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West Haven, CT, United States

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. Source


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. Source


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. Source


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. Source


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. Source

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