Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ from<i>Pseudomonas aeruginosa</i>

Matsuyama, Bruno Y.; Krasteva, Petya V.; Baraquet, Claudine; Harwood, Caroline S.; Sondermann, Holger; Navarro, M.V.A.S.

doi:10.1073/pnas.1523148113

Cited by 155 publications

(212 citation statements)

References 63 publications

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“…In Streptomyces, c-di-GMP mediates the switch from vegetative growth to sporulation through its binding to the transcriptional repressor BldD (20). In Pseudomonas, c-di-GMP binding to the NtrC-like protein FleQ results in stimulation of pel transcription (21)(22)(23). In Klebsiella pneumoniae, the PilZ domain-containing transcriptional activator MrkH activates genes for fimbriae synthesis upon c-di-GMP binding (24).…”

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confidence: 99%

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Schäper

Steinchen

Krol

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

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confidence: 99%

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Schäper

Steinchen

Krol

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, a full activity of FlrA to repress the transcription of bpfA involves both of the binding sites of box 1 and box 2. c-di-GMP interferes with the binding of FlrA to the promoter of bpfA. FleQ, an FlrA ortholog in Pseudomonas aeruginosa, functions as a c-di-GMP-responsive regulator, and its residues involved in the biding of c-di-GMP have been characterized (30). FlrA also possesses these conserved residues (Fig.…”

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confidence: 99%

“…FlrA also possesses these conserved residues (Fig. S5) (30), which suggests that the activity of FlrA may be tuned by c-di-GMP, a second messenger found in many bacteria (31). We first tested the effect of c-di-GMP on the binding of FlrA to the FlrA box ib bpfA promoter region in vitro via fluorescence polarization (FP).…”

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confidence: 99%

FlrA Represses Transcription of the Biofilm-Associated bpfA Operon in Shewanella putrefaciens

Cheng

et al. 2017

Appl Environ Microbiol

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Manipulation of biofilm formation in Shewanella is beneficial for application to industrial and environmental biotechnology. BpfA is an adhesin largely responsible for biofilm formation in many Shewanella species. However, the mechanism underlying BpfA production and the resulting biofilm remains vaguely understood. We previously described the finding that BpfA expression is enhanced by DosD, an oxygen-stimulated diguanylate cyclase, under aerobic growth. In the present work, we identify FlrA as a critical transcription regulator of the bpfA operon in Shewanella putrefaciens CN32 by transposon mutagenesis. FlrA acted as a repressor of the operon promoter by binding to two boxes overlapping the Ϫ10 and Ϫ35 sites recognized by 70 . DosD regulation of the expression of the bpfA operon was mediated by FlrA, and cyclic diguanylic acid (c-di-GMP) abolished FlrA binding to the operon promoter. We also demonstrate that FlhG, an accessory protein for flagellum synthesis, antagonized FlrA repression of the expression of the bpfA operon. Collectively, this work demonstrates that FlrA acts as a central mediator in the signaling pathway from c-di-GMP to BpfA-associated biofilm formation in S. putrefaciens CN32.IMPORTANCE Motility and biofilm are mutually exclusive lifestyles, shifts between which are under the strict regulation of bacteria attempting to adapt to the fluctuation of diverse environmental conditions. The FlrA protein in many bacteria is known to control motility as a master regulator of flagellum synthesis. This work elucidates its effect on biofilm formation by controlling the expression of the adhesin BpfA in S. putrefaciens CN32 in response to c-di-GMP. Therefore, FlrA plays a dual role in controlling motility and biofilm formation in S. putrefaciens CN32. The cooccurrence of flrA, bpfA, and the FlrA box in the promoter region of the bpfA operon in diverse Shewanella strains suggests that bpfA is a common mechanism that controls biofilm formation in this bacterial species.KEYWORDS FlrA, Shewanella, biofilm, cyclic di-GMP, transcriptional factor B iofilm formed by bacteria is a multicell architecture for adaptation to diverse niches (1). Shewanella can form biofilm on a variety of surfaces, such as ferric oxides, electrodes, stainless steel, and glass (2-4). This genus is also renowned for an extracellular respiration ability to reduce iron oxide, electrodes, and other extracellular electron acceptors, including heavy metal ions. Such an ability has diverse potential applications in bioengineering and bioremediation (5). Biofilm formation in Shewanella benefits close contact with solid electron acceptors, thereby accelerating iron oxide reduction and improving current output, as well as inducing spatially stratified metabolic responses during contaminant exposure (4, 6, 7). Biofilm formed by Shewanella prevents microbially induced corrosion of steel and cast iron pipes (8, 9). On the other hand, biofilm formation of Shewanella also causes problems in some circumstances. For

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“…Because several PilR-type transcription regulators, e.g., FleQ from P. aeruginosa (29,30) and XbmR from Xanthomonas citri (31), have been shown to bind c-di-GMP directly, (25). ΔpilR(pURF047), the ΔpilR mutant containing an empty vector; ΔpilR(pilR-cp2), the ΔpilR mutant with plasmid pilR-pUFR047, containing the intact pilR gene.…”

Section: Resultsmentioning

confidence: 99%

“…The PilR-mediated regulation of HSAF production turned out to be indirect and independent of the regulation of T4P genes. Because several NtrC-type RRs to which PilR belongs, including P. aeruginosa FleQ (29,30) and X. citri XbmR (31), bind c-di-GMP directly, in this work we tested L. enzymogenes PilR for c-di-GMP binding; however, no binding was detected. Intriguingly, we found that the pilR deletion resulted in elevated intracellular c-di-GMP levels, which proved to be inhibitory for HSAF production.…”

Section: Discussionmentioning

confidence: 99%

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway

Chen

Xia

et al. 2017

Appl Environ Microbiol

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Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenes.

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Mechanistic insights into c-di-GMP–dependent control of the biofilm regulator FleQ fromPseudomonas aeruginosa

Cited by 155 publications

References 63 publications

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

FlrA Represses Transcription of the Biofilm-Associated bpfA Operon in Shewanella putrefaciens

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway

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