A mutant of Bacillus megaterium relieved from catabolite repression has been used to clone ccpA from B. megaterium by complementation. ccpA is the first gene of a presumed operon, in which it is followed by the motA homologue ORF1 and the motB homologue ORF2. The mutation maps in the 3'-terminal region of ccpA, where an in-frame duplication of 84 nucleotides located between two 9 bp direct repeats leads to an insertion of 28 amino acids near the C-terminus of CcpA. An in-frame deletion of 501 bp in ccpA exhibits the same phenotype as the 84 bp duplication. Deletion of ORF1 and ORF2 does not yield an apparent phenotype. A single-copy ccpA::lacZ transcriptional fusion is constitutively expressed, independent of whether the growth medium triggers catabolite repression or not. The ccpA mutation leads to relief of catabolite repression exerted by glucose, fructose, mannitol, glucitol and glycerol, whereas only smaller effects were found with ribose, citrate and glutamate. The respective growth rates on these carbon sources are uniformly reduced to a generation time of about 90 min in the ccpA mutant. Catabolite repression of a plasmid-encoded xylA::ccpA fusion is less efficient than that of a xylA::lacZ fusion in the same vector. Furthermore, overproduction of CcpA decreases catabolite repression of a single-copy xylA::lacZ fusion approximately twofold. Thus, overexpression of CcpA may be counterproductive for catabolite repression, supporting the hypothesis that CcpA by itself may not bind sufficiently strongly to the cis-active catabolite-responsive element to exert catabolite repression.

The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xyloseresponsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon. Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source. Expression of XylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre. Northern analysis of the xyl mRNA reveals that a potential stem‐loop structure located in the non‐translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5′‐xylA portion is very abundant, while the 3′‐xylBT portion constitutes only a fraction of it. XylT has an apparent Michaelis constant (KM) of approx. 100 μM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40 min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.

Bacillus subtilis 168 is unable to effectively utilize xylose as sole carbon source. We demonstrate here that this strain cannot actively transport xylose into the cell. After leaving B. subtilis 168 for a few days on minimal plates with xylose as sole carbon source large colonies arise with a frequency of 1 × 10−6/cell. These mutants grow well on xylose and efficiently take up that sugar. This new property is not inducible by xylose, indicating that the mutation is neither in the xyl nor in the xyn operon.