Streptomyces Lavendulae Research Articles

The carbohydrate-containing cell wall polymers of some species from the cluster "Streptomyces lavendulae"

The type strains of the cluster "Streptomyces lavendulae" species with a low level of DNA-DNA relatedness were found to contain different cell-wall carbohydrate polymers, whereas the species of this cluster with a level of DNA-DNA relatedness of about 60% contain similar or identical carbohydrate polymers. The type strains Streptomyces katrae VKM Ac-1220T and S. polychromogenes VKM Ac-1207T synthesize mannan with different amounts of alpha-1,2- and alpha-1,3-substituted mannopyranose units and a small number of 1,3-poly(glycerophosphate) chains. The cell walls of S. lavendulocolor VKM Ac-215T and Streptomyces sp. VKM Ac-2117 were found to contain a hitherto unknown teichuronic acid, whose repeating unit is a disaccharide consisting of diaminomannuronic acid and N-acetylgalactosamine:-->4)-beta-D-ManpNAc3NAcA-(1-->3)-alpha-D- GalpNAc-(1-->. In addition, the cell walls of these two streptomycetes contain beta-glucosylated 1,5-poly(ribitol phosphate). The cell walls of S. virginiae VKM Ac-1218T and S. flavotricini VKM Ac-1277T contain the same poly(glucosyl-glycerophosphate). The results presented in this paper are in accordance with the DNA-DNA relatedness data published earlier and indicate a taxonomic significance of the structure of the cell-wall polysaccharides for the delineation of pheneticall/related Streptomyces species.

The Kinetic Mechanism of Penicillin V Acylase from Streptomyces Lavendulae

Penicillin Vacylase isolated from Streptomyces lavendulae is an extracellular enzyme with interesting properties for the industrial production of 6-APA. The enzyme hydrolysed penicillin V and synthetic analogues such as 2-nitro-5-(phenoxyacetarnido)-benzoic acid (NTPOAB). The corresponding kinetic parameters were KM = 3 mM and V max = 92 μmol min-1 mg-1 for penicillin V; KM = 15.3 mM and V max = 14.5 μmol. min-1 mg-1 for NIPOAB. Studies of inhibition by products, alternative products and dead-end inhibition supported an ordered uni bi mechanism that could involve an acyl-enzyme intermediate as has been described for penicillin G acylases and other amidases.

Activation and stabilization of penicillin V acylase from streptomyces lavendulae in the presence of glycerol and glycols.

Penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae showed both enhanced activity and stability in mixed water/glycerol and water/glycols solvents. The catalytic activity was increased up to a critical concentration of these cosolvents, but further addition of the latter led to a gradual protein deactivation. The highest stabilizing effect was achieved in the presence of glycerol. Thermal stability was increased proportionally to the concentration of glycerol and glycols in the reaction mixture only if the amount added is below the threshold concentration. Reaction conditions that allow simultaneously enhanced activity and stability in the hydrolysis of penicillin V catalyzed by penicillin V acylase from S. lavendulae could be established.

Enhanced production of penicillin V acylase from Streptomyces lavendulae.

A 28 degrees C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 degrees C to 55 degrees C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

Involvement of amfC in physiological and morphological development in Streptomyces coelicolor A3(2).

amfC plays a regulatory role in aerial mycelium formation in Streptomyces griseus and is distributed widely among Streptomyces species. Disruption of the chromosomal amfC gene in Streptomyces coelicolor A3(2) severely reduced formation of aerial hyphae, indicating that amfC is important in morphological development. In addition, the disruption caused S. coelicolor A3(2) M130 to produce much less actinorhodin, and to produce undecylprodigiosin at a later stage of growth, indicating that amfC also regulates secondary metabolism. S1 nuclease mapping showed that transcription of actII-ORF4, the pathway-specific transcriptional activator in the act gene cluster, was greatly reduced in the amfC disruptants. The defect in secondary metabolite formation was suppressed or overcome by a mutation in sre-1. Consequently, an amfC-disrupted strain derived from S. coelicolor A3(2) M145, an actinorhodin-overproducing strain due to the sre-1 mutation, still produced a large amount of actinorhodin. Extra copies of amfC in strains M130 and M145 did not change spore-chain morphology or secondary metabolite formation. However, the spores in these strains remained white even after prolonged incubation. Since only spore pigmentation was affected, all known whi genes, except whiE, responsible for the polyketide spore pigment formation, were assumed to function normally. S1 nuclease mapping showed that transcription of whiEP1, one of the promoters in the whiE locus, was reduced in S. coelicolor A3(2) containing extra copies of amfC. Introducing amfC into several other Streptomyces species, such as Streptomyces lividans, Streptomyces lavendulae and Streptomyces lipmanii, also abolished spore pigment formation. An increase in the amount of AmfC appeared to disturb the maturation of spores.

