Blanka 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 aprE leader is a determinant of extreme mRNA stability in Bacillus subtilis.

The Bacillus subtilis aprE gene encodes subtilisin, an extracellular proteolytic enzyme produced in stationary phase. The authors examined the stability of aprE mRNA and aprE leader-lacZ fusion mRNA. Both mRNAs were found to be unusually stable, with half-lives longer than 25 min, demonstrating that the aprE leader contains a determinant for extreme mRNA stability. The half-lives were the same in growing and stationary-phase cells. This contrasts with the findings of O. Resnekov et al. (1990) [Proc Natl Acad Sci USA 87, 8355-8359], which suggested a growth-phase-dependent mechanism for decay of aprE mRNA. The discrepancy is explained by the techniques used. Substitution of two bases or deletion of 25 nucleotides in the aprE leader led to a major difference in its predicted secondary structure and resulted in a fivefold reduction of the half-life of aprE mRNA. The authors also determined the half-life of amyE mRNA, which encodes alpha-amylase, another stationary-phase, excreted enzyme and found it to be around 5 min. This shows that extreme stability is not a general property of stationary-phase mRNAs encoding excreted enzymes.

Expression of the gene encoding glycerol‐3‐phosphate dehydrogenase (glpD) in Bacillus subtilis is controlled by antitermination

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 start‐point 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.

Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of Bacillus subtilis to grow on glycerol.

Although glycerol is not taken up via the phosphotransferase system (PTS) in Bacillus subtilis, some mutations that affect the general components of the PTS impair the ability of cells to grow on glycerol. Five revertants of a pts deletion mutant that grow on glycerol were analysed. They were shown to carry mutations in the glycerol kinase gene. These are missense mutations located in parts of the glpK gene that could encode regions important for the activity of glycerol kinase. The results strongly suggest that the main effect of the PTS on glycerol utilization in B. subtilis is mediated via glycerol kinase.

Antiterminator protein GlpP of Bacillus subtilis binds to glpD leader mRNA

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate (G3P) dehydrogenase. Expression of glpD is mainly controlled by termination/antitermination of transcription at an inverted repeat in the glpD leader. Antitermination is mediated by the antiterminator protein GlpP in the presence of G3P. In this paper, interaction between GlpP and glpD leader mRNA in vivo and in vitro is reported. In vivo, the antiterminating effect of GlpP can be titrated in a strain carrying the glpD leader on a plasmid. GlpP has been purified and gel shift experiments have shown that it binds to glpD leader mRNA in vitro. GlpP is not similar to other known antiterminator proteins, but database searches have revealed an Escherichia coli ORF which has a high degree of similarity to GlpP.

Characterization of a Pleiotropic Succinate Dehydrogenase-negative Mutant of Bacillus subtilis

A succinate dehydrogenase-negative mutant of Bacillus subtilis is described which lacks all three subunits of the membrane-bound succinate dehydrogenase complex: flavoprotein, iron protein, and cytochrome b558. The corresponding mutation is revertible and it maps at one extreme of the sdh region. The results presented suggest that the structural genes for the subunits of the succinate dehydrogenase complex are part of one operon.

Different Processing of an mRNA Species in Bacillus subtilis and Escherichia coli

Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds to glpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of the glpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of the glpD mRNA. In the presence of GlpP, the glpD mRNA is stabilized. glpD leader-lacZ fusions were integrated into the chromosomes of B. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by the glpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA in B. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that the glpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target of E. coli RNase III.

Escherichia coli RNase E and RNase G cleave a Bacillus subtilis transcript at the same site in a structure-dependent manner

The decay of Bacillus subtilis aprE leader-lacZ mRNA was examined in Escherichia coli wild-type and in mutants deficient in RNase E, RNase G, or both. Two versions of the mRNA were studied: the wild-type mRNA, which has a stem-loop at the 5′ end, and a mutant mRNA, with a single-stranded 5′ end. The half-life of both transcripts was determined by RNase E, the half-life of the mutant transcript being one-third of that of the wild-type transcript. RNase G cleaved both transcripts at a site within an AU-rich sequence in the stem-loop region, but cleavage was much more efficient when the stem-loop was destabilized. RNase E cleaved at the same site, but less efficiently and only in the mutant transcript.