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Zwir I.,Yale University | Zwir I.,University of Granada | Zwir I.,University of Washington | Zwir I.,Howard Hughes Medical Institute | And 9 more authors.
Molecular Microbiology | Year: 2012

The DNA-binding protein PhoP controls virulence and Mg 2+ 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. © 2012 Blackwell Publishing Ltd.

Yeo W.-S.,Yale University | Yeo W.-S.,Howard Hughes Medical Institute | Yeo W.-S.,University of Washington | Zwir I.,Yale University | And 10 more authors.
Molecular Cell | Year: 2012

PhoP and PhoQ comprise a two-component system in the bacterium Salmonella enterica. PhoQ is the sensor kinase/phosphatase that modifies the phosphorylation state of the regulator PhoP in response to stimuli. The amount of phosphorylated PhoP surges after activation, then declines to reach a steady-state level. We now recapitulate this surge invitro by incubating PhoP and PhoQ with ATP and ADP. Mathematical modeling identified PhoQ's affinity for ADP as the key parameter dictating phosphorylated PhoP levels, as ADP promotes PhoQ's phosphatase activity toward phosphorylated PhoP. The lid covering the nucleotide-binding pocket of PhoQ governs the kinase to phosphatase switch because a lid mutation that decreased ADP binding compromised PhoQ's phosphatase activity invitro and resulted in sustained expression of PhoP-dependent mRNAs invivo. This feedback mechanism may curtail futile ATP consumption because ADP not only stimulates PhoQ's phosphatase activity but also inhibits ATP binding necessary for the kinase reaction. © 2012 Elsevier Inc.

Chen H.D.,University of Washington | Chen H.D.,Howard Hughes Medical Institute | Jewett M.W.,University of Washington | Jewett M.W.,University of Central Florida | And 3 more authors.
PLoS Genetics | Year: 2012

Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties. © 2012 Chen et al.

Kato A.,Howard Hughes Medical Institute | Kato A.,University of Washington | Kato A.,Kinki University | Chen H.D.,Howard Hughes Medical Institute | And 8 more authors.
Molecular Cell | Year: 2012

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 Fe3+ binding to the bacterial cell. Because Fe3+ 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. © 2012 Elsevier Inc.

Raghavan V.,University of Washington | Lowe E.C.,Northumbria University | Townsend G.E.,Howard Hughes Medical Institute | Townsend G.E.,Yale University | And 5 more authors.
Molecular Microbiology | Year: 2014

Summary: Cells respond to nutrient availability by expressing nutrient catabolic genes. We report that the regulator controlling utilization of chondroitin sulphate (CS) in the mammalian gut symbiont Bacteroides thetaiotaomicron is activated by an intermediate in CS breakdown rather than CS itself. We determine that the rate-determining enzyme in CS breakdown is responsible for degrading this intermediate and establish that the levels of the enzyme increase 100-fold, whereas those of the regulator remain constant upon exposure to CS. Because enzyme and regulator compete for the intermediate, B. thetaiotaomicron tunes transcription of CS utilization genes to CS catabolic rate. This tuning results in a transient increase in CS utilization transcripts upon exposure to excess CS. Constitutive expression of the rate-determining enzyme hindered activation of CS utilization genes and growth on CS. An analogous mechanism regulates heparin utilization genes, suggesting that the identified strategy aids B. thetaiotaomicron in the competitive gut environment. © 2014 John Wiley & Sons Ltd.

Lee E.-J.,Howard Hughes Medical Institute | Lee E.-J.,Yale Microbial Diversity Institute | Groisman E.A.,Howard Hughes Medical Institute
Molecular Microbiology | Year: 2012

