Gene cloning and characterization of a second alanine racemase from Bacillus subtilis encoded by yncD.

Alanine racemase activity was investigated in Bacillus subtilis. A putative second alanine racemase gene (yncD) was cloned in parallel with the previously identified alanine racemase gene, dal. Each of the B. subtilis genes, dal and yncD complemented the Escherichia coli Alr- DadX- double mutant alanine auxotrophic strain MB2159 in vivo, restoring the prototrophic phenotype. Alanine racemase activity was also detected in vitro in cell-free extracts prepared from cultures of E. coli MB2159 harboring plasmids expressing either of the cloned B. subtilis genes and preliminary characterization of enzyme activity is presented.

Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

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-beta-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-beta-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.

Characterisation of a glucose phosphotransferase system in Clostridium acetobutylicum ATCC 824.

The transport of glucose by the solventogenic anaerobe Clostridium acetobutylicum was investigated. Glucose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on glucose and extract fractionation revealed that both soluble and membrane components are required for activity. Glucose PTS activity was inhibited by the analogue methyl alpha-glucoside, indicating that the PTS enzyme II belongs to the glucose-glucoside (Glc) family of proteins. Consistent with this conclusion, labelled methyl alpha-glucoside was phosphorylated by PEP in cell-free extracts and this activity was inhibited by glucose. A single gene encoding a putative enzyme II of the glucose family, which we have designated glcG, was identified from the C. acetobutylicum ATCC 824 genome sequence. In common with certain other low-GC gram-positive bacteria, including Bacillus subtilis, the C. acetobutylicum glcG gene appears to be associated with a BglG-type regulator mechanism, as it is preceded by a transcription terminator that is partially overlapped by a typical ribonucleic antiterminator (RAT) sequence, and is downstream of an open reading frame that appears to encode a transcription antiterminator protein. This is the first report of a glucose transport mechanism in this industrially important organism.

Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101.

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.

Analysis of the elements of catabolite repression in Clostridium acetobutylicum ATCC 824.

The PTSH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a PTSH mutant of Escherichia coli. The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein. Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C. Acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus Subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. Subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. Acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.