DNA relatedness among the Streptomyces fulvissimus and Streptomyces griseoviridis phenotypic cluster groups

As a continuation of an ongoing study of DNA relatedness among red-spored streptomycetes, the homology between the four species comprising the Streptomyces fulvissimus phenotypic cluster (S. fulvissimus, Streptomyces aureoverticillatus, Streptomyces longispororuber and Streptomyces spectabilis) and the four species comprising the Streptomyces griseoviridis phenotypic cluster (S. griseoviridis, Streptomyces chryseus, Streptomyces daghestonicus and Streptomyces murinus) was measured spectrophotometrically from C0t05 determinations. All strains were also compared to 12 strains representing previously determined DNA-relatedness clusters in the Streptomyces lavendulae phenotypic cluster, including the type strain NRRL B-1230T. The strains segregated into 15 cluster groups at DNA relatedness <80%, including 10 single-membered clusters. S. griseoviridis and S. daghestonicus were synonymous, as were S. chryseus and S. longispororuber. S. spectabilis exhibited species-level homology with strain NRRL B-2402, which had been received as S. lavendulae. S. aureoverticillatus exhibited significant DNA relatedness to Streptomyces flavotricini. None of the strains from the S. fulvissimus or S. griseoviridis clusters exhibited significant homology to the type strain of S. lavendulae.

Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae.

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an Mr of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.

Microbial Transformation of Artemether

Microbial transformation of the semisynthetic sesquiterpene artemether, using Cunninghamella elegans and Streptomyces lavendulae, resulted in the isolation of five microbial metabolites that were characterized on the basis of their 1H and 13C NMR spectral data. These metabolites were identified as 9β-hydroxyartemether, 9α-hydroxyartemether, ring-rearranged 9β-hydroxyartemether, 3α-hydroxydeoxyartemether, and 14-hydroxyartemether. The latter three compounds are new and are being reported here for the first time.

Bacterial ribonuclease: mutagenic effect in microbial test-systems.

Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, AraR-assay; the prophage induction test; and the DNA-repair test. The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward AraR-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution). Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity. Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coli strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain. The importance of the roles of both of enzyme catalytic activity and native structure is emphasized. A proposed mechanism for exogenous ribonuclease action is discussed. Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis.

Protein tyrosine phosphorylation in streptomycetes

Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

Protein tyrosine phosphorylation in streptomycetes

Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

DNA Relatedness among Strains of the Streptomyces lavendulae Phenotypic Cluster Group

DNA relatedness was evaluated among the type strains of Streptomyces lavendulae and the 11 species which were designated as subjective synonyms in volume 4 of Bergey’s Manual of Systematic Bacteriology. Three S. lavendulae subspecies, five putative strains of S. lavendulae, and the type strain of “S. majorciensis” were also included in the present study. The 21 strains studied segregated into 14 clusters (average linkage) when grouped at >70% DNA relatedness, including 10 single-member clusters. S. lavendulae, S. lavendulae subsp. avirens, S. lavendulae subsp. grasserius, and S. columbiensis were found to be synonymous at >79% DNA relatedness. The other clusters, on the basis of their relatively low DNA relatedness to S. lavendulae, appear to be valid species rather than subjective synonyms of S. lavendulae.

Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei

We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point ( tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi,K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929–1937], even though the nt sequencesof the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502

L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 ( Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.

Molecular Analysis of -Lactamases from Four Species of Streptomyces: Comparison of Amino Acid Sequences with those of other -clactamases

Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase 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 beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.

Synthesis of mimocin, an isoquinolinequinone antibiotic from Streptomyces lavendulae, and its congeners.

Mimocin, an isoquinolinequinone antibiotic from Streptomyces lavendulae No.314, and its congeners were synthesized.

A new pigment from Streptomyces lavendulae.

A new pigment from Streptomyces lavendulae.

Purification and biochemical characterization of streptothricin acetyltransferase coded by the cloned streptothricin-resistance gene of Streptomyces lavendulae.

Streptothricin acetyltransferase was purified from Streptomyces lividans harboring a plasmid which carried the streptothricin-resistance gene cloned from a streptothricin-producing strain, Streptomyces lavendulae No. 1080. Some properties of the enzyme were determined and the reaction product was identified to be N beta-acetylstreptothricin by NMR spectroscopy.

Immunodiffusion Analyses of Phenetically Defined Strains of Streptomyces, Streptoverticillium and Nocardiopsis

Summary Seventeen strains of Streptomyces , 13 of Streptoverticillium and two of Nocardiopsis dassonvillei were studied by the immunodiffusion technique and the results were compared with those of a previous numerical phenetic study. Generally there was good agreement between the serological and phenetic data, and the former supported the view that there is much synonymy among the described species of Streptomyces and Streptoverticillium . Thus, the number of species of these genera can be substantially reduced. The immunological results also underlined the close relationships between these genera, this being particularly marked between streptoverticillia and the Streptomyces lavendulae cluster. The relationships of Nocardiopsis dassonvillei were ambiguous as one strain showed clear serological affinities with streptomycetes but the other did not.

Isolation and structural elucidation of new saframycins Y3, Yd-1, Yd-2, Ad-1, Y2b and Y2b-d.

A new series of saframycins Y3, Yd-1, Yd-2, Ad-1, Y2b and Y2b-d was produced by directed biosynthesis employing resting cells of saframycin producer, Streptomyces lavendulae No. 314. Their structures were determined by comparison with the spectral data of UV, IR, and 1H and 13C NMR of saframycin A, whose structure has already been established. Saframycin Y3 is 25-deoxy-25-aminosaframycin A, while saframycin Yd-1 is 26-homosaframycin Y3, and saframycin Y2b is a dimer of saframycin Y3. Saframycin Ad-1 is 26-homosaframycin A. Saframycin Yd-2 is 26-demethylsaframycin Y3, and saframycin Y2b-d is a dimer of saframycin Yd-1.