Two new extracellular serine proteases from Streptomyces fradiae.

1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. Differential keratinolytic substrate specificity among these two microbes are probable due to a difference in disulfide reduction capacity.     

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-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 beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-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 beta-lactamase structural gene. The beta-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 beta-lactamase than the original S. cacaoi.     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.     

Cell-bound and extracellular carboxylesterases from Streptomyces: hydrolytic and synthetic activities

AIM: The distribution of cell-bound and extracellular carboxylesterases was investigated among the genus Streptomyces using 420 strains. METHODS AND RESULTS: Primary screening was carried out on solid media using tributyrin, triolein and Tween 60 as current substrates. Eleven representative strains were selected and grown in submerged cultures for evaluating their cell-bound and extracellular hydrolytic activity independently on various naphthyl and aliphatic esters. The best lipolytic strain was lyophilized and used as dry mycelium for catalysing the synthesis of various aliphatic esters in heptane, with molar conversions ranging from 28 to 78% after 3 days. CONCLUSIONS: Carboxylesterase activities can easily be found among the Streptomyces, often being cell-bound and also employable for catalysing esterification in organic solvent. SIGNIFICANCE AND IMPACT OF THE STUDY: A wide screening among Streptomyces, a genus poorly studied for the production of carboxylesterases, has allowed the selection of several strains with interesting enzymatic activities to be used in commercially valuable biotransformation.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

Valine dehydrogenase from Streptomyces fradiae: purification and properties

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.     

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.     

beta-lactamase from Streptomyces cacaoi. Purification and properties

A beta-lactamase was purified to an apparently homogeneous state from Streptomyces cacaoi. The molecular weight calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000. pI was 4.7 and the optimal pH was 6.5. The optimum temperature was found to be between 40 degrees C and 45 degrees C, but the enzyme lost activity above 50 degrees C. N-Bromosuccinimide was the strongest inhibitor among the reagents tested, followed by iodine. p-Chloromercuribenzoate showed a weak inhibitory effect. Diisopropylfluorophosphate and sodium chloride did not show any inhibitory effect on the enzyme. The beta-lactamase catalyzed the hydrolysis of methicillin and cloxacillin at two-thirds to one-third the rate of benzylpenicillin. On the other hand, the enzyme hydrolyzed cephalosporins and 7-methoxycephalosporin only slowly. With benzylpenicillin as a substrate, the Km increased sharply with decreasing pH and the pK alpha estimated from the Km versus pH curve was 6.5 to 7.0. In contrast, with cloxacillin as a substrate, the Km showed a minimum at pH 7.5. The Vmax changed with pH in a bell-shaped curve in the case of benzylpenicillin, but the Vmax for cloxacillin changed only within a small range. In addition, the ratio of the hydrolysis rate of benzylpenicillin and cloxacillin at 30 degrees C and 20 degrees C (V30 degrees/V20 degrees) was found to be 1.23 and 1.55, respectively. These results indicate that the S. cacaoi beta-lactamase behaves differently toward benzylpenicillin and cloxacillin, although both are penicillins. S. cacaoi seems to release beta-lactamase into the culture medium soon after its biosynthesis without retaining it in the membrane and the soluble fraction. The possible relationships between beta-lactamases from Streptomyces and those from pathogenic bacteria are discussed.     

Cloning and analysis of the beta-lactamase gene from epsilon-poly-L-lysine-producing actinomycete Streptomyces albulus IFO14147

Streptomyces albulus IFO14147 produces epsilon-poly-L-lysine, which exhibits antimicrobial activity. It is necessary for its molecular breeding to develop host-vector systems. We recently found a novel cryptic plasmid, pNO33, in this strain. As part of a search for a selectable marker gene for pNO33, we report here the isolation and analysis of the beta-lactamase gene of this strain, which can grow on ampicillin-containing plates. It was shown that the beta-lactamase production in S. albulus was induced by ampicillin. By introducing a genomic library of S. albulus into Escherichia coli, a 3.6-kbp fragment was identified as the region involved in ampicillin resistance. It contained three open reading frames, all of which are highly homologous to the beta-lactamase (the blaL product) and its regulatory proteins (the blaA and blaB products) of S. cacaoi. The growth phenotypes and enzyme assaying of E. coli and S. lividans showed that the blaL homologue (blaSa) encodes a beta-lactamase required for ampicillin resistance. The beta-lactamae gene can be utilized as a selectable marker in a cloning vector of S. albulus. However, the beta-lactamase activity was decreased in E. coli and repressed in S. lividans by the blaA and blaB homologues (blaASa and blaBSa). It appears as if the blaASa product is a repressor of blaSa instead of an activator as in S. cacaoi.     

Purification and partial characterization of a cholesterol oxidase from Streptomyces fradiae

An extracellular cholesterol oxidase from Streptomyces fradiae (PTCC 1121) was purified in one step using DEAE-Sepharose. The purified enzyme had a molecular weight of 60 KDa. The optimum pH and temperature for activity was found to be 7 and 70 degrees C, respectively. This cholesterol oxidase was stable in pHs between 4-10 at 4 degrees C until 4 h. Thermal stability experiments showed that it has high stability and retains its full activity at 50 degrees C for 90 min. K(m) value for cholesterol oxidase was obtained to be about 7.06 x 10(-)(5) Mol.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).     

Cloning of tlrD, a fourth resistance gene, from the tylosin producer, Streptomyces fradiae

In addition to tlrA, tlrB and tlrC, which were previously cloned by others, a fourth antibiotic-resistance gene (tlrD) has been isolated from Streptomyces fradiae, a producer of tylosin (Ty), and cloned in Streptomyces lividans. Like tlrA, tlrD encodes an enzyme that methylates the N6-amino group of the A2058 nucleoside within 23S ribosomal RNA. However, whereas the tlrA protein dimethylates that nucleoside, the tlrD product generates N6-monomethyladenosine. The genes also differ in their mode of expression: tlrA is inducible, whereas tlrD is apparently expressed constitutively, and it has been confirmed that the tlrA-encoded enzyme can add a second methyl group to 23S rRNA that has already been monomethylated by the tlrD-encoded enzyme. Presumably, that is what happens in S. fradiae.