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, 48 (3), 930-9

Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces Clavuligerus

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Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces Clavuligerus

Kapil Tahlan et al. Antimicrob Agents Chemother.

Abstract

Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and beta-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upstream of pah1 on the chromosome. Furthermore, sequence analysis of the region downstream of pah1 revealed a second copy of a gene encoding ornithine acetyltransferase (oat1), thus indicating the presence of a cluster of paralogue genes. ceaS1, bls1, and oat1 display 73, 60, and 63% identities, respectively, at the nucleotide level to the original ceaS2, bls2, and oat2 genes from the clavulanic acid gene cluster. Single mutants defective in ceaS1, bls1, or oat1 were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The ceaS1/ceaS2 and the bls1/bls2 mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand, oat1/oat2 double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the argJ gene in S. clavuligerus, which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants.

Figures

FIG. 1.
FIG. 1.
Early steps of clavulanic acid and 5S clavam metabolite biosynthesis.
FIG. 2.
FIG. 2.
Southern analysis of genomic DNA from wild-type S. clavuligerus after EcoRI digestion. The fractionated and blotted genomic DNA was probed with a ceaS2-specific probe.
FIG. 3.
FIG. 3.
Southern analysis of cosmids 6G9 and 14E10 with a ceaS2-specific probe. Lane 1, ceaS2 probe (control); lanes 2 and 3, EcoRI-digested 6G9 and 14E10, respectively.
FIG. 4.
FIG. 4.
Diagram of the genes encoded by the paralogue gene cluster thought to be involved in the early stages of clavulanic acid and 5S clavam metabolite biosynthesis. Only restriction sites referred to in the text are shown. EcoRI* denotes an EcoRI site arising from the multiple cloning site of the cosmid vector, pWE15. (The diagram is not to scale.)
FIG. 5.
FIG. 5.
Southern analysis of the ceaS2-Fs mutant. (A) Diagram of the ceaS2 region of the S. clavuligerus chromosome in the wild type, the ceaS2::apr mutant, and the ceaS2-Fs mutant. The gray arrow represents the apr disruption cassette, and the open arrow represents ceaS2 with the direction of transcription represented by the direction of the arrowheads. The solid bar represents the frame shift mutation, and the fine lines represent the rest of the S. clavuligerus chromosome. (B) Southern analysis of EcoRI- and NruI-digested genomic DNA from S. clavuligerus wild-type and ceaS2 mutant strains. DNA from the wild type, from ceaS2::apr mutants 4B and 4B-C, and from ceaS2-Fs mutants FS3, FS7, FS8, and FS10 was probed with a ceaS2-specific probe and an apr-specific probe.
FIG. 6.
FIG. 6.
Similarities between regions of β-LS1 and β-LS2. Heavy shading indicates conserved residues, and light shading indicates similar residues. Amino acids that have been shown to be involved in substrate binding, either directly or indirectly, are indicated by dots. The boxed residues represent a loop thought to form a part of the active site of β-LS2.

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