Catabolite Regulation of the Bacillus subtilis ctaBCDEF Gene Cluster

Abstract

ABSTRACT Bacillus subtilis cytochrome c oxidase caa 3 is encoded by the ctaCDEF genes at the ctaABCDEF locus, with the ctaBCDEF genes organized as an operon-like unit. A dyad symmetry sequence and a catabolite response element homolog can be recognized in the 240-bp intercistronic region between ctaB and ctaC. ctaB′-lacZ and ctaBCD′-lacZ transcriptional fusions integrated at the native locus were used to study catabolite effects on transcription of the ctaB and ctaCDEF genes. In Schaeffer’s medium lacking glucose, ctaBCD′-lacZ was expressed at a very low level during the exponential phase, and expression increased about 30-fold 2 h after entry into the stationary phase. In the presence of 0.5% glucose, ctaBCD′-lacZ expression was totally repressed. In contrast to ctaBCD′-lacZ , ctaB′-lacZ was constitutively expressed regardless of carbon source. The ctaCDEF genes were separated from ctaB by insertion of plasmids carrying selectable markers in such a way that the ctaCDEF and ctaB transcription units remained intact. Enzymatic assays of caa 3 with these constructs, showed that ctaCDEF was not expressed independently of ctaB . Also, when a ′ ctaB-ctaC′-lacZ fusion (containing the ctaB-ctaC intercistronic region) was placed at a remote nonessential locus, β-galactosidase activity could not be detected. The absence of a promoter in the ctaB-ctaC intercistronic space also was indicated by the inability to detect ctaC -specific transcripts with RNase protection assays, primer extension, and rapid amplification of 5′ cDNA ends. Direct mRNA measurements showed that, in the presence of 0.5% glucose, ctaBCDEF transcripts terminated at the 3′ end of the putative stem-loop structure and the distal portion was down-regulated. A possible mechanism for ctaCDEF gene regulation is suggested. Catabolite repression of ctaBCD′-lacZ was partly dependent on CcpA but was independent of HPr. The expression of ctaBCDEF also appears to require the strC , ctaA , and resD-resE gene products.