The mgtCBR operon from Salmonella enterica serovar Typhimurium specifies the virulence protein MgtC, the Mg2+ transporter MgtB and the regulatory peptide MgtR. The mgtCBR transcript includes a long leader region harbouring two short open reading frames (ORFs). Translation of these ORFs is anticipated to impact the formation of particular stem-loop structures and control transcription of the coding region by an attenuation-like mechanism. We previously reported that ORF mgtM enables Salmonella to promote transcription of the mgtC and mgtB coding regions when experiencing a rise in cytoplasmic ATP levels. We now show that the proline codon-rich ORF mgtP mediates an increase in transcription of the mgtC and mgtB coding regions under conditions predicted to decrease the levels of proline-charged tRNAPro. The high ATP and low proline signals act independently in an additive form. Replacing conserved mgtP proline codons with codons specifying other amino acids abolished the response to proline limitation but had no effect on the response to ATP. Substitution of conserved adenine nucleotides in mgtM abolished the response to ATP but had no effect in the response to proline limitation. This provides a singular example of a leader mRNA with tandem attenuators responding to different signals. Copyright © 2012 Blackwell Publishing Ltd.

Lee E.-J.,Howard Hughes Medical Institute | Lee E.-J.,Yale University | Lee E.-J.,Yale Microbial Diversity Institute | Lee E.-J.,Kyung Hee University | And 5 more authors.
Cell | Year: 2013

Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F 1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1F o ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein. © 2013 Elsevier Inc.

Groisman E.A.,Yale University | Groisman E.A.,Howard Hughes Medical Institute | Groisman E.A.,Yale Microbial Diversity Institute | Hollands K.,Yale University | And 13 more authors.
Annual Review of Genetics | Year: 2013

Organisms must maintain physiological levels of Mg2+ because this divalent cation is critical for the stabilization of membranes and ribosomes, for the neutralization of nucleic acids, and as a cofactor in a variety of enzymatic reactions. In this review, we describe the mechanisms that bacteria utilize to sense the levels of Mg2+ both outside and inside the cytoplasm. We examine how bacteria achieve Mg2+ homeostasis by adjusting the expression and activity of Mg2+ transporters and by changing the composition of their cell envelope. We discuss the connections that exist between Mg2+ sensing, Mg2+ transport, and bacterial virulence. Additionally, we explore the logic behind the fact that bacterial genomes encode multiple Mg2+ transporters and distinct sensing systems for cytoplasmic and extracytoplasmic Mg2+. These analyses may be applicable to the homeostatic control of other cations. © 2013 by Annual Reviews. All rights reserved.

Park S.-Y.,Yale University | Park S.-Y.,Howard Hughes Medical Institute | Park S.-Y.,Yale Microbial Diversity Institute | Groisman E.A.,Yale University | And 2 more authors.
Molecular Microbiology | Year: 2014

The two-component system PhoP/PhoQ controls a large number of genes responsible for a variety of physiological and virulence functions in Salmonella enterica serovar Typhimurium. Here we describe a mechanism whereby the transcriptional activator PhoP elicits expression of dissimilar gene sets when its cognate sensor PhoQ is activated by different signals in the periplasm. We determine that full transcription of over half of the genes directly activated by PhoP requires the Mg2+ transporter MgtA when the PhoQ inducing signal is low Mg2+, but not when PhoQ is activated by mildly acidic pH or the antimicrobial peptide C18G. MgtA promotes the active (i.e. phosphorylated) form of PhoP by removing Mg2+ from the periplasm, where it functions as a repressing signal for PhoQ. MgtA-dependent expression enhances resistance to the cationic antibiotic polymyxin B. Production of the MgtA protein requires cytoplasmic Mg2+ levels to drop below a certain threshold, thereby creating a two-tiered temporal response among PhoP-dependent genes. © 2013 John Wiley & Sons Ltd.

May J.F.,Yale University | May J.F.,Yale Microbial Diversity Institute | Groisman E.A.,Yale University | Groisman E.A.,Yale Microbial Diversity Institute | Groisman E.A.,Howard Hughes Medical Institute
Molecular Microbiology | Year: 2013

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O-antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram-negative bacteria. We determined that activation of the PmrA/PmrB two-component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA-activated gene wzzst, which encodes the protein that determines long O-antigen chain length and confers resistance to complement-mediated killing. By contrast, this growth defect did not require the wzzfepE gene, which controls production of very long O-antigen, or other PmrA-activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O-antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer-membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O-antigen in its LPS to respond to the different conditions it faces during infection. © 2013 John Wiley & Sons Ltd.

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