Martin Tangney

Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

ABSTRACT The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity inB. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.

Analysis of the Mechanism and Regulation of Lactose Transport and Metabolism in Clostridium acetobutylicum ATCC 824

ABSTRACT Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-β-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-β-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.

Maltose uptake and its regulation in Bacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsI6 null-mutation. A strain carrying the temperature-sensitive ptsI1 mutation accumulated maltose normally at 37 degrees C but, in contrast to the wild-type, was devoid of maltose transport activity at 47 degrees C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.

Cloning and nucleotide sequence of a thermostable cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 and its expression in Escherichia coli

A gene, cgtA, encoding an extremely thermostable cyclodextrin glycosyltransferase (CGTase) was cloned from a thermophilic anaerobe, Thermoanaerobacter sp. ATCC 53627, and expressed in Escherichia coli. DNA and protein sequencing revealed that the mature enzyme of 683 amino acid residues (MW 75 kDa) was preceded by a signal peptide of 27 amino acid residues. The sequence of the Thermoanaerobacter CGTase was similar to sequences of Bacillus CGTases, with more than 58% identity, and very similar (89% identity) to a CGTase enzyme from Thermoanaerobacterium thermosulfurogenes.

Cloning and Sequencing of an Alkaline Protease Gene from Bacillus lentus and Amplification of the Gene on the B. lentus Chromosome by an Improved Technique

ABSTRACT A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Maltose transport in Bacillus licheniformis NCIB 6346

Bacillus licheniformis NCIB 6346 utilized glucose in preference to maltose when both sugars were present in the growth medium. Addition of glucose to a culture growing on maltose resulted in inhibition of maltose uptake and an immediate cessation of maltose metabolism. The mechanism of maltose transport was examined in whole cells and cell extracts. Phosphoenolpyruvate did not stimulate phosphorylation of maltose, indicating the absence of a phosphotransferase system. However, the presence of a maltose phosphorylase enzyme was suggested by phosphorylation of the sugar in the presence of inorganic phosphate. Maltose accumulation was strongly inhibited by proton conducting uncouplers, and was driven by an artificial transmembrane pH gradient, inside alkaline. These results imply that maltose is transported by a proton symport mechanism in this bacterium.

Development of a shuttle vector for screening of strong promoter sequences

A new promoter probe plasmid, pMOL618, has been specifically designed to be selective for strong promoter sequences. The plasmid contains two origins of replication which allow it to replicate both in Bacillus subtilis and Escherichia coli, as well as an indicator gene which also functions in both backgrounds. The plasmid is therefore useful in the screening of promoter sequences in both organisms. The stringency of the promoter selection is demonstrated using a known strong promoter.

Integration and amplification of a cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 on the Bacillus subtilis chromosome

A plasmid was constructed containing the replication functions of pUC19, and the cgtA gene from Thermoanaerobacter sp. ATCC 53627, flanked by the dal gene from Bacillus subtilis and a sequence downstream from this gene. This was transformed into a Dal- B. subtilis strain, selecting for Dal+ transformants, which contained the cgtA gene in single copy integrated in the B. subtilis chromosome. The gene was subsequently amplified by a method which ensured that there was no functional plasmid replication system on the integrated DNA. The amplified structure was stable in the absence of selection pressure.