ABSTRACT
DW-116 is a new fluoroquinolone antimicrobial agent with a broad spectrum. In order to elucidate the resistance mechanism to DW-116 in Acinetobacter spp. bacteria, total chromosomal DNA was isolated from 10 strains of Acinetobacter spp. resistant to DW-116. Quinolone resistance determinant region (QRDR) of DNA gyrase gene was amplified by PCR. The 345 bp nucleotide fragment yielded was inserted into pKF 3 which was used as the vector. Comparisons of the DNA sequences of 8 strains with that of the wild type strain revealed a Ser-83 to Leu mutation in mutants and all ten strains contained one silent mutation(T-->G) in QRDR. From Acinetobacter MB4-8 strain, DNA gyrase was isolated and purified, through no-vobiocin-sepharose, heparin-sepharose affinity column chromatography. The enzyme was composed of two subunits and the molecular mass of subunits A and B were 75.6 and 51.9 kDa, respectively. The supercoiling activity of the reconstituted DNA gyrase composed of subunit A from Acinetobacter MB4-8 and subunit B from E. coli was not inhibited by 128 micrograms/ml of ciprofloxacin. It might be said that one of the resistance mechanisms to DW-116 in A-cinetobacter MB4-8 was subunit A alteration of DNA gyrase.
Subject(s)
Acinetobacter/drug effects , Anti-Infective Agents/pharmacology , Fluoroquinolones , Piperazines/pharmacology , Quinolones/pharmacology , Acinetobacter/enzymology , Acinetobacter/genetics , Acinetobacter Infections/microbiology , Base Sequence , Electrophoresis, Polyacrylamide Gel , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Topoisomerase II InhibitorsABSTRACT
This study is to predict the possible roles of the arylsulfate sulfotransferase (ASST) in the microorganism. At first we studied the spectrum of a distribution of the ASST enzyme through about 1,300 bacteria and the several selected strains were compared with Klebsiella K-36 previously reported in the level of DNA homology using the Southern blot method. From this study, we could predict that this enzyme would not exist in specific bacteria and it might not be a critical enzyme for the life of bacteria.
Subject(s)
Arylsulfotransferase/metabolism , Bacteria/enzymology , Bacteria/genetics , Bacteria/metabolism , Blotting, Southern , DNA, Bacterial/analysis , Gram-Negative Bacteria/enzymology , Gram-Negative Bacteria/genetics , Gram-Positive Bacteria/enzymology , Gram-Positive Bacteria/genetics , Klebsiella/enzymology , Klebsiella/genetics , Sequence HomologyABSTRACT
A procedure has been developed for the overexpression and purification of milligram quantities of the Klebsiella K-36 arylsulfate sulfotransferase (ASST). The structural gene was amplified by means of a polymerase chain reaction (PCR) technique and inserted into the plasmid vector pGEX-3X. The plasmid pGEX-100, carrying the Klebsiella K-36 astA structural gene under the control of the Escherichia coli tac promoter, was transformed into the E. coli strain BL21 (DE3). The ASST was produced in E. coli as a fusion with glutathione S-transferase. Conditions for protein production, isolation on glutathione Sepharose 4B, and Xa cleavage to generate active ASST were developed. The purification yielded approximately 0.7 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties almost the same as those of the enzyme purified from Klebsiella K-36 cells. The purification procedure was very rapid and is suitable for obtaining considerable amounts of enzyme at a relatively high yield compared with its purifying method from the culture of the Klebsiella K-36 strain.
Subject(s)
Arylsulfotransferase/biosynthesis , Klebsiella/enzymology , Amino Acid Sequence , Arylsulfotransferase/isolation & purification , Arylsulfotransferase/metabolism , Base Sequence , Chromatography, Affinity , Cloning, Molecular , DNA Primers , Escherichia coli , Genes, Bacterial , Glutathione Transferase/biosynthesis , Kinetics , Klebsiella/genetics , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
A gene-encoding arylsulfate sulfotransferase (ASST) was cloned from a Klebsiella K-36 genomic library. ASST transfers a sulfate group from phenolic sulfate esters to a phenolic acceptor substrate. The gene, designated astA, was subcloned into vector pGEM3Zf(-) and sequenced. Recombinant clone-harbouring astA was directly identified using a fluorescent product. The nucleotide sequencing revealed an open reading frame (ORF) of 2,082 bp encoding a protein of 694 amino acids with a secretory signal sequence. A protein of similar size was visualized after in vitro transcription and translation using a plasmid carrying the cloned 3.1-kb fragment as a template. The N-terminal amino acid sequence of the purified processed protein was found to be identical to that predicted from the gene sequence. When searching the database for astA nucleotide or its deduced amino acid sequence, no significant homology to any sequence was found.
