Investigation of the Role of Electrostatic Charge in Activation of the
            <i>Escherichia coli</i>
            Response Regulator CheY

Smith, Jenny G.; Latiolais, Jamie A.; Guanga, Gerald P.; Citineni, Sindhura; Silversmith, Ruth E.; Bourret, Robert B.

doi:10.1128/jb.185.21.6385-6391.2003

Cited by 20 publications

(30 citation statements)

References 55 publications

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“…1) (42). Changes of aspartate 52 to alanine (D52A), glutamine (D52Q), and asparagine (D52N) are not tolerated (42), whereas a change to glutamate (D52E) results in a mutant VicR with low-level activity, consistent with analogous Asp3Glu mutations that constitutively activate other RRs (29,54). We also showed previously that the essential target of the VicRK TCS is the pcsB gene, which mediates murein biosynthesis and appears to be strongly positively regulated by VicRK under some conditions (41,42).…”

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confidence: 71%

Regulation of thepspAVirulence Factor and EssentialpcsBMurein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator inStreptococcus pneumoniae

2005

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The VicRK (YycFG) two-component regulatory system (TCS) is required for virulence of the human respiratory pathogen Streptococcus pneumoniae (pneumococcus). The VicR (YycF) response regulator (RR) is essential through its positive regulation of pcsB, which encodes an extracellular protein that mediates murein biosynthesis. To determine other genes that are regulated by VicR, we performed microarray analyses on a unique ⌬vicR deletion mutant, which was constructed by uncoupling regulation of pcsB. Results from these microarray experiments support the idea that the VicR RR exerts strong positive regulation on the transcription of a set of genes encoding important surface proteins, including the PspA virulence factor, two proteins (Spr0096 and Spr1875) containing LysM peptidoglycan-binding domains, and a putative membrane protein (Spr0709) of unknown function. To demonstrate direct regulation, we performed band shift and footprinting experiments using purified unphosphorylated VicR and phosphorylated VicR-P, which was prepared by reaction with acetyl phosphate. VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, spr1875, and spr0709. Phosphorylation of VicR to VicR-P increased the apparent strength and changed the nature of binding to these regions. DNase I footprinting of VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, and spr1875 showed protection of extended regions containing a degenerate sequence related to a previously proposed consensus. These combined approaches did not support autoregulation of the vicRKX operon or substantive direct regulation of fatty acid biosynthesis by VicR or VicR-P. However, the ⌬vicR mutant required fatty acids in some conditions, which supports the notion that the VicRK TCS may mediate membrane integrity as well as murein biosynthesis and virulence factor expression in S. pneumoniae.Streptococcus pneumoniae (pneumococcus) is an opportunistic extracellular human respiratory pathogen that causes several serious diseases, including pneumonia, otitis media (ear infection), sinusitis, meningitis, and septicemia (40,62,64). Invasive pneumococcal disease results in a high mortality and morbidity rate (as many as 1 million deaths annually worldwide), especially among young, elderly, debilitated, and immunosuppressed individuals (25,26), and resistance to a range of antibiotics is increasing at an alarming rate among clinical isolates of S. pneumoniae (1,30). As in other bacterial pathogens (17,23,57), two-component signal transduction systems (TCSs) are thought to play key roles in pneumococcal colonization and virulence. The genomes of S. pneumoniae serotype 2 and 4 strains encode 13 complete TCSs, each containing a presumed cognate response regulator (RR) and histidine kinase (HK) pair, and one orphan response regulator (21,31,60,61). Several of these TCSs have recently been found to be required for full pneumococcal pathogenesis, possibly through the regulation of virulence factor genes. These TCSs include ComDE (quorum sensing and competence) (3,16,32),...

