BDSF Is a Degradation-Prone Quorum-Sensing Signal Detected by the Histidine Kinase RpfC of Xanthomonas campestris pv. campestris.

Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species, and it is the predominant in planta quorum-sensing signal in Xanthomonas campestris. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF does not seem to be a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC-RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase activity to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl coenzyme A ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects. IMPORTANCE The diffusible signal factor (DSF) family consists of quorum-sensing signals employed by Gram-negative bacteria. These signals are a group of cis-2-unsaturated fatty acids, such as DSF, BDSF, IDSF, CDSF, and SDSF. However, the functional divergence of various DSF signals remains unclear. The present study demonstrates that though BDSF is a low active quorum-sensing signal, it binds histidine kinase RpfC with a higher affinity and activates RpfC autophosphorylation to the similar level as DSF. Rather than regulation of enzymatic activities of RpfC and its cognate response regulator RpfG encoding a c-di-GMP hydrolase, BDSF is prone to degradation in bacterial cells by RpfB, which effectively avoided the inhibition of bacterial growth by accumulating high concentrations of BDSF. Therefore, our study sheds new light on the functional differences of quorum-sensing signals and shows that bacteria balance quorum sensing and growth by fine-tuning concentrations of signaling chemicals.

Author Correction: BsmR degrades c-di-GMP to modulate biofilm formation of nosocomial pathogen Stenotrophomonas maltophilia.

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BsmR degrades c-di-GMP to modulate biofilm formation of nosocomial pathogen Stenotrophomonas maltophilia.

c-di-GMP is a cellular second messenger that regulates diverse bacterial processes, including swimming, biofilm formation and virulence. However, in Stenotrophomonas maltophilia, a nosocomial pathogen that frequently infects immunodeficient or immunoincompetent patients, the regulatory function of c-di-GMP remains unclear. Here we show that BsmR is a negative regulator of biofilm development that degrades c-di-GMP through its EAL domain. Increasing BsmR expression resulted in significant increase in bacterial swimming and decrease in cell aggregation. BsmR regulates the expression of at least 349 genes. Among them, 34 involved in flagellar assembly and a flagellar-assembly-related transcription factor (fsnR) are positively regulated. Although BsmR is a response regulator of the two-component signaling system, its role in biofilm formation depends on the expression level of its respective gene (bsmR), not on the protein's phosphorylation level. A transcription factor, BsmT, whose coding gene is located in the same tetra-cistronic operon as bsmR, was shown to directly bind to the promoter region of the operon and, through a positive regulatory loop, modulate bsmR transcription. Thus, our results revealed that the c-di-GMP signaling pathway controls biofilm formation and swimming in S. maltophilia, suggesting c-di-GMP signaling as a target in the development of novel antibacterial agents to resist this pathogen.

Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.