MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

Schuhmacher, Jan S.; Roßmann, Florian; Dempwolff, Felix; Knauer, Carina; Altegoer, Florian; Steinchen, Wieland; Dörrich, Anja K.; Klingl, Andreas; Stephan, Milena; Linne, Uwe; Thormann, Kai M.; Bange, Gert

doi:10.1073/pnas.1419388112

Cited by 94 publications

(148 citation statements)

References 49 publications

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“…Our present results suggest a potential interaction between FlrA and FlhG to control the transcription of the bpfA operon; however, we still cannot exclude the possibility that other mechanisms may be involved. It has been reported that FlhG affects the number and location of flagella not as a result of FlrA but through direct interaction with flagellar C-ring proteins (38).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Proteins were produced in BL21 (DE3) (Novagen). After cell lysis by a Microfluidizer (M110-L, Microfluidics), cell debris was removed by high-speed centrifugation and proteins were purified by Ni-ion affinity-and size-exclusion chromatography, as described recently (43). The SEC buffer consisted of 20 mM Hepes-Na (pH 7.5), 200 mM NaCl, 20 mM KCl, and 20 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein

Altegoer

Rensing

Bange

2016

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

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Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5′UTR of an mRNA occludes the Shine–Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal β-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5′UTR from the CsrA–RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein.

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“…FleQ works in concert with another protein, FleN, which is an ATPase with a deviant Walker A motif (24,25). FleN is required for full expression of biofilm-related genes in the presence of c-di-GMP (4), but its exact biological role in biofilm gene expression is not clear.…”

Section: Figmentioning

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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MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

Cited by 94 publications

References 49 publications

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

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

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein

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

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