Producer Streptomyces Research Articles

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains

A p-aminobenzoic synthase gene ( pabS) from Streptomyces griseus IMRU 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains were never before described as polyene producers.

Chromophore activating enzyme involved in the biosynthesis of the mikamycin B antibiotic etamycin from Streptomyces griseoviridus.

A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP. SDS-polyacrylamide gel electrophoresis indicates that the enzyme is a single polypeptide chain with a Mr between 56,000 and 58,000. The molecular mass of the native enzyme was in the same range. In addition to 3-HPA, the enzyme catalyzes the formation of adenylates from picolinic acid, nicotinic acid, and 2-pyrazinecarboxylic acid. Nicotinic acid and picolinic acid when added externally to etamycin producing S. griseoviridus cultures gave rise to the formation of etamycin analogues each containing nicotinic acid or picolinic acid instead of the genuine 3-HPA. The data strongly suggest that the enzyme is involved in the biosynthesis of the chromopeptide lactone etamycin and possibly in that of other mikamycin B antibiotics.

The Streptomyces aureofaciens plasmid pIMB R8 and its use for shuttle vector construction

The cryptic low copy plasmid designated as pIMB R8 was isolated from an industrial strain of the chlortetracycline producer Streptomyces aureofaciens R8/26. Using restriction endonucleases the pIMB R8 plasmid was characterized to have 15 kb. Subcloning of the Bam HI fragments of pIMB R8 into replication probe vector resulted in the identification of the replication part. The 0.7 kb Bc/I--Bg/I fragment is sufficient for normal replication, but produces about fourty times high plasmid copy numbers than the original pIMB R8. Using the replication part of pIMB R8 was constructed several shuttle cloning vectors for the E. coli-streptomycete system.

Purification and properties of a prokaryote type glutamine synthetase from the bialaphos producer Streptomyces hygroscopicus SF1293

A prokaryote type glutamine synthetase (GS) was purified from a bialaphos (BA)-producing organism, Streptomyces hygroscopicus SF1293 (SF1293). The GS (GS I) consisted of a 55,000 dalton subunit, and its N-terminal amino acid sequence was similar to that of S. coelicolor GS. GS I was highly sensitive to GS inhibitor phosphinothricin (PPT). An increase of GS activity was observed accompanied by BA accumulation.

Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae.

The aminoglycoside phosphotransferase gene (aph) from the neomycin producer Streptomyces fradiae encodes an enzyme (APH) that phosphorylates, and thereby inactivates, the antibiotic neomycin. Two promoters were identified upstream of and oriented toward the aph coding sequence. One promoter (aphp1) initiated transcription at the A of the ATG translational initiation codon, or one to two bases upstream. Mutations made in this promoter region identified functionally important nucleotides and verified that the aphp1 transcript was translated to yield the APH protein, despite the lack of a conventional ribosome binding site. A second aph promoter, aphp2, initiated transcription 315 bp upstream of the translational initiation codon but gave transcripts that appeared to terminate before reaching the coding sequence. Multiple transcriptional initiation sites (pA1-pA5) were identified also in the aph regulatory region oriented in the opposite direction to aph transcription. Promoters for the pA2 and pA4 transcripts overlap with aphp1 such that down-promoter mutations in aphp1 also reduce transcription from the overlapping pA promoters.

Studies of precursor-directed biosynthesis with streptomyces. Part 2. New and unusual manumycin analogues produced by Streptomyces parvulus

The application of a new type of precursor-directed biosynthesis, in which artificial aromatic starter molecules were fed to the manumycin producer Streptomyces parvulus, resulted in new and unusual manumycin analogues for which structures are reported. The new compounds can be divided into three classes; the artificial starter molecule elongated (i) with the triene chain including the C5N moiety, (ii) with the chiral manumycin C13-side chain only, and (iii) with both substituents. Based on these results it is plausible to differentiate between an amidase linking the chiral C13-side chain to the artificial precursor, and a CoA-transferase activating the aromatic carboxy group for further elongation via the polyketide pathway. The specificity of the enzymes involved with regard to the structure of the aromatic precursor is discussed.

