Relatedness of a periplasmic, broad-specificity RNase from Aeromonas hydrophila to RNase I of Escherichia coli and to a family of eukaryotic RNases.
The isolation, sequencing, and characterization of a periplasmic RNase gene from Aeromonas hydrophila AH1133 is described. Following subcloning of the gene on a 2.7-kb PstI fragment, its direction of transcription and approximate location were determined. Analysis of the nucleotide sequence reveals that the gene is 645 bp long, coding for 215 amino acid residues with a total molecular weight of 24,215. A typical leader sequence is present at the beginning of the corresponding protein. Computer analysis revealed strong local similarities to Escherichia coli RNase I and to the active site of a family of eukaryotic RNases. Expression studies indicate that the RNase natural promoter functions poorly in E. coli. In this organism, the enzyme is mainly localized in the cytoplasm and periplasm, although high levels of expression lead to significant release into the extracellular medium. Functional and physical characterizations further indicate that the periplasmic and cytoplasmic enzymes of A. hydrophila are likely to be the counterparts of E. coli RNase I and its cytoplasmic form RNase I*: as for the E. coli enzymes, the A. hydrophila RNase forms have similar sizes and show broad specificity, and the periplasmic form is more active towards natural polymer RNA than its cytoplasmic counterpart. Both forms are relatively thermosensitive and are reversibly inactivated by up to 0.6% sodium dodecyl sulfate. Southern hybridization revealed homology to E. coli K-12 and Shigella sp. genomic DNA, a finding which correlates with the presence of secreted RNases in these organisms. In contrast, species of phylogenetically closer genera, such as Vibrio and Plesiomonas, did not hybridize to the A. hydrophila RNase gene.
Favre
D
D
Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Ngai
P K
PK
Timmis
K N
KN
D016980:Aeromonas hydrophila / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D012329:RNA, Bacterial / Q000235:genetics; D015183:Restriction Mapping; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003062:Codon; D012329:RNA, Bacterial; D012967:Sodium Dodecyl Sulfate; D012260:Ribonucleases