Bacterial nucleoid-associated protein uncouples transcription levels from transcription timing.

The histone-like nucleoid-structuring (H-NS) protein binds to horizontally acquired genes in the bacterium Salmonella enterica serovar Typhimurium, silencing their expression. We now report that overcoming the silencing effects of H-NS imposes a delay in the expression of genes activated by the transcriptional regulator PhoP. We determine that PhoP-activated genes ancestral to Salmonella are expressed before those acquired horizontally. This expression timing reflects the in vivo occupancy of the corresponding promoters by the PhoP protein. These results are surprising because some of these horizontally acquired genes reached higher mRNA levels than ancestral genes expressed earlier and were transcribed from promoters harboring PhoP-binding sites with higher in vitro affinity for the PhoP protein. Our findings challenge the often-made assumption that for genes coregulated by a given transcription factor, early genes are transcribed to higher mRNA levels than those transcribed at later times. Moreover, they provide a singular example of how gene ancestry can impact expression timing. Importance: We report that gene ancestry dictates the expression behavior of genes under the direct control of the Salmonella transcriptional regulator PhoP. That is, ancestral genes are transcribed before horizontally acquired genes. This reflects both the need to overcome silencing by the H-NS protein of the latter genes and the architecture of the corresponding promoters. Unexpectedly, transcription levels do not reflect transcription timing. Our results illustrate how a bacterium can exhibit an elaborate temporal expression behavior among genes coregulated by a transcription factor even though the products encoded by the target genes do not participate in a morphological or developmental pathway.

Reciprocal control between a bacterium's regulatory system and the modification status of its lipopolysaccharide.

Gram-negative bacteria often modify their lipopolysaccharide (LPS), thereby increasing resistance to antimicrobial agents and avoidance of the host immune system. However, it is unclear how bacteria adjust the levels and activities of LPS-modifying enzymes in response to the modification status of their LPS. We now address this question by investigating the major regulator of LPS modifications in Salmonella enterica. We report that the PmrA/PmrB system controls expression of a membrane peptide that inhibits the activity of LpxT, an enzyme responsible for increasing the LPS negative charge. LpxT's inhibition and the PmrA-dependent incorporation of positively charged L-4-aminoarabinose into the LPS decrease Fe(3+) binding to the bacterial cell. Because Fe(3+) is an activating ligand for the sensor PmrB, transcription of PmrA-dependent LPS-modifying genes is reduced. This mechanism enables bacteria to sense their cell surface by its effect on the availability of an inducing signal for the system regulating cell-surface modifications.

The promoter architectural landscape of the Salmonella PhoP regulon.

The DNA-binding protein PhoP controls virulence and Mg²âº homeostasis in the Gram-negative pathogen Salmonella enterica serovar Typhimurium. PhoP regulates expression of a large number of genes that differ both in their ancestry and in the biochemical functions and physiological roles of the encoded products. This suggests that PhoP-regulated genes are differentially expressed. To understand how a bacterial activator might generate varied gene expression behaviour, we investigated the cis-acting promoter features (i.e. the number of PhoP binding sites, as well as their orientation and location with respect to the sites bound by RNA polymerase and the sequences that constitute the PhoP binding sites) in 23 PhoP-activated promoters. Our results show that natural PhoP-activated promoters utilize only a limited number of combinations of cis-acting features--or promoter architectures. We determine that PhoP activates transcription by different mechanisms, and that ancestral and horizontally acquired PhoP-activated genes have distinct promoter architectures.

Evolution of a bacterial regulon controlling virulence and Mg(2+) homeostasis.

Related organisms typically rely on orthologous regulatory proteins to respond to a given signal. However, the extent to which (or even if) the targets of shared regulatory proteins are maintained across species has remained largely unknown. This question is of particular significance in bacteria due to the widespread effects of horizontal gene transfer. Here, we address this question by investigating the regulons controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+) homeostasis in several bacterial species. We establish that the ancestral PhoP protein directs largely different gene sets in ten analyzed species of the family Enterobacteriaceae, reflecting both regulation of species-specific targets and transcriptional rewiring of shared genes. The two targets directly activated by PhoP in all ten species (the most distant of which diverged >200 million years ago), and coding for the most conserved proteins are the phoPQ operon itself and the lipoprotein-encoding slyB gene, which decreases PhoP protein activity. The Mg(2+)-responsive PhoP protein dictates expression of Mg(2+) transporters and of enzymes that modify Mg(2+)-binding sites in the cell envelope in most analyzed species. In contrast to the core PhoP regulon, which determines the amount of active PhoP and copes with the low Mg(2+) stress, the variable members of the regulon contribute species-specific traits, a property shared with regulons controlled by dissimilar regulatory proteins and responding to different signals.

