Galactose utilization in Lactobacillus helveticus: isolation and characterization of the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes.
By complementing appropriate gal lesions in Escherichia coli K802, we were able to isolate the galactokinase (galK) and galactose-1-phosphate uridyl transferase (galT) genes of Lactobacillus helveticus. Tn10 transposon mutagenesis, together with in vivo complementation analysis and in vitro enzyme activity measurements, allowed us to map these two genes. The DNA sequences of the genes and the flanking regions were determined. These revealed that the two genes are organized in the order galK-galT in an operonlike structure. In an in vitro transcription-translation assay, the galK and galT gene products were identified as 44- and 53-kDa proteins, respectively, data which corresponded well with the DNA sequencing data. The deduced amino acid sequence of the galK gene product showed significant homologies to other prokaryotic and eukaryotic galactokinase sequences, whereas galactose-1-phosphate uridyl transferase did not show any sequence similarities to other known proteins. This observation, together with a comparison of known gal operon structures, suggested that the L. helveticus operon developed independently to a translational expression unit having a different gene order than that in E. coli, Streptococcus lividans, or Saccharomyces cerevisiae. DNA sequencing of the flanking regions revealed an open reading frame downstream of the galKT operon. It was tentatively identified as galM (mutarotase) on the basis of the significant amino acid sequence homology with the corresponding Streptococcus thermophilus gene.
Mollet
B
B
Nestlé Research Center, Nestlé Ltd., Lausanne, Switzerland.
Pilloud
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005686:Galactokinase / Q000235:genetics*; D005690:Galactose / Q000378:metabolism*; D005798:Genes, Bacterial; D005838:Genotype; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics*
D005686:Galactokinase; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D005690:Galactose