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confidence: 71%

Regulation of thepspAVirulence Factor and EssentialpcsBMurein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator inStreptococcus pneumoniae

2005

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“…E. coli BL21(DE3) harboring pASK-IBA7 ϩ ::cheD was induced with 200 ng/ml of anhydrotetracycline. Expression and purification of E. coli CheA and B. burgdorferi His-CheY3 and His-CheX proteins were described previously (33,52,53). B. burgdorferi mcp3, mcp4, and mcp5 open reading frames were cloned separately in the expression vector pMAL c2x (NEB Inc.) to generate MBP-MCP3, MBP-MCP4, and MBP-MCP5 recombinant proteins, respectively.…”

Section: Methodsmentioning

confidence: 99%

Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

2016

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b Borrelia burgdorferi possesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized the B. burgdorferi CheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies. Bacillus subtilis CheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates that B. burgdorferi CheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of the cheD mutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tickmouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability of cheD mutants in ticks is marginally reduced compared to that of the wild-type or complemented cheD spirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis of B. burgdorferi. We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.

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“…Phosphorylation occurs on Asp57 (9), while two additional Asp residues, at positions 12 and 13, chelate a Mg 2ϩ ion necessary for transfer of the activating phosphoryl group from the donor His (81). Three other amino acids, Thr87, Tyr106, and Lys109, convey the phosphorylation-associated conformational change that facilitates effector function (6,12,37,38,70).…”

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confidence: 99%

Two-Component Response Regulators ofVibrio fischeri: Identification, Mutagenesis, and Characterization

et al. 2007

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Two-component signal transduction systems are utilized by prokaryotic and eukaryotic cells to sense and respond to environmental stimuli, both to maintain homeostasis and to rapidly adapt to changing conditions. Studies have begun to emerge that utilize a large-scale mutagenesis approach to analyzing these systems in prokaryotic organisms. Due to the recent availability of its genome sequence, such a global approach is now possible for the marine bioluminescent bacterium Vibrio fischeri, which exists either in a free-living state or as a mutualistic symbiont within a host organism such as the Hawaiian squid species Euprymna scolopes. In this work, we identified 40 putative two-component response regulators encoded within the V. fischeri genome. Based on the type of effector domain present, we classified six as NarL type, 13 as OmpR type, and six as NtrC type; the remaining 15 lacked a predicted DNA-binding domain. We subsequently mutated 35 of these genes via a vector integration approach and analyzed the resulting mutants for roles in bioluminescence, motility, and competitive colonization of squid. Through these assays, we identified three novel regulators of V. fischeri luminescence and seven regulators that altered motility. Furthermore, we found 11 regulators with a previously undescribed effect on competitive colonization of the host squid. Interestingly, five of the newly characterized regulators each affected two or more of the phenotypes examined, strongly suggesting interconnectivity among systems. This work represents the first large-scale mutagenesis of a class of genes in V. fischeri using a genomic approach and emphasizes the importance of two-component signal transduction in bacterium-host interactions.The symbiosis between the Hawaiian squid Euprymna scolopes and the bacterium Vibrio fischeri serves as a model for symbiotic bacterium-host interactions. Previous studies have revealed a number of bacterial factors required for host colonization (24, 56), including motility (25, 49, 57) and luminescence (78). Many of these factors were identified by generating and testing specific hypotheses developed from an understanding of the general colonization process. To date, however, large-scale studies of colonization factors have been hampered by a deficit of genetic tools needed for bacterial mutant construction. Recently, however, the genome sequence of V. fischeri has been published (65). In addition, the availability of a useful suicide vector (17) has greatly facilitated mutant construction (73, 90). Thus, it is now possible to approach the investigation of V. fischeri biology from a genomic perspective.

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Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY

Cited by 20 publications

References 55 publications

Regulation of thepspAVirulence Factor and EssentialpcsBMurein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator inStreptococcus pneumoniae

Regulation of thepspAVirulence Factor and EssentialpcsBMurein Biosynthetic Genes by the Phosphorylated VicR (YycF) Response Regulator inStreptococcus pneumoniae

Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

Two-Component Response Regulators ofVibrio fischeri: Identification, Mutagenesis, and Characterization

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