Digest: Structuring interactions in Streptomyces*

For bacteria growing in colonies, spatial structure can allow maintenance of costly traits such as the production of antibiotics. Using spatially structured environments, Westhoff et al. examined the benefits of streptomycin production for the bacterium Streptomyces griseus in competition with a streptomycin-susceptible strain. Streptomyces griseus outcompeted susceptible competitors, but the benefit of its antibiotic decreased as competitor resistance to streptomycin increased. Spatial structure also increased the ability of S. griseus to invade susceptible competitor populations from low starting densities. These results demonstrate that spatially structured environments can both provide and amplify benefits of antibiotics to antibiotic-producing bacteria on a microbial scale.     

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.     

Involvement of 16S ribosomal RNA in resistance of the aminoglycoside-producers Streptomyces tenjimariensis, Streptomyces tenebrarius and Micromonospora purpurea

Summary Resistance to aminoglycoside antibiotics in Micromonospora purpurea (the producer of gentamicin C complex), Streptomyces tenebrarius (the nebramycin producer) and Streptomyces tenjimariensis (which makes istamycin) occurs at the level of the ribosome. Reconstitution analysis has revealed, in each case, that 16S rRNA plays a critical role in determining such resistance.     

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid     

Classification of novel soil streptomycetes as Streptomyces aureus sp. nov., Streptomyces laceyi sp. nov. and Streptomyces sanglieri sp. nov

The taxonomic positions of soil isolates known as Streptomyces groups A, B and C were clarified. Comparative 16S rDNA sequence studies indicated that representatives of all three taxa formed distinct phyletic lines within the Streptomyces tree though the group A strains were shown to be related to Streptomyces griseus and associated validly described species. The taxonomic integrity of all three groups was highlighted by DNA:DNA relatedness and ribotype data though the group A strains encompassed a higher degree of genetic variation than the group B and C strains. In light of these and earlier phenotypic data it is proposed that Streptomyces groups A, B and C be given species status as Streptomyces sanglieri sp. nov., Streptomyces aureus sp. nov. and Streptomyces laceyi sp. nov., respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato

Aim:  To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. Methods and Results:  In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. Conclusions:  Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. Significance and Impact of the Study:  The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.     

The <Emphasis Type="Italic">Streptomyces violaceusniger</Emphasis> clade: a home for streptomycetes with rugose ornamented spores

The taxonomic status of 16 strains received as Streptomyces hygroscopicus, Streptomyces melanosporofaciens, Streptomyces sparsogenes, Streptomyces sporoclivatus and Streptomyces violaceusniger was evaluated in a polyphasic study. Eleven of the organisms formed a distinct clade in the Streptomyces 16S rRNA gene tree with the type strains of Streptomyces asiaticus, Streptomyces cangkringensis, Streptomyces indonesiensis, Streptomyces javensis, Streptomyces malaysiensis, Streptomyces rhizosphaericus, Streptomyces yatensis and Streptomyces yogyakartensis, the members of this group produced rugose ornamented spores in spiral spore chains. The eleven strains were assigned to three established and four novel species, namely Streptomyces albiflaviniger sp. nov., Streptomyces demainii sp. nov., Streptomyces geldanamycininus sp. nov., Streptomyces griseiniger sp. nov., and Streptomyces hygroscopicus, Streptomyces melanosporofaciens and Streptomyces violaceusniger. It is also proposed that S. sporoclivatus becomes a subjective synonym of S. melanosporofaciens. S. sparsogenes NRRL 2940^T, which produced ridged ornamented spores in spiral spore chains, formed a distinct phyletic line in the Streptomyces 16S rRNA gene tree and was readily distinguished from the other strains using a range of phenotypic properties. S. violaceusniger strains NRRL 8097, NRRL B-5799, NRRL 2834 and ISP 5182 fell outside the S. violaceusniger 16S rRNA gene clade and formed either smooth or ridged ornamented spores in either flexuous or spiral spore chains. These organisms were distinguished from one another and from their closest phylogenetic neighbors and were considered to merit species status as Streptomyces auratus sp. nov., Streptomyces phaeoluteichromatogenes sp. nov., Streptomyces phaeogriseichromatogenes sp. nov., and Streptomyces phaeoluteigriseus sp. nov., respectively. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the tested strains are S. albiflaviniger NRRL B-1356^T (AJ391812), S. auratus NRRL 8097^T (AJ391816), S. geldanamycininus NRRL 3602^T (DQ334781), S. griseiniger NRRL B-1865^T (AJ391818), S. hygroscopicus NRRL 2387^T (AJ391820), NRRL 2339 (AJ391821) and NRRL B-1477 (AJ391819), S. demainii NRRL B-1478^T (DQ334782), S. melanosporofaciens NRRL B-12234^T (AJ391837), S. phaeogriseichromatogenes NRRL 2834^T (AJ391813), S. phaeoluteichromatogenes NRRL B-5799^T (AJ391814), S. phaeoluteigriseus ISP 5182^T (AJ391815), S. sparsogenes NRRL 2940^T (AJ391817), S. sporoclivatus NRRL B-24330^T (AJ 781369), S. violaceusniger ISP 5563^T (AJ 391823) and NRRL B-1476^T (AJ 391822).     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin

