The glpPKD region of the Bacillus subtilis chromosome was cloned in its natural host in plasmid pHPl3. The glpPKD region contains genes required for glycerol catabolism: glpK coding for glycerol kinase, glpD coding for glycerol-3-phosphate (G3P) dehydrogenase and glpP, proposed to code for a positively acting regulatory protein.The cloned 7 kb fragment carries wild-type alleles of glpK, glpD and glpP. It can also complement a strain deleted for the entire glpPKD region. The wild-type alleles were mapped to different subfragments, establishing the gene order glpP-glpK-glpD. The nucleotide sequence of glpK and glpD was determined. Immediately upstream of glpK, an additional open reading frame was found, possibly being part of the same operon. Putative transcription terminators were found in the region between glpK and glpD and downstream of glpD. In a coupled in vi&u transcriptionltranslation system, two proteins were found, corresponding in size to those predicted from the deduced amino acid sequences of glycerol kinase and G3P dehydrogenase (54 kDa and 63 kDa, respectively).

The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P). The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined. glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP. A aA-type promoter and the transcriptional start point for glpFK were identified. In the 5' untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found. The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase. Expression of glpFK and glpD requires the inducer G3P and the glpP gene product. A 2.9 kb B. subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7. pLUM7 contains a functional glpP gene as shown by its ability to complement various gZpP mutants. Immediately upstream of glpP an open reading ffame is found (Owl). Disrupting ORFl by plasmid integration in the B. subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source. With the present report all B. subtilis glp genes located at 75" on the chromosomal map have been identified.

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate (G3P) dehydrogenase. A sigma A type promoter and the transcriptional startpoint for glpD were identified. Between the transcriptional startpoint and glpD there is an inverted repeat followed by a run of T residues. The inverted repeat prevents expression of a reporter gene, xylE, when positioned between this gene and a constitutive promoter. Expression of xylE, like expression of glpD, is induced by G3P and repressed by glucose. Induction also requires the product of the glpP gene. Our results suggest that glpD expression is controlled by antitermination of transcription. The inverted repeat appears to be a target for induction by G3P and GlpP. We speculate that glucose repression is mediated via an inhibitory effect on synthesis or activity of GlpP.

The Bacillus subtilis glpD gene, encoding glycerol-3-phosphate (G3P) dehydrogenase, is preceded by a promoter and an inverted repeat which is located between the promoter and the glpD coding region. The inverted repeat acts as a transcriptional terminator in vitro. Expression of glpD is induced by G3P in the presence of the glpP gene product. Full-length glpD transcripts can be detected only in glycerol-induced cells. The major glpD transcript is initiated from the glpD promoter but minor amounts of larger transcripts, possibly initiated at upstream glp promoters, can also be found. In uninduced cells short transcripts are present, corresponding to initiation at the glpD promoter and termination at the inverted repeat. Upon induction, these short transcripts disappear and are replaced by full-length glpD transcripts. The 3'-ends of full-length glpD transcripts were mapped to an inverted repeat located immediately downstream of glpD. These results show that glpD of B. subtilis is regulated by termination/antitermination of transcription.

A 3.5 kb fragment of Bacillus subtilis DNA which contains wild type alleles of mutations in glpK (glycerol kinase) and glpD (glycerol-3-phosphate [G3P] dehydrogenase) was cloned in plasmid pHV32 in Escherichia coli. The cloned fragment expresses glycerol kinase in B. subtilis mutants carrying the mutations glpK11 and recE4 after induction with glycerol or G3P whereas it does not express G3P dehydrogenase. The cloned fragment thus contains the complete glpK but probably only part of glpD.