Molecular cloning and characterization of the pyrB gene of Lactobacillus leichmannii encoding aspartate transcarbamylase.
The Lactobacillus leichmannii pyrB gene, encoding pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase), was cloned from a partial genomic library lying on a 1468 bp Sa/I/BstXI fragment. The predicted polypeptide sequence extending over 351 amino acid residues (M(r) 39 855 Da) was compared to those of various other organisms revealing clear identities towards them and important conservative stretches, implying that these proteins are closely related. Transcriptional initiation was mapped by primer extension and occurred 54 bp upstream of the pyrB open reading frame (ORF). Northern blot analysis indicates that the pyrB gene is transcribed as a single mRNA and not together with the following overlapping pyrC gene as a bicistronic mRNA. At high copy number the pyrB gene of L leichmannii seems to be lethal for its E coli host; inserted in a low copy vector it complements the uracil auxotrophy of an E coli pyrB mutant which shows distinct ATCase activity in the cell extract. With an excess of uracil in the growth medium the gene is apparently repressed and no ATCase activity can be measured.
Becker
J
J
Institut für Mikrobiologie, JW Goethe-Universität, Frankfurt/Main, Germany.
Brendel
M
M
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; C030986:pyrimidine