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

Abstract

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

Partially Reciprocal Replacement of FlrA and FlrC in Regulation of Shewanella oneidensis Flagellar Biosynthesis

“…Studies of P. aeruginosa FleN, an ortholog of FlhG, have established that FleN inhibits FleQ (the FlrA ortholog) by direct interaction (32)(33)(34). For Shewanella spp., a similar mechanism for the antagonistic effect of FlhG against FlrA was proposed recently (35). Despite this, given that overproduction of FlrA or FlrC results in the same phenotype, we reasoned that FlhG and FlhFG may influence the expression of FlrA and FlrC.…”

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

“…Indeed, the bpfA operon, encoding proteins involved in biofilm formation in several Shewanella species, was shown to be controlled by binding of the FlrA repressor in its promoter region. This binding is abolished by c-di-GMP, leading to increased bpfA expression (Theunissen et al, 2010;Zhou et al, 2015;Cheng et al, 2017). S. oneidensis strains containing this fusion as well as the pBpdgA, pBpdgB or pBdgcQ plasmid were incubated under pellicle assay conditions in the presence or absence of the arabinose inducer and β-galactosidase activities were measured after 2 and 4 h. As shown on Fig.…”

Mechanism and Role of Globin-Coupled Sensor Signalling

“…Both BpfA and AggA are required for DosD-dependent effects on biofilm formation, suggesting that, similarly to Ec DosC, DosD serves to sense O 2 levels and regulate expression of key proteins involved in bacterial biofilm formation by regulating c-di-GMP levels. Given the broad importance of c-di-GMP in regulating biofilm formation, it is likely that all DGC-containing GCS proteins will be found to play some role in O 2 -dependent expression of biofilm-related genes (Cheng et al, 2016; Wu et al, 2013). …”