Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins.
Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme. A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported. We now present a molecular study of the gene, ansB, encoding L-asparaginase II. This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence. The nucleotide sequence of ansB, including 5'- and 3'-untranslated regions, was determined. The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence. The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues. A single region of sequence similarity is observed when ansA and ansB are compared. The transcriptional start site in ansB was determined, allowing the identification of the promoter region. The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter. Regulation by cyclic AMP receptor protein and anaerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated.
Division of Science and Technology, Griffith University, Brisbane, Queensland, Australia.
M P
MP
Jennings
I R
IR
Beacham
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes; D011953:Receptors, Cyclic AMP; D014157:Transcription Factors; D001215:Asparaginase