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PubMed1988

Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli.

Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23. The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tü494. Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment. This fragment was shown to include the Ptt-resistance promoter. Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations. Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase.

Acetyltransferases↗
PubMed1990

Resistance to pactamycin in clones of Streptomyces lividans containing DNA from pactamycin-producing Streptomyces pactum.

A pactamycin (Pc)-resistance determinant (pct) from Streptomyces pactum has been isolated on a 4.9-kb KpnI fragment. The original construct involving plasmid pIJ702 was highly unstable in Streptomyces lividans, leading to deletion of the pct gene from the vector. Subcloning of pct into an alternative vector (pOJ160) led to the generation of a more stable clone which possessed Pc-resistant ribosomes, and reconstitution analysis established that 16S rRNA was responsible for such resistance. Post-transcriptional modification of rRNA is probably the mechanism of resistance since the cloned DNA fragment did not appear to encode 16S rRNA.

Cloning, Molecular↗
PubMed1993

Cloning, characterization, and expression in Streptomyces lividans 66 of an extracellular lipase-encoding gene from Streptomyces sp. M11.

A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.

Amino Acid Sequence↗
PubMed2006

Streptomyces coelicolor oxidase (SCO2837p): a new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2).

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.

Amino Acid Sequence↗
PubMed1998

Expression of the Streptomyces alboniger pur cluster in Streptomyces lividans is dependent on the bldA-encoded tRNALeu.

Streptomyces lividans 1326-9, a bldA+ strain, and its bldA39 mutant derivative J1725 were transformed with a cosmid containing the pur cluster, which determines the puromycin biosynthetic pathway from Streptomyces alboniger. bldA+ transformants produced puromycin in typical amounts, whereas bldA39 transformants did so at drastically decreased levels. Transformation of low producers with the wild-type bldA gene reverted this phenotype to normal production. These data, in addition to the presence of a TTA codon in the amino-terminal coding region of the pur10 and pur6 genes of the pur cluster, suggest that the puromycin biosynthetic pathway is translationally dependent on the bldA gene product, a tRNALeu.

Acetyltransferases↗
PubMed2001

Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans.

BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Anthelmintics↗
PubMed1991

Opposite facial specificity for two hydroquinone epoxidases: (3-si,4-re)-2,5-dihydroxyacetanilide epoxidase from Streptomyces LL-C10037 and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase from Streptomyces MPP 3051.

(3-si,4-re)-2,5-Dihydroxyacetanilide epoxidase (DHAE I), a key enzyme in the biosynthesis of the epoxysemiquinone antibiotic LL-C10037 alpha by Streptomyces LL-C10037 [Gould, S.J., & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686], and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase (DHAE II) isolated from Streptomyces MPP 3051--which yields the (3R,4S)-epoxyquinone mirror image product of DHAE I--are described. DHAE I was purified 640-fold. Gel permeation chromatography indicated an Mr of 117,000 +/- 10,000; SDS-PAGE gave a major band of 22,300 daltons, indicating that DHAE I is either a pentamer or hexamer in solution. The enzyme had a pH optimum of 6.5, a Km of 8.4 +/- 0.5 microM, and a Vmax of 3.7 +/- 0.2 mumol min-1 mg-1. DHAE II was purified 1489-fold. The enzyme was shown to be a dimer of Mr 33,000 +/- 2000, with 16,000-dalton subunits, with a pH optimum of 5.5 and a Km of 7.2 +/- 0.4 microM. Both enzymes required only O2 and substrate; flavin and nicotinamide coenzymes had little or no effect. Neither catalase nor EDTA affected the activity of either enzyme, but complete inhibition of both was obtained with 1,10-phenanthroline. The activity of the purified DHAE I could be enhanced, but only by Mn2+ (relative V = 246 at 0.04 mM), Ni2+ (relative V = 266 at 0.2 mM), or Co2+ (relative = 498 at 0.2 mM). Reconstitution from a DHAE I apoenzyme, generated by treatment with 1,10-phenanthroline followed by Sephadex G-25 chromatography, occurred only by addition of one of these three metals.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoenzymes↗
PubMed2001

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn4811 of Streptomyces lividans 66.

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.

Base Sequence↗
PubMed2002

Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface.

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (DeltardlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the DeltardlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.

Bacterial Proteins↗
PubMed2001

The variant tuf3 gene of Streptomyces coelicolor A3(2) encodes a real elongation factor Tu, as shown in a novel Streptomyces in vitro translation system.

In Streptomyces coelicolor, the regular and abundant elongation factor (EF)-Tu1 is encoded by tuf1, while the actual function of the highly divergent tuf3 gene product is not yet known. Expression of the latter could so far only be detected on the transcriptional level under stress conditions. In this paper we demonstrate the presence of low levels of EF-Tu3 in strains of the J1501 lineage. Enhanced expression was observed for J1501 glkA and relA deletion mutants, which lack glucose kinase and ribosome-bound ppGpp synthetase, respectively. To assess the putative translational capacities of EF-Tu3, a novel Streptomyces in vitro translation assay was designed, based on the full elimination by Ni2+ affinity adsorption of chromosomally encoded (His)6-tagged EF-Tu1 from a S. coelicolor cell-free extract. Translational activity of this system is totally dependent on the addition of purified EF-Tu species or on the presence of an additional elongation factor Tu in the extract, e.g. encoded by a plasmid-borne tuf gene. Using this EF-Tu-dependent translation system, we have established that S. coelicolor EF-Tu3 has translational capacities despite its striking deviation from the common prokaryotic EF-Tu sequence at positions involved in either aminoacyl-tRNA binding or interaction with the guanine-nucleotide exchange factor EF-Ts.

