Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons

Abstract

The chromosomally encoded galactose utilization (gal) operons of Salmonella typhimurium and S. typhi were each cloned on similar 5.5-kilobase HindIII fragments into pBR322 and were identified by complementation of Gal- Escherichia coli strains. Restriction endonuclease analyses indicated that these Salmonellae operons share considerable homology, but some heterogeneities in restriction sites were observed. Subcloning and exonuclease mapping experiments showed that both operons have the same genetic organization as that established for the E. coli gal operon (i.e., 5' end, promoter, epimerase, transferase, kinase, and 3' end). Two gal operator regions (oE and oI) of S. typhimurium, identified by repressor titration in an E. coli superrepressor [galR(Sup)] mutant, were sequenced and found to flank the promoter region. This promoter region is identical to the -10 and -35 regions of the E. coli gal operon. Minicell studies demonstrated that the three gal structural genes of S. typhimurium encode separate polypeptides of 39 kilodaltons (kDa) (epimerase, 337 amino acids [aa's]), 41 kDa (transferase, 348 aa's), and 43 kDa (kinase, 380 aa's). Despite functional and organizational similarities, DNA sequence analysis revealed that the S. typhimurium gal genes show less than 70% homology to the E. coli gal operon. Because of codon degeneracy, the deduced amino acid sequences of these polypeptides are highly conserved (greater than 90% homology) as compared with those of the E. coli gal enzymes. These studies have defined basic genetic parameters of the gal genes of two medically important Salmonella species, and our findings support the hypothesized divergent evolution of E. coli and Salmonella spp. from a common ancestral parent bacterium.