A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the i-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichwa coil. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the ,B-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium 3-galactosidase gene in E. coli was not subject to glucose repression. By using transposon TnS mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for P-galactosidase expression in E.coi. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and KkbsieUa pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus H, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but TnS insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of 13-galactosidase in E. coli.Clostridium acetobutylicum is a gram-positive obligate anaerobe that is able to utilize a variety of carbohydrates as substrates for production of acetone, butanol, and ethanol, commercially important solvents, by way of the ABE fermentation (32). It has been shown that several strains of C. acetobutylicum are able to utilize whey or deproteinized whey, containing lactose, as a substrate for solvent production (16, 36). There are two major pathways for the uptake and utilization of lactose in bacteria, one involving uptake of lactose by ,-galactoside permease followed by hydrolysis of the lactose to glucose and galactose by P-galactosidase and the other involving uptake of the lactose by a lactose phosphoenol pyruvate-dependent phosphotransferase system followed by hydrolysis of the lactose to glucose and galactose-6-phosphate by phospho-,B-galactosidase (12, 43). The latter pathway is common in gram-positive bacteria such as streptococci (8) and staphylococci (12,43). Recent work has shown that C. acetobutylicum strains grown on lactose have both 0-galactosidase and phospho-,-galactosidase activities, each showing a different pattern of induction during the ABE fermentation (63). Levels of phospho-,B-galactosidase were highest during the early acidogenic phase of the ABE fermentation, whereas the ,B-galactosidase was induced later in the ABE fermentation, with maximum levels coinciding w...

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