Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes

Reyrat, Jean‐Marc; David, M.; Blonski, Casimir; Boistard, P.; Batut, Jacques

doi:10.1128/jb.175.21.6867-6872.1993

Cited by 91 publications

(88 citation statements)

References 32 publications

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“…4) studies suggest that CsrA can activate csrB expression independently of BarA. UvrY is a member of the FixJ family of response regulators, and promoter binding by purified FixJ has been shown elsewhere to require phosphorylation (2,13,44). In addition, BarA has been shown elsewhere to phosphorylate UvrY (42).…”

Section: Discussionmentioning

confidence: 99%

Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli

et al. 2002

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The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA-lacZ expression. The uvrY defect was severalfold more severe than that of barA. Both csrA and uvrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY-lacZ and csrB-lacZ fusions but did not affect csrA-lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms.

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Section: Discussionmentioning

confidence: 99%

Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli

et al. 2002

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“…This second possibility appears to be more likely, because sequence analyses indicate that NodW is most similar to the FixJ class of regulator proteins (14,43), which appear to function as monomers. Both the phosphorylated and unphosphorylated forms of FixJ have been demonstrated to bind to their target promoter DNAs, with phosphorylation of FixJ stimulating the transcription of its target genes (32). Phosphorylation in the N-terminal receiver domain is proposed to alleviate the repressive effect that the N-terminal domain of FixJ has on the protein effector (i.e., C-terminal) domain.…”

Section: Discussionmentioning

confidence: 99%

NodV and NodW, a second flavonoid recognition system regulating nod gene expression in Bradyrhizobium japonicum

1997

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In Bradyrhizobium japonicum, members of two global regulatory families, a LysR-type regulator, NodD 1 , and a two-component regulatory system, NodVW, positively regulate nod gene expression in response to plantproduced isoflavone signals. By analogy to other two-component systems, NodV and NodW are thought to activate transcription via a series of phosphorylation steps. These include the phosphorylation of NodV in response to the plant signal and the subsequent activation of NodW via the transfer of the phosphoryl group to an aspartate residue in the receiver domain of NodW. In this study, we demonstrated that NodW can be phosphorylated in vitro by both acetyl phosphate and its cognate kinase, NodV. In addition, in vivo experiments indicate that phosphorylation is induced by genistein, a known isoflavone nod gene inducer in B. japonicum. By using site-directed mutagenesis, a NodWD70N mutant in which the aspartate residue at the proposed phosphorylation site was converted to an asparagine residue was generated. This mutant was not phosphorylated in either in vitro or in vivo assays. Comparisons of the biological activity of both the wild-type and mutant proteins indicate that phosphorylation of NodW is essential for the ability of NodW to activate nod gene expression.

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“…In these cases, the N-terminal domain modulates the activity of the C-terminal domain by means of several modifications in response to different environmental cues. For example, some proteins, like LuxR of Vibrio fischeri, bind quorum-sensing signaling molecules on their N-terminal domain (28); others, like E. coli NarL (21) and Sinorhizobium meliloti FixJ (1,58), are phosphorylated at a conserved Asp residue; E. coli MalT binds maltotriose (56) and ATP (59); and GerE is the smallest member of the family lacking an N-terminal domain (20). Interestingly, the N terminus of EcpR does not share similitude with any of these proteins, nor contains any conserved functional domains, and thus represents a novel member of this protein family.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Regulation of theecpOperon by EcpR, IHF, and H-NS in Attaching and Effacing Escherichia coli

et al. 2012

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ABSTRACTEnteropathogenic (EPEC) and enterohemorrhagic (EHEC)Escherichia coliare clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. TheE. colicommon pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenicE. colistrains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of theecpoperon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of theecpgenes and the role of EcpR as a transcriptional regulator. EHEC and EPECecpRmutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. Theecpgenes are transcribed as an operon from a promoter located 121 bp upstream of the start codon ofecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of theecppromoter, thus enhancing expression of downstreamecpgenes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activateecpexpression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on theecppromoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

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Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes

Cited by 91 publications

References 32 publications

Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli

Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems ofEscherichia coli

NodV and NodW, a second flavonoid recognition system regulating nod gene expression in Bradyrhizobium japonicum

Transcriptional Regulation of theecpOperon by EcpR, IHF, and H-NS in Attaching and Effacing Escherichia coli

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