Lars Rutberg

Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD).

The glpPKD region of the Bacillus subtilis chromosome was cloned in its natural host in plasmid pHP13. 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 vitro transcription/translation 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).

A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability

The Bacillus subtilis glpD gene encodes glycerol‐3‐phosphate dehydrogenase. This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator. Expression of glpD is controlled by antitermination of transcription at the inverted repeat. Antitermination is effected by the glpP gene product in conjunction with glycerol‐3‐phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source. We have isolated a number of glycerol‐positive revertants of GlpP mutants. Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol‐3‐phosphate dehydrogenase constitutively. Unlike wild‐type bacteria, they are not sensitive to glucose repression of glpD. A few of the revertants are temperature sensitive, i.e. they grow on glycerol at 32°C but not at 45°C and produce glycerol‐3‐phosphate dehydrogenase only at 32°C. Northern blot analyses demonstrated that the temperature‐sensitive expression of glpD is due to destabilization of glpD mRNA. Furthermore, introduction of the wild‐type glpP gene into the revertants stabilized the glpD mRNA. This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA. Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability. Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein.

The importance of the 5′‐region in regulating the stability of sdh mRNA in Bacillus subtilis

The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB. In exponentially growing cells, the entire sdh mRNA seems to decay with an ‘all or nothing’ mechanism and with a uniform half‐life of 2–3 min for all cistrons. In stationary‐phase cells, the half‐life of the 5′‐part had dropped to about 0.6 min whereas that of the 3′‐part was about 1.2 min. Decay of sdh mRNA was also measured in exponentially growing cells containing a ‘down‐mutation’ in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90%. The mutation has a moderate effect on expression of the downstream cistron sdhA. In this mutant, the half‐life of the 5′‐part of sdh mRNA was about 0.5 min (i.e. the same as in stationary phase wild‐type cells) and the half‐life of the 3′‐part about 1.3 min. Also, analysis of the decay of an sdh‐cat fusion transcript revealed that the sdh (5′) part decayed more rapidly than the cat part and this difference was more pronounced in stationary‐phase cells compared to exponentially growing cells. The results of these experiments demonstrate the importance of the 5‐ segment of sdh mRNA in controlling the stability of the transcript under different growth conditions.

An inverted repeat preceding the Bacillus subtilis glpD gene is a conditional terminator of transcription

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 glpD 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.