Expression and nucleotide sequence of the Clostridium acetobutylicum β-galactosidase gene cloned in Escherichia coli

Abstract

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the β-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indoly-β-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the β-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium β-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for β-galactosidase expression in E. coli. 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 Klebsiella 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 II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 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 β-galactosidase in E. coli.