Production of a streptomycin-Park nucleotide complex by Streptomyces griseus

A compound (compound X) with antibacterial activity was isolated from early-exponential-phase cultures of the streptomycin producer Streptomyces griseus and from protoplast cultures of the same strain. The protoplast cultures produced a larger amount of compound X than did the young hyphae. Both the mycelia and the protoplasts incorporated 14C-labeled myo-inositol, a precursor of streptomycin, into compound X, which has an amino acid content related to that of the cell wall peptidoglycan of the producer strain. Compound X may contain a streptomycin molecule covalently bound to a cell wall precursor unit, i.e., to a Park nucleotide (J. T. Park, J. Biol. Chem. 194:897-904, 1952), through the glutamic acid of the pentapeptide component.

Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate

Genetic and biochemical evidence was obtained for lysine catabolism via cadaverine and delta-aminovalerate in both the beta-lactam producer Streptomyces clavuligerus and the nonproducer Streptomyces lividans. This pathway is used when lysine is supplied as the sole source of nitrogen for the organism. A second pathway for lysine catabolism is present in S. clavuligerus but not in S. lividans. It leads to alpha-aminoadipate, a precursor for beta-lactam biosynthesis. Since it does not allow S. clavuligerus to grow on lysine as the sole nitrogen source, this pathway may be used exclusively to provide a precursor for beta-lactam biosynthesis. beta-Lactam producers were unable to grow well on alpha-aminoadipate as the only nitrogen source, whereas three of seven species not known to produce beta-lactam grew well under the same conditions. Lysine epsilon-aminotransferase, the initial enzyme in the alpha-aminoadipate pathway for lysine catabolism, was detected in cell extracts only from the beta-lactam producers. These results suggest that synthesis of alpha-aminoadipate is exclusively a secondary metabolic trait, present or expressed only in beta-lactam producers, while genes governing the catabolism of alpha-aminoadipate are present or fully expressed only in beta-lactam nonproducers.

The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy.

Two novel compounds, 2-methyl, 4-carboxy, 5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A] and 2-methyl, 4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B] have been identified in the pool of Streptomyces parvulus by in vivo and in vitro studies. 13C and 15N were introduced into the compounds by feeding S. parvulus with 15N- and 13C-labeled L-glutamate. High resolution 13C and 15N NMR have been applied to elucidate their structure and biosynthesis in S. parvulus. The splitting patterns and coupling constants of adjacent nitrogen-carbon molecular fragments enable us to unravel their molecular structure. Two different glutamate pools are responsible for their biosynthesis, THP(A) carbon skeleton derives from the extracellular L-[13C]glutamate, whereas THP(B) stems from D-fructose via the intracellular glutamate. During cell growth, THP(A) is synthesized and becomes the major constituent of the intracellular pool. It is consumed after THP(B) is accumulated intracellularly. The onset of THP(A) and -(B) synthesis seems correlated to the time of actinomycin D synthesis. Their high cellular concentrations during actinomycin D synthesis suggest that they may function as nitrogen storage. Other possible functions of THP molecules within the cell are discussed.

Effects of reduced oxygen on growth and antibiotic production inStreptomyces clavuligerus

The effects of reduced oxygen on growth and antibiotic production were studied in batch cultures ofStreptomyces clavuligerus in defined media. Antibiotic levels were unaffected by reduced oxygen for the first 50 hours of fermentation. After growth ceased, antibiotic concentrations dropped by a factor of about three under reduced oxygen, while the antibiotic concentration was stable when air was used. This suggests that enzymes for hydrolyzing antibiotics may be regulated by the aeration conditions.

Translational attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae.

An inducible resistance determinant, ermSF, from the tylosin producer Streptomyces fradiae NRRL 2338 has been cloned, sequenced, and shown to confer inducible macrolide-lincosamide-streptogramin B resistance when transferred to Streptomyces griseofuscus NRRL 23916. From mapping studies with S1 nuclease to locate the site of transcription initiation, the ermSF message contains a 385-nucleotide 5' leader sequence upstream from the 960-nucleotide major open reading frame that encodes the resistance determinant. On the basis of the potential secondary structure that the ermSF leader can assume, a translational attenuation model similar to that for ermC is proposed. The model is supported by mutational analysis involving deletions in the proposed attenuator. By analysis with restriction endonucleases, ermSF is indistinguishable from the tlrA gene described by Birmingham et al. (V. A. Birmingham, K. L. Cox, J. L. Larson, S. E. Fishman, C. L. Hershberger, and E. T. Seno, Mol. Gen. Genet. 204:532-539, 1986) which comprises one of at least three genes from S. fradiae that can confer tylosin resistance when subcloned into S. griseofuscus. When tested for inducibility, ermSF appears to be strongly induced by erythromycin, but not by tylosin.

