Abstract
Complex 3′-5′-cyclic diguanylic acid (c-di-GMP) responsive regulatory networks that are modulated by the action of multiple diguanylate cyclases (DGC; GGDEF domain proteins) and phosphodiesterases (PDE; EAL domain proteins) have evolved in many bacteria. YfgF proteins possess a membrane-anchoring domain (MASE1), a catalytically inactive GGDEF domain and a catalytically active EAL domain. Here, sustained expression of the Salmonella enterica spp. Enterica ser. Enteritidis YfgF protein is shown to mediate inhibition of the formation of the aspartate chemotactic ring on motility agar under aerobic conditions. This phenomenon was c-di-GMP-independent because it occurred in a Salmonella strain that lacked the ability to synthesize c-di-GMP and also when PDE activity was abolished by site-directed mutagenesis of the EAL domain. YfgF-mediated inhibition of aspartate chemotactic ring formation was impaired in the altered redox environment generated by exogenous p-benzoquinone. This ability of YfgF to inhibit the response to aspartate required a motif, 213Lys-Lys-Glu215, in the predicted cytoplasmic loop between trans-membrane regions 5 and 6 of the MASE1 domain. Thus, for the first time the function of a MASE1 domain as a redox-responsive regulator of bacterial responses to aspartate has been shown.
Highlights
Enteritidis 3934 and a derivative Salmonella DXII which has deletions of all 12 genes encoding GGDEF proteins [5] were used in the investigation of c-di-GMP-independent regulation of aspartate chemotaxis by YfgF
Enteritidis YfgF protein was achieved by expression of the corresponding genes under the control of the pBAD promoter in the indicated plasmids
The results presented here used YfgF protein with a C-terminal FLAG-tag, but it should be noted that similar results were obtained with YfgF carrying a C-terminal Histag, suggesting that the presence of the C-terminal tag does not impair YfgF function
Summary
Enteritidis 3934 and a derivative Salmonella DXII which has deletions of all 12 genes encoding GGDEF proteins [5] were used in the investigation of c-di-GMP-independent regulation of aspartate chemotaxis by YfgF. Enteritidis YfgF protein (and site-directed variants thereof) was achieved by expression of the corresponding genes under the control of the pBAD promoter in the indicated plasmids (all YfgF proteins were expressed with a C-terminal FLAG-tag for immunoassays). The pGS2421 plasmid that encoded the wild-type yfgF gene was the template for site-directed mutagenesis using the Quikchange (Stratagene) protocol with appropriate oligonucleotides to express the YfgF variants studied here.
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