Nudeotide sequence of the gene encoding the Streptomyces albus G )9-lactamase precursor (original) (raw)
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Journal of general microbiology
A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine Q-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces liuidans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of P-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by p-iodopenicillanate) the overproduced S. liuidans ML 1 Q-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of j3-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the p-lactamase structural gene. The Q-lactamase gene was located within a 1-8 NcoI-BclI fragment but when this fragment was cloned in S . lividans pIJ702, the resulting strain produced hardly any more Q-lactamase than the original S. cacaoi.
Journal of General Microbiology
Genes encoding extracellular p-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces Zauendulae were cloned and mapped in Streptomyces liuiths. DNA sequence analysis of the plactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the p-lactamases including their signal peptides was deduced. The four p-lactamases exhibited homology to each other and to class A plactamases from other bacterial genera. We suggest that Strepfomyces p-lactamases are representatives of a superfamily of genes, from which class A p-lactamases of Gram-negative bacteria may have evolved. P-lactamases of this genus. The genes for the fllactamases of S. badius, S. cacaoi and S. fradiae have been cloned previously in S. lividans (Jaurin et al., 1988a) and the DNA sequence encoding the S. cacaoi enzyme has been established (Forsman et al., 1989). Here we report the primary structure of p-lactamases of S. badius, S. fradiae and S. lavendulae, and show that Streptomyces p-lactamases are representatives of a superfamily of genes from Gram-positive bacteria, from which it is possible that class A /I-lactamases of Gram-negative bacteria have evolved. Methods Bacterial strains, plasmids, media and culture conditions. S. lividans 1326 (Lomovskaya et al., 1972) was used as the host for all Streptomyces 0001-5706 O 1990 SGM
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
Genes encoding extracellular fl-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The fl-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi fl-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The fl-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the fl-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three fl-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi fl-lactamases exhibited a 10-100-times lower activity in S. lividans, whereas the S. fradiae fl-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.
Physicochemical properties of a β-lactamase from streptomyces UCSM-104
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1985
Streptomyces UCSM-104 shows the presence of three subforms when stained for protein and/or for activity after polyacrylamide gel electrophoresis or after electrofocusing. 2. The pI values of the three subforms were 5.45, 5.30 and 5.10, respectively. 3. The respective electrophoretic mobilities were 4.6 x 10-5, 5.2 x 10-5 and 5.9 x 10-Sm2/sV. 4. Relative molecular mass of 14,900 was determined. 5. The amino acid composition was established. Cysteine was not detected. A fairly high proline content (8.3~o) differentiates this enzyme from other fl-lactamases. 6. Lysine was the only N-terminal amino acid detected after dansylation. 7. The possibld origin of the subforms is discussed.
Induction of a Streptomyces cacaoi β-lactamase gene cloned in S. lividans
Molecular and General Genetics MGG, 1992
The previously cloned class A /Mactamase gene (bla) of Streptomyees caeaoi was shown to be inducible by /Mactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30-to 60-fold decrease in the basal (non-induced) level of/%lactamase production. ORFI codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C /%lactamase) genes in several Enterobacteria.
Two different β-lactamase genes are present in Streptomycees cacaoi
FEMS Microbiology Letters, 1992
Two /3-1actamase genes called blaL and blaU have been cloned independently in Liege and in Ume~], from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of /3-1actamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Liege and in Ume~. 2. INTRODUCTION The cloning of a/3-1actamase gene from Streptomyces cacaoi into Streptornyces liuidans has been reported independently by two laboratories in
FEBS Letters, 1992
The nucleotide scqucncc of the pci~'I,N gene from toc/orocc.rt.s Iurrb erlcoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated ilf, of 95,368, which aprccs with the apparent M,of95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopcptidasc. The amino acid sequence of the aminopeptidase of L. fuctis wss found to be similar to the corresponding cnzymcs of human, rat and mouse, with host 30% of the residues identical. Also. a highly conserved arca was identified which has similarity with the uctivc site of thcrmolysin. A zinc-binding site, as well as the catalytic site for RpN, is predicted to lie within this conscrvcd stretch, Putative promoter regions upstream of PcpN were confirmed by primer extension analysis. Aminopctxidasc N; Lacrococar,r lurris; Mammalian _ Correspondcc uddress: WN:. Koninys, Dcpartmcnt of Microbiology. University uf Groningen. Kcrklaon 30.9751 NN Harcn, The Netherlands. Fax: (3 I) (50) 635 205.