Streptomyces longisporoflavus produces the poly-ketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug. A genomic library of S. longisporoflavus DN A was cloned in Streptomyces Uvldans and screened to identify tetronasin-resistance determinants. The inclusion of 0.2 M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen. Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified. The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus. The tnrB determinant was further localized, by subcloning, to a 2.8 kb Kpnl fragment. DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3). The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (A TP b inding c assette) superfamily of transport-related proteins. The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter. The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.     

Random insertion of Tn4560 in Streptomyces lividans and Streptomyces avermitilis

Summary Pulsed-field gel electrophoresis (PFGE) was used to show that insertions of transposon Tn4560 in Streptomyces lividans 66 and the avermectin-producer S. avermitilis are randomly distributed. DNA from the latter strain suffered degradation during electrophoresis that could be avoided by modification of the buffer. Inverse PCR primers were developed for Tn4560.     

Isolation and characterization of halotolerant Streptomyces radiopugnans from Antarctica soil

An actinomycete wild strain PM0626271 (= MTCC 5447), producing novel antibacterial compounds, was isolated from soil collected from Antarctica. The taxonomic status of the isolate was established by polyphasic approach. Scanning electron microscopy observations and the presence of LL‐Diaminopimelic acid in the cell wall hydrolysate confirmed the genus Streptomyces. Analysis of 16S rRNA gene sequence showed highest sequence similarity to Streptomyces radiopugnans (99%). The phylogenetic tree constructed using near complete 16S rRNA gene sequences of the isolate and closely related strains revealed that although the isolate fell within the S. radiopugnans gene subclade, it was allocated a different branch in the phylogenetic tree, separating it from the majority of the radiopugnans strains. Similar to type strain, S. radiopugnans R97^T, the Antarctica isolate displayed thermo tolerance as well as resistance to ⁶⁰Co gamma radiation, up to the dose of 15 kGy. However, media and salt tolerance studies revealed that, unlike the type strain, this isolate needed higher salinity for its growth. This is the first report of S. radiopugnans isolated from the Antarctica region. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces radiopugnans MTCC 5447 is JQ723477 .

Significance and Impact of the Study

The study presents the first report of isolation of Streptomyces radiopugnans from Antarctica. To date, there is only one publication regarding S. radiopugnans R97^T isolated from radiation‐polluted soil. Like the type strain, Antarctica isolate was thermotolerant and radiotolerant, but in addition, it required salts for growth and did not degrade phenol. We envisaged that metabolic pattern of the same species varies based on acclimatization in its native ecological habitat. Additionally, Antarctica isolate had produced novel antibacterial compounds (patent‐US2012/0156295). The study highlighted that least explored extreme regions like Antarctica are rich resources of novel microbial strains producing novel bioactive compounds.     

Genetic instability in Streptomyces niveus plasmid pSN2: in vivo formation of deletion derivatives

A plasmid designated pSN2 (molecular size 32.0 kb) was isolated from the wild type of Streptomyces niveus ATCC 19793. To permit phenotypic identification of pSN2 the 1.9 kb BclI fragment was replaced in vitro by the 1.1 kb BclI fragment of pIJ702 carrying the thiostrepton resistance (tsr) gene to form the plasmid pSN3. pSN3 transforms S. lividans to thiostrepton resistance at high frequency and is stably maintained. However, when used to transform S. niveus pSN3 was unstable and produced a 5.5 kb thiostrepton resistant deletion derivative pLG5. pLG5 is also stable and expresses thiostrepton resistance in S. lividans but on transformation of S. niveus was unstable and produced a further thiostrepton resistant derivative, pLG10, of 6.5 kb. pLG5 and pLG10 like pSN3 transform S. lividans at high frequency and produce pocks. DNA hybridizations with a probe derived from pLG5 confirm that pLG5 is derived from DNA sequences present on pSN2 and pSN3.Abbreviations SDS sodium dodecyl sulphate - PEG polyethylene glycol     

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.     

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus

Summary The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic productions in strains othersise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 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.     

High production of a class III lantipeptide AmfS in Streptomyces griseus

AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.     

Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans.

Summary The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.     

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X₂ even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X₂ has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5–100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA–DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.