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Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

ABSTRACT: Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp SalI fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp SalI fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed delta 1-dehydrogenase in Escherichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacIq plasmid pRG1 and pTEK21, the expression of delta 1-dehydrogenase was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Delta 1-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction. The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases.

SUBMITTER: Plesiat P

PROVIDER: S-EPMC209228 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

Plesiat P P Grandguillot M M Harayama S S Vragar S S Michel-Briand Y Y

Journal of bacteriology 19911101 22

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. ...[more]

PMID: 1657885

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Project description:Comamonas testosteroni delta 4(5 alpha)- and delta1-dehydrogenases [delta4(5alpha)- and delta1DH] are key enzymes in the degradation of steroids having an A:B ring fusion in a trans configuration. We previously reported the isolation of the delta1dh gene (P. Plesiat, M. Grandguillot, S. Harayama, S. Vragar, and Y. Michel Briand, J. Bacteriol. 173:7219-7227, 1991). In this study, the gene encoding delta 4(5 alpha)DH was cloned in Escherichia coli on a 16-kbp BamHI fragment by screening a genomic bank of C. testosteroni ATCC 17410 with a probe derived from delta1dh. Subcloning experiments in plasmid pUC19 mapped delta 4(5 alpha)dh immediately downstream of delta1dh. The enzyme was overexpressed 18-fold in cells of E. coli JM109 carrying a 2.5-kbp cloned fragment (plasmid pXE25). However, much higher levels of enzymatic activity (264-fold) were obtained in Pseudomonas putida KT2440, using pMMB208 as an expression vector. Studies with crude lysates of KT2440 showed that delta4(5alpha)DH exhibits higher specificity and higher activity toward delta l-androstene-3,17-dione than toward the saturated derivative 5 alpha-androstane-3,17-dione. The reaction was found to be irreversible and to use efficiently typical flavoprotein electron acceptors; optimal conditions for the enzyme activity were pH 8 and 40 degrees C. Analysis of the nucleotide sequence of the insert of pXE25 revealed an open reading frame of 1,593 bp preceded by a putative ribosome-binding site and followed by a potential transcription terminator. The amino acid sequence of the deduced peptide showed a typical flavin adenine dinucleotide-binding site in its N-terminal region, confirming the flavoproteinic structure of delta 4(5 alpha)DH. The predicted molecular mass was consistent with that of the enzyme expressed in a T7 polymerase system (60 kDa). Alignment between delta 4(5 alpha)dh and delta1dh indicated that both genes, though coding for functionally related enzymes, do not derive from a common ancestor.

Project description:Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of S. suis type 2 and used to screen an S. suis gene library in an effort to identify protective antigen(s) and antigens of diagnostic importance. A clone that produced a 45-kDa S. suis-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 45-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size as the recombinant protein in S. suis strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally infected with a virulent strain of S. suis type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect S. suis infection. Activity staining showed that the S. suis GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha-ketoglutarate as the substrate. Highly virulent strains of S. suis type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the S. suis GDH protein is exposed at the surface of intact cells.

Dataset Information

Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

Publications

Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

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