Overcoming H-NS-mediated transcriptional silencing of horizontally acquired genes by the PhoP and SlyA proteins in Salmonella enterica.

The acquisition of new traits through horizontal gene transfer depends on the ability of the recipient organism to express the incorporated genes. However, foreign DNA appears to be silenced by the histone-like nucleoid-structuring protein (H-NS) in several enteric pathogens, raising the question of how this silencing is overcome and the acquired genes are expressed at the right time and place. To address this question, we investigated transcription of the horizontally acquired ugtL and pagC genes from Salmonella enterica, which is dependent on the regulatory DNA-binding proteins PhoP and SlyA. We reconstituted transcription of the ugtL and pagC genes in vitro and determined occupancy of their respective promoters by PhoP, H-NS, and RNA polymerase in vivo. The SlyA protein counteracted H-NS-promoted repression in vitro but could not promote gene transcription by itself. PhoP-promoted transcription required SlyA when H-NS was present but not in its absence. In vivo, H-NS remained bound to the ugtL and pagC promoters under inducing conditions that promoted RNA polymerase recruitment and transcription of the ugtL and pagC genes. Our results indicate that relief of H-NS repression and recruitment of RNA polymerase are controlled by different regulatory proteins that act in concert to express horizontally acquired genes.

The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica.

The sigma factor RpoS regulates the expression of many stress response genes and is required for virulence in several bacterial species. We now report that RpoS accumulates when Salmonella enterica serovar Typhimurium is growing logarithmically in media with low Mg(2+) concentrations. This process requires the two-component regulatory system PhoP/PhoQ, which is specifically activated in low Mg(2+). We show that PhoP controls RpoS protein turnover by serving as a transcriptional activator of the iraP (yaiB) gene, which encodes a product that enhances RpoS stability by interacting with RssB, the protein that normally delivers RpoS to the ClpXP protease for degradation. Mutation of the phoP gene rendered Salmonella as sensitive to hydrogen peroxide as an rpoS mutant after growth in low Mg(2+). In Escherichia coli, low Mg(2+) leads to only modest RpoS stabilization, and iraP is not regulated by PhoP/PhoQ. These findings add the sigma factor RpoS to the regulatory proteins and two-component systems that are elevated in a PhoP/PhoQ-dependent fashion when Salmonella face low Mg(2+) environments. Our data also exemplify the critical differences in regulatory circuits that exist between the closely related enteric bacteria Salmonella and E. coli.

An RNA sensor for intracellular Mg(2+).

Most RNA molecules require Mg(2+) for their structure and enzymatic properties. Here we report the first example of an RNA serving as sensor for cytoplasmic Mg(2+). We establish that expression of the Mg(2+) transporter MgtA of Salmonella enterica serovar Typhimurium is controlled by its 5' untranslated region (5'UTR). We show that the 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg(2+) levels, which determine whether transcription reads through into the mgtA coding region or stops within the 5'UTR. We could recapitulate the Mg(2+)-regulated transcription using a defined in vitro transcription system with RNA polymerase as the only protein component. The initiation of mgtA transcription responds to extracytoplasmic Mg(2+) and its elongation into the coding region to cytoplasmic Mg(2+), providing a singular example in which the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription.

Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium.

Drug efflux systems play a major role in resistance to a wide range of noxious compounds in several Gram negative species. Here, we report the drug resistance and virulence phenotypes of Salmonella mutants defective in either resistance-nodulation-division (RND)-type systems and/or in drug efflux systems belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATP-binding cassette (ABC) superfamilies. We determined that nine potential drug transporters contribute to drug resistance of Salmonella and found that the Salmonella-specific MdsABC system conferred resistance to a variety of toxic compounds. The RND-type MdsAB system could function with either MdsC, which is encoded in the same operon, or TolC as the outer membrane component. Although the Salmonella EmrAB, MdfA and MdtK are 90% identical in their amino acid sequences to their Escherichia coli homologues, the drug specificity of Salmonella transporters was different from that reported for equivalent E. coli transporters. Deletion of the macAB genes attenuated Salmonella virulence and a strain lacking all drug efflux systems was avirulent when mice were inoculated by the oral route. The promoter region of the macAB drug efflux system genes harbours a binding site for the response regulator PhoP, which functions to repress macAB transcription. The PhoP/PhoQ two-component system is a major regulator of Salmonella virulence, which underscores the connection between drug efflux systems and virulence.

Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica.

Genetic and genomic approaches have been successfully used to assign genes to distinct regulatory networks. However, the present challenge of distinguishing differentially regulated genes within a network is particularly hard because members of a given network tend to have similar regulatory features. We have addressed this challenge by developing a method, termed Gene Promoter Scan, that discriminates coregulated promoters by simultaneously considering both multiple cis promoter features and gene expression. Here, we apply this method to probe the regulatory networks governed by the PhoP/PhoQ two-component system in the enteric bacteria Escherichia coli and Salmonella enterica. Our analysis uncovered members of the PhoP regulon and interactions with other regulatory systems that were not discovered in previous approaches. The predictions made by Gene Promoter Scan were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, regulates acid resistance determinants, and is a central element in a highly connected network.

Transcriptional regulation of the 4-amino-4-deoxy-L-arabinose biosynthetic genes in Yersinia pestis.

Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe(3+), which is sensed by PmrA cognate sensor PmrB, and by low Mg(2+), in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg(2+)-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins.

Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins.

The PhoP/PhoQ two-component system is a master regulator that governs the ability of Salmonella to cause a lethal infection in mice, the adaptation to low Mg(2+) environments, and resistance to a variety of antimicrobial peptides. We have recently established that the PhoP-activated ugtL gene is required for resistance to the antimicrobial peptides magainin 2 and polymyxin B. Here we report that ugtL transcription requires not only the PhoP protein but also the virulence regulatory protein SlyA. The PhoP protein footprinted two regions of the ugtL promoter, mutation of either one of which was sufficient to abolish ugtL transcription. Although the SlyA protein is a transcriptional activator of the ugtL gene, it footprinted the ugtL promoter at a region located downstream of the transcription start site. The PhoP protein footprinted the slyA promoter, indicating that it controls slyA transcription directly. The slyA mutant was hypersensitive to magainin 2 and polymyxin B, suggesting that the virulence attenuation exhibited by slyA mutants may be caused by hypersensitivity to antimicrobial peptides. We propose that the PhoP and SlyA proteins control ugtL transcription using a feed-forward loop design.

Signal-dependent requirement for the co-activator protein RcsA in transcription of the RcsB-regulated ugd gene.

The RcsC/YojN/RcsB phosphorelay system controls gene expression in response to a variety of signals, including changes in temperature, osmolarity, and overproduction of membrane proteins. Transcription of certain RcsB-activated genes, such as the capsule synthesis cps operon, requires the co-activator protein RcsA, whereas expression of other RcsB-activated genes is RcsA-independent. We have established previously that a tolB mutation induces transcription of the Salmonella UDP-glucose dehydrogenase ugd gene in an RcsA- and RcsB-dependent manner. This induction is independent of the two-component systems PhoP/PhoQ and PmrA/PmrB, which are required for ugd expression in response to low Mg2+. We now report that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in expression of both cps and ugd genes. However, whereas cps transcription remained RcsA-dependent, ugd transcription became RcsA-independent but dependent on the PhoP protein. S1 mapping experiments demonstrated that RcsA-dependent and -independent transcription of the ugd gene use the same promoter. DNase footprinting analysis identified a PhoP-binding site in the ugd promoter. Yet, PhoP-mediated ugd transcription required either the RcsC/YojN/RcsB or the PmrA/PmrB systems.

Closing the loop: the PmrA/PmrB two-component system negatively controls expression of its posttranscriptional activator PmrD.

A fundamental question in biology is how an organism integrates multiple signals to mediate an appropriate cellular response. The PmrAPmrB two-component system of Salmonella enterica can be activated independently by Fe(3+), which is sensed by the PmrB protein, and in low Mg(2+), which is sensed by the PhoQ protein. The low-Mg(2+) activation requires pmrD, a PhoPPhoQ-activated gene that activates the response regulator PmrA at a posttranscriptional level. We now report that pmrD expression is negatively regulated by the PmrAPmrB system. Conditions that activate the PmrA protein independently of pmrD, such as exposure to Fe(3+), resulted in lower levels of pmrD transcription. The PmrA protein footprinted the pmrD promoter upstream of the PhoP-binding site but did not interfere with binding of the PhoP protein. Mutation of the PmrA-binding site in the pmrD promoter abolished PmrA-mediated repression. Negative regulation of the PhoPPhoQ-activated pmrD gene by the PmrAPmrB system closes a regulatory circuit designed to maintain proper cellular levels of activated PmrA protein and constitutes a singular example of a multicomponent feedback loop.