Cloning and nucleotide sequence of a hsp70 gene from Streptomyces griseus

The cultivation of Streptomyces griseus 2247 at the growth-limited temperature (37°C) or in liquid medium containing 5% ethanol (toxic for growth) revealed the presence of heat-induced proteins in the total cellular proteins. Among them, a 70 kDal protein was isolated and its N-terminal amino acid sequence was determined. The 70 kDal protein possessed a possible ATP-binding site in the N-terminus, which was conserved among the HSP70 family. A DNA fragment encoding the HSP70 homologue was isolated from a genomic library of S. griseus 2247 strain using an oligonucleotide probe based on the N-terminal amino acid sequence of the 70 kDal protein. DNA sequence analysis of the cloned gene revealed an open reading frame consisting of 618 amino acid residues. The deduced amino acid sequence is highly homologous to the HSP70 family proteins; it is 59.8 % identical to Clostridium perfringens HSP70, 59.7% to the Bacillus megaterium DnaK protein, 58.4% to the Methanosarcina mazei DnaK protein, 58.1% to Synechocystis HSP70, 52.8% to the DnaK protein of Escherichia coli, and about 50% to some of the mitochondrial heat shock proteins. The cloned gene could encode the HSP70 of S. griseus.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

Cloning and nucleotide sequence of the N-acetylmuramidase M1-encoding gene from Streptomyces globisporus

The gene (acm) encoding N-acetylmuramidase M1 (ACM) was cloned of Streptomyces globisporus ATCC No. 21553. The nucleotide sequence of the acm gene was determined and found to code for an ORF of 294 amino acids (aa). Comparison of aa sequence deduced from the acm gene with the N-terminal sequence of the extracellular enzyme suggests that ACM is synthesized with a 77-aa leader peptide. A comparison of the ACM aa sequence with the aa sequences of other proteins in the NBRF data base reveals that ACM has strong similarity to the N-O-diacetylmuramidase secreted by the fungus Chalaropsis.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Cloning and nucleotide sequence of the mycodextranase gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an alpha-glucanase that cleaves alpha-1,4-bonds of alternating alpha-1,3- and alpha-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.     

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.     

Cloning and sequence of a gene encoding macrotetrolide antibiotic resistance from Streptomyces griseus.

A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S. lividans TK64. The sequence (3326 bp) of the cloned DNA revealed three complete open reading frames (ORFs) and one incomplete ORF encoded on one strand of the DNA. The nonR gene (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases. A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring. The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyl-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.     

Cloning and nucleotide sequence of the isoamylase gene from a strain of Pseudomonas sp

A strain of Pseudomonas sp., SMP1, isolated from a soil sample collected in the Monterotondo area (Rome), secreted isoamylase activity into the culture medium. The enzyme was purified and optimal reaction and stability conditions were determined by varying pH and temperature. The chemico-physical properties of the enzyme were similar to those of the isoamylase purified in Japan more than 20 years ago from 'Pseudomonas amyloderamosa' strain SB15. A genomic library of SMP1 was prepared in Escherichia coli using pUC12 as vector. Two isoamylase-producing colonies were identified out of 6300 screened. The hybrid plasmids isolated from the two clones showed common restriction patterns. The chromosomal portion of one of these plasmids (pSM257) was completely sequenced. Comparison between the deduced amino acid sequence of the isoamylase and the published sequences of other amylolytic enzymes showed the presence of conserved domains.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

Cloning and nucleotide sequence of the urea amidolyase gene from Candida utilis

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida utilis CA(u)-37 (DUR1,2^c) was cloned by plaque hybridization, and the nucleotide sequences of DUR1, 2^c and its flanking regions were determined. DUR1, 2^c was found to be composed of 5,490 base pairs and 1,830 amino acid residues. Using Edman degradation of the purified enzyme, it was revealed that the amino-terminal residue (methionine) was processed for maturation. A TATA-box like sequence was found 112 bases upstream from the translation start site (ATG). The site of the poly (A) tail was found 54 bases downstream from the translation stop site (TGA), since cDNA of DUR1, 2^c was synthesized from mRNA and sequenced. The nucleotide sequences of the urea amidolyase gene from Saccharomyces cerevisiae and DUR1, 2^c were very similar to each other (65.3%), as were the deduced amino acid sequences (67.2%). The molecular weight of DUR1, 2^c was calculated to be 200,700. This value corresponded to the result obtained from SDS-polyacrylamide gel electrophoresis of the purified enzyme. The enzyme functions in a dimeric form. Three important regions were found in the amino acid sequence of urea amidolyase through the homology search. It was predicted that each region was equivalent to the active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of the DUR1, 2^c gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.     

Cloning and nucleotide sequence of the chimpanzee c-myc gene

A DNA fragment covering the chimpanzee c-myc locus was cloned from the DNA of peripheral blood lymphocytes, sequenced, and compared to its human c-myc counterpart. The two nucleotide sequences were found to be highly homologous (99%). The divergence rate between the two species was 0.4% in exons and 1.7% in introns. The different TATA-boxes described in the human myc gene were also identified in the chimpanzee sequence and an open reading frame (ORF) was observed which overlaps the chimpanzee c-myc first exon. This latter ORF contained three silent mutations with regard to the human region, whereas the chimpanzee Myc oncoprotein coded by exons 2 and 3 differed by two amino acids from the human one.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.     

Cloning of a nosiheptide-resistant gene from Streptomyces actuosus

The cloning of the nosiheptide resistance gene (nos) from S. actuosus, which produces this antibiotic, in plasmid pS10147 was carried out in S. lividans. The nosipheptide resistant clones contained the plasmid pNS1. It deletion derivative plasmid, pNS2, confers nosiheptide resistance to both S. ambofaciens and S. lividans.