Regulation of the rhaEWRBMA Operon Involved in l -Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

Abstract

The Bacillus subtilis rhaEWRBMA (formerly yuxG-yulBCDE) operon consists of four genes encoding enzymes for L-rhamnose catabolism and the rhaR gene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of the rhaEW gene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited by L-rhamnulose-1-phosphate, an intermediate of L-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control in B. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site. In vivo analysis of the rhaEW promoter-lacZ fusion in the background of ccpA deletion showed that the L-rhamnose-responsive induction of the rhaEW promoter was negated by the disruption of rhaA or rhaB but not rhaEW or rhaM, whereas rhaR disruption resulted in constitutive rhaEW promoter activity. These in vitro and in vivo results clearly indicate that RhaR represses the operon by binding to the operator site, which is detached by L-rhamnulose-1-phosphate formed from L-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, the lacZ reporter analysis using the strains with or without the ccpA deletion under the background of rhaR disruption supported the involvement of CcpA in the carbon catabolite repression of the operon. IMPORTANCESince L-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested that L-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance of L-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system of Escherichia coli. In this study, we show that, in Bacillus subtilis, one of the plant growth-promoting rhizobacteria, the rhaEWRBMA operon for L-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms of L-rhamnose catabolism in other bacterial species. Bacillus subtilis is a soil-dwelling bacterium that has been extensively and closely studied as a Gram-positive model bacterium. B. subtilis species have often been isolated from the rhizosphere of various terrestrial plants; many of them have been shown to be plant growth-promoting rhizobacteria whose association with the plant roots enhances the adaptive potential of plants and increases their growth (1). Interestingly, B. subtilis is also a saprophytic bacterium that secretes various degrading enzymes, incl...

Disruption of l -Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

“…Further, the rhaEWRBMA rhamnose catabolism operon is lacking in the genome of S. mutans and other closely related streptococcal species (27). In contrast, this genetic locus has been well characterized in the Gram-positive organism Bacillus subtilis, where rhamnose degradation is tightly linked to metabolism (40). Unlike streptococci that incorporate rhamnose into their cell wall polysaccharides, the WTAs of B. subtilis are devoid of any rhamnose, nor does it produce any other known structures into which rhamnose is incorporated.…”

Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis

“…The assimilation of carboxylic acids by B. subtilis mainly relies on active transport systems, whose expression is induced via two-component systems (e.g., maeN by MalK/R [ 6 ]) by the transported carbon source ( 7 ). Besides carboxylic acids, B. subtilis is capable of utilizing a wide variety of carbon sources, including plant materials such as pectin, galactan, polygalacturonan, and rhamnogalacturonan ( 8 – 10 ). Indeed, B. subtilis has recently been suggested to be an epiphyte ( 11 ).…”

YsbA and LytST are essential for pyruvate utilization in Bacillus subtilis

“…Its genome encodes many genes associated with adaptation to this environment as it is able to swim, swarm and form complex biofilms. Furthermore, B. subtilis is capable of utilizing a wide variety of carbon sources including plant material composed of rhamnose and arabinose (S a-Nogueira et al, 1997;Hirooka et al, 2015). Since its first discovery, B subtilis has become a widely used model organism, and many gene functions have been unraveled.…”