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-␤-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-␤-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.The acetone-butanol-ethanol (ABE) fermentation of Clostridium acetobutylicum was a classical method to produce the commercially important solvents acetone and butanol, which operated successfully at an industrial scale in many countries during the first half of the 20th century. After the Second World War, the fermentation process declined because of the emergence of the competitive petrochemical-based synthesis of the solvents (18). Nevertheless, oil is a finite commodity and global oil prices are on the rise, and this, coupled with a general worldwide interest in exploiting renewable resources, has stimulated research on the biochemistry and physiology of solventogenic clostridia in recent years, with the aim of exploring the potential for revival of the ABE fermentation (17, 22). A major consideration in any bioconversion process is the availability of a cost-effective and high-product-yielding growth medium, whereby there is maximal conversion of the available carbon into the commercial end product. The substrate of choice in the traditional industrial ABE fermentation, molasses, accounted for more than 60% of the overall cost of the process (19). However, the clostridia are metabolically versatile with respect to carbohydrate utilization and the potential therefore exists to exploit alternative, cheaper, low-grade substrates.Several clostridial strains are able to produce solvents by fermentation of whey (21), which has been shown to be economically superior to the traditional process (19). Despite low reactor productivity, an attractive feature of these fermenta...

M . T AN G NE Y, C . R OU S SE , M . YA ZD A NI AN A ND W. J . M IT C HE LL . 1998. Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetonebutanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed.

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 ␥-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 in B. 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. Strains of Bacillus licheniformis andBacillus subtilis may synthesize a water-soluble, viscous slime material containing Dand L-glutamic acid residues. This polyglutamic acid (PGA) is polymerized via amide linkages between the ␣-amino and ␥-carboxylic groups of the amino acid residues. PGA is the principal component of "Itohiki-natto," a traditional Japanese food prepared from steamed soybean by the biological action of PGA-producing strains of B. subtilis, which are generally referred to as "Bacillus natto" or B. subtilis (natto) (17). PGA has other biotechnological applications in cosmetics, medicines, and foods. However, the synthesis of even small amounts of PGA can be a problem in the fermentation industry, most notably in the production of extracellular enzymes from bacilli, where PGA accumulation causes increased viscosity of the fermentation broth, reduced enzyme yield, uncontrollable foaming, and complications in product recovery. The unpredictable nature of PGA synthesis is particularly troublesome.Cultural conditions affecting PGA biosynthesis have been studied using various poorly identified strains of B. licheniformis and B. subtilis in complex, ill-defined media, and these studies have resulted in conflicting conclusions. In general, strains have been classified into two categories: (i) those that require exogenous L-glutamate for PGA synthesis, for example, B. subtilis strains IFO 3335 (6) and F-2-01 and B. licheniformis 9945 (16) (although Birrer et al. reported that strain 9945 does not require exogenous glutamate for PGA synthesis [2]), and (ii) strains that do not require exogenous glutamate (often described as de novo synthesis), such as B. licheniformis A35 (3) and B. subtilis TAM-4 (10).The mechanisms of PGA biosynthesis in B. licheniformis and B. subtilis have been elusive until recently. A membranous synthetase complex from B. licheniformis which catalyzes the activation, racemization, and polymerization of L-glutamate into exclusively poly-D-glutamate has been partially characterized (5), but B. subtilis (natto) has been studied more extensively in this context. A gene originally thought to code for ␥-glutamyltranspeptidase (GGT) (8) but later referred to as a PGA "stimulating ...

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.

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.