Amino Acid Sequence↗
PubMed2004

BOX-pCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus.

The type strains of 451 validly described Streptomyces species were screened using the BOX-PCR fingerprint technique. Of the different primers tested, the BOX primer yielded the most robust and reproducible band patterns. The majority of the species (350) had an unique pattern each. A total number of 30 clusters were delineated comprising species with nearly identical patterns. Four of these clusters grouped synonymous Streptomyces species known from previous studies. A high correlation exists between BOX-PCR fingerprinting and DNA-DNA homology data. In the present study, the latter was confirmed for 7 additional BOX clusters and led to the following emended descriptions: S. cochleatus is a subjective synonym of S. cinereorectus, S. roseoflavus of S. fradiae, S. roseodiastaticus of S. tricolor, S. roseosporus of S. filamentosus, S. distallicus of S. colombiensis, S. arabicus of S. vinaceus and S. phaeoviridis of S. phaeopurpureus. Although not yet confirmed by DNA-DNA pairing studies, rep-PCR data suggested the presence of at least 33 additional junior synonyms.

DNA Fingerprinting↗
PubMed1999

High performance in refolding of Streptomyces griseus trypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor.

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 microg/ml of SGT did not exceed 15%. This low yield was assumed to be due mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refolding media were examined for their ability to minimize misfolding. As a result, 1 M Tris, 1 M diethanolamine, and 1 M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding. The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.

Bacterial Proteins↗
PubMed1993

Interaction of Streptomyces subtilisin inhibitor (SSI) with Streptomyces griseus metallo-endopeptidase II (SGMP II).

We have unexpectedly found that Streptomyces subtilisin inhibitor (SSI) and some other similar serine protease inhibitors produced by Streptomycetes strongly inhibit Streptomyces griseus metallo-endopeptidase II (SGMP II) [Kajiwara, K. et al. (1991) J. Biochem. 110, 350-354]. In order to elucidate the mode of their unusual interaction with SGMP II in more detail, we prepared twelve kinds of SSI analogues, in which one or two amino acid residues in the peptide segment from Thr64 to Val74 of wild-type SSI had been replaced or deleted by site-directed mutagenesis, and determined the dissociation constants of their complexes with SGMP II. Six analogues among them showed dissociation constants one order of magnitude lower than that of the wild type. Three had higher values. The results suggest that at least some residues in this segment are interacting with SGMP II in the complex. We also prepared an SSI mutant in which the disulfide bridge between Cys71 and Cys101 had been eliminated by replacing the two Cys residues with Ser residues. This mutated SSI inhibited SGMP II as strongly as the wild-type SSI did. While peptide bonds in the wild-type molecule did not suffer from the hydrolytic action of SGMP II except those at the amino-terminal fragile portion, the Pro72-Met73 bond of the mutant was specifically cleaved by the enzyme. This peptide bond, therefore, seems to play the role of the reactive site in the interaction of SSI with SGMP II.

Amino Acid Sequence↗
PubMed1977

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.

Bacterial Proteins↗
PubMed1985

Interactions of Streptomyces subtilisin inhibitor with Streptomyces griseus proteases A and B. Enzyme kinetic and computer simulation studies.

Streptomyces subtilisin inhibitor (SSI), a dimeric protein that strongly inhibits subtilisins, was shown to form tight inhibitory complexes with Streptomyces griseus proteases A and B (SGPA and SGPB). The apparent dissociation constants of the SGPA-SSI and SGPB-SSI complexes were found to be orders of magnitude less than those of subtilisin-SSI complexes. Using the known atomic coordinates for SGPA and SSI, the highly complementary nature of the surface geometries of the two proteins was confirmed by a computer graphics study, which led to a proposed structure for the SGPA-SSI complex. Kinetic studies further suggested that the SSI dimer can bind two molecules of either SGPA or SGPB, and the 2:1-complexes (consisting of one inhibitor dimer and one enzyme molecule) apparently possess lower intrinsic dissociation constants than the 2:2-complexes. It was also shown that both of SGPA and SGPB are inhibited by both soybean trypsin inhibitor (Kunitz) and bovine pancreatic trypsin inhibitor (Kunitz), but far less strongly than by SSI.

Aspartic Acid Endopeptidases↗
PubMed1998

Streptomyces thermogriseus, a new species of the genus Streptomyces from soil, lake and hot-spring.

Many thermophilic actinomycetes were isolated from samples collected from a hot-spring, lake and soil in Yunnan, China. Chemical and molecular classification of four selected strains of thermophilic Streptomyces with an upper limited growth temperature of 65-68 degrees C and autolytic characteristics was carried out. A new species, Streptomyces thermogriseus sp. nov. is described. The type strain is Y-14046T (= CCTCC AA97014T).

China↗
PubMed1987

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.

Cloning, Molecular↗
PubMed1991

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus.

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Anti-Bacterial Agents↗