high. Subsequent genetic analysis revealed that this phenotype is dependent on phosphorylation of the response regulator CpxR and that activation of CpxA in wild-type cells results in similar regulation of porin expression. Using DNase I footprinting, we demonstrated that CpxR binds upstream of both the ompF and ompC promoters. It thus appears that two distinct two-component systems, CpxA-CpxR and EnvZ-OmpR, converge at the porin promoters. Within the context of envelope stress, outer membrane beta-barrel proteins have generally been associated with the sigma E pathway. However, at least for the classical porins OmpF and OmpC, our results show that the Cpx envelope stress response system plays a role in regulating their expression

Salmonella enterica Response Regulator SsrB Relieves H-NS Silencing by Displacing H-NS Bound in Polymerization Mode and Directly Activates Transcription*

The response regulator SsrB activates expression of genes encoded within and outside of a pathogenicity island (SPI-2), which is required for systemic infection of Salmonella. SsrB binds upstream of the sifA, sifB, and sseJ effector genes and directly regulates transcription. SsrB also relieves gene silencing by the nucleoid protein H-NS. Single molecule experiments with magnetic tweezers demonstrated that SsrB displaces H-NS from DNA only when it is bound in a polymerization (stiffening) mode and not when H-NS is bound to DNA in the bridging mode. Thus, in contrast to previous views, the polymerization binding mode of H-NS is the relevant form for counter-silencing by SsrB. Our results reveal that response regulators can directly activate transcription and also relieve H-NS silencing. This study adds to the repertoire of mechanisms by which NarL/FixJ subfamily members regulate transcription. Because SsrB-dependent promoters are diversely organized, additional mechanisms of transcriptional activation at other loci are likely