SummaryAntitermination of transcription mediated by proteins interacting with mRNA sequences is described for nine operons/regulons. Eight of the systems are catabolic, while the ninth, the Klebsiella pneumoniae nas regulon, is involved in the assimilation of nitrate and nitrite. Six of the catabolic operons/regulons are found in Bacillus subtilis, one is found in Escherichia coli, and one in Pseudomonas aeruginosa. The antitermination system of five of the operons/regulons (E. coli blg, and sacPA, sacB, bgl, and lic from B. subtilis) are assigned to the bgl-sac family on the basis of extensive similarities with regard to antiterminator proteins and the sequences of the antiterminators. Other members of the bgl-sac family are the arb operon of Erwinia chrysanthemi and a presumed bgl operon of Lactococcus lactis. The antitermination systems of the other four operons/regulons (B. subtilis glp, B. subtilis hut, P. aeruginosa ami, and K. pneumoniae nas) seem to be unrelated both to the bgl-sac family and to each other. The antiterminator protein of the B. subtilis glp regulon has been found not only to cause antitermination but also to stabilize the resultant mRNA and to mediate glucose repression. If other antiterminator proteins, and antitermination factors, also prove to have additional functions, it will broaden the impact of antitermination as a means of controlling gene expression.

The Bacillus subtilis aprE leader is a determinant of extreme mRNA stability.The authors examined what properties of the aprE leader confer stability on an mRNA. The secondary structure of the aprE leader mRNA was analysed in vitro and in vivo, and mutations were introduced into different domains of an aprE leader-lacZ fusion. The half-lives of the corresponding transcripts were determined and β-galactosidase activities were measured. Removal of a stem-loop structure at the 5' end or diminishing the strength of the RBS reduced the half-lives from more than 25 min to about 5 min. Interfering with translation by abolishing the start codon or creating an early stop codon had no or little effect on mRNA stability. The authors conclude that a 5' stem-loop and binding of ribosomes are necessary for the stability of aprE leader mRNA. The present results, together with a number of other data, suggest that translation of a B. subtilis mRNA is generally not important for its stability ; the situation seems different in Escherichia coli. It is further concluded that the calculated strength of a B. subtilis RBS cannot be used to predict the stability of the corresponding transcript.

The genetic location of mutations affecting the citric acid cycle and the properties of mutants of Bacillus subtilis possessing these mutations have been examined. Genes coding for the component enzymes of the cycle were found to be unlinked to each other and thus do not form an operon. The mutational defect in a mutant lacking fumarase mapped between thr-5 and cysB3. Mutations causing inability to produce isocitrate dehydrogenase and succinate dehydrogenase were found to map between argAIl and leu-1. The a-ketoglutarate dehydrogenase mutations were mapped at the terminal end of the B. subtilis chromosome through a weak linkage in phage PBS-1 transduction of one class of these mutations to ilvA2 and metB4. A second class of a-ketoglutarate dehydrogenase mutations mapped closer to ilvA2 and metB4 but still terminal with respect to these markers. Aconitaseless mutants possessed mutations that could not be linked to any of the known transducing segments of the chromosome. An effect of mutation conferring loss of one enzyme of the cycle on the specific activity of the other enzymes in the cycle was observed.

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).