novel lipase/chaperone pair from Ralstonia sp. M1: analysis of the folding interaction and evidence for gene loss in R. solanacearum

• Published in: Molecular genetics and genomics : MGG 2004-12, Vol.272 (5), p.538-549
• Main Authors: Quyen, D.T, Nguyen, T.T, Le, T.T.G, Kim, H.K, Oh, T.K, Lee, J.K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; aminoacyl tRNA ligases; bacterial proteins; Base Sequence; Binding sites; Burkholderia; Cloning, Molecular; DNA Primers; E coli; Electrophoresis, Polyacrylamide Gel; enzyme activity; Escherichia coli; Evolution, Molecular; Gene Deletion; Gene Expression; Genes; glutaminyl tRNA synthetase; lipA gene; Lipase - genetics; Lipase - metabolism; lipB gene; molecular chaperones; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Molecular Sequence Data; nucleotide sequences; Plasmids - genetics; Protein Folding; Proteins; Ralstonia; Ralstonia - genetics; Ralstonia solanacearum; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology; soil bacteria; Soil Microbiology; transmembrane proteins; triacylglycerol lipase; Vietnam
• ISSN: 1617-4615; 1617-4623
• DOI: 10.1007/s00438-004-1084-7

A microbial strain (referred to as M1) that produces an extracellular lipase was isolated from a soil sample in Vietnam, and identified as a Ralstonia species by partial sequencing of its 16S rDNA. A genomic library was constructed from Pst I fragments, and a colony showing lipase activity was selected for further analysis. Sequencing of the 4.7-kb insert in this clone (named M1-72) revealed one incomplete and three complete ORFs, predicted to encode a partial hypothetical glutaminyl tRNA synthetase (304 aa), a hypothetical transmembrane protein (500 aa), a lipase (328 aa) and a lipase chaperone (352 aa), respectively. Alignment of the insert sequence with the corresponding region of the genome of R. solanacearum GMI1000 (GenBank Accession No. AL646081) confirmed the presence in the latter of the genes for the hypothetical transmembrane protein and glutaminyl tRNA synthetase, which exhibited 89-91% identity to their counterparts in M1. However, R. solanacearum GMI1000 lacks the complete lipase-encoding gene and the major part of the chaperone-encoding gene, creating a so-called "black hole". The deduced amino acid sequences of the products of the lipase gene lipA and chaperone gene lipB from strain M1 shared 49.3-60.3% and 23.9-32.7% identity, respectively, with those of the Burkholderia lipase/chaperone subfamily I.2. lipB is located downstream of lipA, and separated from it by only 9 bp, and each gene has a putative ribosome binding site. The mature lipase LipA, a His-tagged derivative (LipAhis), the tagged full-length chaperone LipBhis and a truncated form (DeltaLipBhis) lacking the 56 N-terminal residues were expressed in Escherichia coli BL21. LipA, LipAhis and DeltaLipBhis could be expressed at high levels (70, 15 and 12 mg/g wet cells, respectively) and were easily purified. However, LipBhis was expressed at a much lower level which precluded purification. The specific activity of purified LipAhis, expressed on its own, was very low (