LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis

Li, Weihui; He, Zheng‐Guo

doi:10.1093/nar/gks923

Cited by 79 publications

(88 citation statements)

References 59 publications

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“…While most early studies documented c-di-GMP regulation at the posttranslational level, recent characterization of the DNA-binding proteins VpsT, BldD, Bcam1349, BrlR, CLP, FleQ/FlrA, LtmA, MrkH, and VpsR (33,57,61,62,(70)(71)(72)(73)(74)(75) provided clear examples of c-di-GMP involvement in transcriptional regulation. Remarkably, while most of these transcriptional regulators bind DNA via standard helix-turn-helix domains, MrkH combines its c-di-GMP-binding PilZ domain with a previously unknown type of the DNA-binding domain, which is related to PilZN/YcgR and PilZNR/YcgR_2 domains (see Fig.…”

Section: Discussionmentioning

confidence: 99%

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

2016

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Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMPbinding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms.

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Section: Discussionmentioning

confidence: 99%

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

2016

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“…Both Clp and Bcam1349 are homologous to the well-studied cyclic AMP (c-AMP)-responsive regulator Crp. LtmA, an apparently new class of regulator from Mycobacterium smegmatis, binds c-di-GMP to positively regulate the expression of lipid transport and metabolism genes (47). Finally, BrlR, a MerR family member from P. aeruginosa, was recently shown to bind c-di-GMP to control gene expression (48).…”

Section: Discussionmentioning

confidence: 99%

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

2016

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The transcription factor FleQ from Pseudomonas aeruginosa derepresses expression of genes involved in biofilm formation when intracellular levels of the second messenger cyclic diguanosine monophosphate (c-di-GMP) are high. FleQ also activates transcription of flagellar genes, and the expression of these genes is highest at low intracellular c-di-GMP. FleQ thus plays a central role in mediating the transition between planktonic and biofilm lifestyles of P. aeruginosa. Previous work showed that FleQ controls expression of the pel operon for Pel exopolysaccharide biosynthesis by converting from a repressor to an activator upon binding c-di-GMP. To explore the activity of FleQ further, we carried out DNase I footprinting at three additional biofilm gene promoters, those of psl, cdrAB, and PA2440. The expression of cdrAB, encoding a cell surface adhesin, was sufficiently responsive to FleQ to allow us to carry out in vivo promoter assays. The results showed that, similarly to our observations with the pel operon, FleQ switches from a repressor to an activator of cdrAB gene expression in response to c-di-GMP. From the footprinting data, we identified a FleQ DNA binding consensus sequence. A search for this conserved sequence in bacterial genome sequences led to the identification of FleQ binding sites in the promoters of the siaABCD operon, important for cell aggregation, and the bdlA gene, important for biofilm dispersal, in P. aeruginosa. We also identified FleQ binding sites upstream of lapA-like adhesin genes in other Pseudomonas species. IMPORTANCEThe transcription factor FleQ is widely distributed in Pseudomonas species. In all species examined, it is a master regulator of flagellar gene expression. It also regulates diverse genes involved in biofilm formation in P. aeruginosa when intracellular levels of the second messenger c-di-GMP are high. Unlike flagellar genes, biofilm-associated genes are not always easy to recognize in genome sequences. Here, we identified a consensus DNA binding sequence for FleQ. This allowed us to survey Pseudomonas strains and find new genes that are likely regulated by FleQ and possibly involved in biofilm formation. Cyclic diguanosine monophosphate (c-di-GMP) is an intracellular second messenger that is produced by bacteria and has diverse effects on bacterial physiology. c-di-GMP binds to riboswitches and effector proteins to modulate transcription, translation, and protein activities (1, 2). In the opportunistic pathogen Pseudomonas aeruginosa, the transcription factor FleQ binds c-di-GMP to derepress expression of genes for biofilm components (3, 4). Biofilms are surface-attached communities of bacteria embedded in a matrix made of exopolysaccharides, proteins, and DNA. Biofilm infections are particularly difficult to treat because bacteria are protected by the biofilm matrix, making them resistant to antimicrobial treatment compared to planktonic cells (5). Genes controlled by FleQ include pel genes, coding for Pel exopolysaccharide; psl genes, coding for Psl exopolys...

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“…2). The presence of FleQ dimers with ATPase activity may signify that (34), and VpsR from V. cholerae, remain to be identified (35). The X. campestris Clp protein and FleQ belong to well-conserved and well-studied families of transcriptional regulators.…”

Section: Discussionmentioning

confidence: 99%

Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ

Baraquet

Harwood

2013

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

160

185

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The transcription factor FleQ is a bacterial AAA+ ATPase enhancerbinding protein that is the master activator of flagella gene expression in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Homologs of FleQ are present in all Pseudomonas species and in many polarly flagellated gamma proteobacteria. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls the transition between planktonic and biofilm modes of growth in bacteria in response to diverse environmental signals. C-di-GMP binds to FleQ to dampen its activity, causing down-regulation of flagella gene expression. This action is potentiated in the simultaneous presence of another protein, FleN. We explored the effect of c-di-GMP and FleN on the ATPase activity of FleQ and found that a relatively low concentration of c-di-GMP competitively inhibited FleQ ATPase activity, suggesting that c-di-GMP competes with ATP for binding to the Walker A motif of FleQ. Confirming this, a FleQ Walker A motif mutant failed to bind c-di-GMP. FleN, whose gene is regulated by FleQ, also inhibited FleQ ATPase activity, and FleQ ATPase activity was much more inhibited by c-di-GMP in the presence of FleN than in its absence. These results indicate that FleN and c-di-GMP cooperate to inhibit FleQ activity and, by extension, flagella synthesis in P. aeruginosa. The Walker A motif of FleQ is perfectly conserved, opening up the possibility that other AAA+ ATPases may respond to c-di-GMP.an ubiquitous second messenger in bacteria, where it affects the transition between a motile planktonic lifestyle and an adhesive biofilm lifestyle by binding to various effector proteins to modulate enzymatic activity, protein-protein interactions, transcription, and translation (1-6). One of these effector proteins is FleQ (PA1097), an enhancer-binding protein (EBP) transcription factor from the opportunistic pathogen Pseudomonas aeruginosa.FleQ contains an N-terminal FleQ domain, a central AAA+ ATPase domain, and a C-terminal helix-turn-helix DNA-binding domain. It activates expression of biofilm-related genes, including genes for Pel and Psl exopolysaccharide (EPS) in response to binding of c-di-GMP at an unknown site (7,8). EBPs typically act in conjunction with σ 54 -RNA polymerase; however, FleQ regulates EPS gene expression independent of σ 54 , most likely with σ 70 (8, 9).In addition to regulating genes for biofilm formation, FleQ has a second, better-known role as a σ 54 -dependent master regulator of P. aeruginosa flagella gene expression (9, 10). FleQ activates expression of the two-component regulatory genes fleSR, as well as genes for the assembly of the flagella export apparatus and for the initiation of flagella basal body assembly. FleSR controls genes to complete the basal body-hook structure (9, 11).The regulation of both flagella and biofilm genes is also under the control of a second protein, FleN. Mutations in fleN (PA1454) lead to an up-regulation of flagella gene expression and a smalldown-regulation of biofilm genes (7, 12). The expression ...

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LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis

Cited by 79 publications

References 59 publications

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ

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