Analysis of the nourseothricin-resistance gene ( nat) of Streptomyces noursei

A gene ( nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNATl. The nat gene was localized on a 1-kb SalI- MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of Nc R in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.

Metabolic products of microorganisms. 249. Tetracenomycins B3 and D3, key intermediates of the elloramycin and tetracenomycin C biosynthesis.

Tetracenomycins B3 and D3, besides tetracenomycin D (D1), were produced by a blocked mutant of the elloramycin producer Streptomyces olivaceus TU 2353. The compounds were isolated as red powders, and their structures were elucidated by comparing their physicochemical data with those of the known tetracenomycins A2, B1, B2, D and E. Tetracenomycin B3 (2), the main compound, and tetracenomycin D (3) were antibiotically inactive against Gram-positive and Gram-negative bacteria, whereas tetracenomycin D3 (1) showed a moderate activity against Bacillus subtilis and Arthrobacter aurescens. Tetracenomycin B3 (2) is the key intermediate where the biosynthesis of the elloramycins branches off from the line leading to tetracenomycin C (5) as the final product of the tetracenomycin biosynthesis branch.

Relation between growth rate and erythromycin production inStreptomyces erythraeus

The relation between specific growth rate and specific rate of product formation was studied in phosphate-limited chemostat cultures ofStreptomyces erythraeus. Specific rates of formation were measured for both the final product, erythromycin A, and several of its biosynthetic precursors. In all cases rates of formation increased with inereasing growth rate, indicating that antibiotic production was strongly growth-linked.

Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria

Previous studies had localised the gene ( mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein ( M r 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene ( tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.

A method to transform the β-lactam antibiotic producer Streptomyces wadayamensis

A method to transform the β-lactam antibiotic producer Streptomyces wadayamensis

Cloning and analysis of the promoter region of the erythromycin-resistance gene ( ermE) of Streptomyces erythraeus

A DNA fragment containing the coding and regulatory sequences of the erythromycin (Er) resistance ( ermE) gene of the Er producer Streptomyces erythraeus was cloned in Streptomyces lividans using the plasmid vector pIJ61. The approximate location and orientation of ermE were deduced from studies of its expression after subcloning in Escherichia coli. Sequences responsible for transcription of ermE in Streptomyces were studied by nucleotide (nt) sequencing, high resolution S1 and exonuclease VII mapping, in vitro transcription and in vivo promoter-probing. Tandemly arranged promoters of typical prokaryotic appearance initiate transcription of the coding region of ermE; a promoter of similar sequence was identified that initiates transcription of a likely coding region running in the opposite direction to ermE. It is suggested that these sites represent a class of vegetatively expressed Streptomyces promoter that is utilised by a form of RNA polymerase holoenzyme that also recognises typical promoters of other bacterial genera.

Elloramycins B, C, D, E and F: minor congeners of the elloramycin producer Streptomyces olivaceus.

Elloramycins B, C, D, E and F: minor congeners of the elloramycin producer Streptomyces olivaceus.

Self-resistance of the nourseothricin-producing strain Streptomyces noursei.

The nourseothricin producer Streptomyces noursei is resistant to its own antibiotic in submerged as well as in surface culture. The strain shows no cross-resistance to miscoding inducing aminoglycoside antibiotics. Cell free extracts of Streptomyces noursei inactivate nourseothricin by enzymatic acetylation. The pattern of cross-resistance of Streptomyces noursei correlates well with the substrate specificity of the nourseothricin acetyltransferase. Furthermore, the acetyltransferase activity parallels the resistance level in nourseothricin-producing strains and nonproducing mutants. The results suggest that the nourseothricin acetyltransferase is important in the self-defence strategy of the nourseothricin-producing strain.

Bacterial resistance to streptothricins.

Resistance to streptothricin was studied in bacteria with different resistance mechanisms. The laboratory-induced streptothricin-resistant mutant E. coli A19 Stcr 2/2/1 showed a high level of cross-resistance to aminoglycosides and other miscoding inducing antibiotics. In contrast, aminoglycosid-resistant E. coli strains with plasmid-determined aminoglycoside modifying enzymes were sensitive to streptothricin. Enzymatic inactivation of streptothricin by acetylation was demonstrated for the streptothricin producing Streptomyces noursei, strain NG13. This strain showed no cross-resistance to miscoding inducing aminoglycosides.