Complete nucleotide sequence and overexpression of cat1 gene cluster, and roles of the putative transcriptional activator CatR1 in Acinetobacter lwoffii K24 capable of aniline degradation.
The aniline-assimilating bacterium Acinetobacter lwoffii K24 has two cat gene clusters (cat1 and cat2). In this study, we completely sequenced 10-kb DNA fragment of cat1 genes of A. lwoffii K24, which had been cloned in plasmid pCD1-1. Sequence analysis revealed that the order of genes in the cat1 operon-containing gene cluster was ORF porin, catR1, catB1C1A1D, ORF1, and ORF2. Two ORFs located immediately downstream catD were most similar with two ORFs in cat gene cluster of Acinetobacter calcoaceticus ADP1 but the gene structure of catR1B1C1A1 was closest to that found in Frateurua sp. ANA-18 or Pseudomonas putida PRS2000. CatA1 gene product was significantly overexpressed and detected in SDS-PAGE when four cat1 genes (catB1C1A1D) were placed under the control of a lac promoter in pUC118 while overexpressions of other cat genes were accomplished under the control of a lac promoter in pET vector system. All gene products were verified by N-terminal amino acid sequencing. Gel retardation assay revealed that the putative regulatory gene activator CatR1 for the catB1C1A1 operon could bind the promoter region of catB2 as well as catB1, suggesting that transcription of catB1 or catB2 might be controlled by the putative gene activator CatR1. However, the promoter regions of catA1 and catA2 were found to have no affinity with catR1.
Kim
S I
SI
Biomolecule Research Team, Korea Basic Science Institute, Daejon, 305-333, Korea. ksi@comp.kbsi.re.kr
Yoo
Y C
YC
Kahng
H Y
HY
D000150:Acinetobacter / Q000235:genetics*; D000814:Aniline Compounds / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000235:genetics*
D000814:Aniline Compounds; D001426:Bacterial Proteins; D015534:Trans-Activators; C023650:aniline