Horizontal transfer of the plant virulence gene, nec1, and flanking sequences among genetically distinct Streptomyces strains in the Diastatochromogenes cluster

Evidence for the horizontal transfer of a pathogenicity island (PAI) carrying the virulence gene nec1 and flanking sequences among Streptomyces strains in the Diastatochromogenes cluster is presented. Plant-pathogenic, thaxtomin-producing Streptomyces strains, previously classified as S. scabiei based on the conventionally used phenotypic characteristics, were found to be genetically distinct from the type strain of S. scabiei based on DNA relatedness and 16S rDNA sequence analysis. Pairwise DNA-DNA hybridizations between some of these strains and the S. scabiei type strain were as low as 36%, a value much below what is conventionally accepted for species identity (70%). The sequence of the nec1 gene, however, was identical in all the S. scabiei and S. scabiei-like strains tested, irrespective of their DNA relatedness to the type strain of S. scabiei, their geographic origin, or the isolation host. Furthermore, a 26-kb DNA fragment including and flanking nec1 was also conserved among these strains based on restriction and Southern analyses. These data indicate that the etiology of potato scab is more complex than previously recognized; this result has important implications for potato scab management strategies. Previous research has suggested that horizontal transfer of a PAI was the mechanism for evolution of pathogenicity in S. acidiscabies and S. turgidiscabies, species that lie outside of the Diastatochromogenes cluster. Data presented here support this model and indicate that PAI transfer also has occurred frequently in species closely related to S. scabiei.     

Biological control agent of common scab disease by antagonistic strain Bacillus sp. sunhua

AIMS: To identify an antagonistic strain against Streptomyces scabiei and to characterize the antibiotic agent. The efficacy of the isolated strain in controlling common scab disease was also evaluated. METHODS AND RESULTS: A bacterial strain antagonistic against S. scabiei was isolated from the soil of a potato-cultivating area. This bacterium was identified as a Bacillus species by 16S rRNA gene sequence analysis and was designated Bacillus sp. sunhua. Antibiotics produced by this strain were proven to be stable within a broad pH range and at high temperatures. The culture broth was extracted with ethyl acetate, and then the crude extract was applied to HPLC. Two compounds were isolated and identified as iturin A and macrolactin A by 1H-NMR, 13C-NMR, HMBC, HMQC and mass spectrometer. The culture broth of Bacillus sp. sunhua had a suppressive effect on common scab disease in a pot assay, decreasing the infection rate from 75 to 35%. This strain also suppressed Fusarium oxysporum, the pathogen of potato dry rot disease. CONCLUSIONS: Bacillus sp. sunhua was shown to inhibit S. scabiei effectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating that macrolactin A and iturin A inhibit S. scabiei. This study demonstrated the possibility of controlling potato scab disease using Bacillus sp. sunhua.     

Detection of the nec1 virulence gene and its correlation with pathogenicity in Streptomyces species on potato tubers and in soil using conventional and real-time PCR

AIMS: To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays. Methods AND RESULTS: Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173(T). The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil. CONCLUSIONS: Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.     

Cell wall teichoic acids of streptomycetes of the phenetic cluster 'Streptomyces fulvissimus'

Structures of the anionic polymers of streptomycetes Streptomyces fulvissimus VKM Ac-994(T), Streptomyces longispororuber VKM Ac-1735(T), Streptomyces aureoveticillatus VKM Ac-48(T) and Streptomyces spectabilis INA 00606 belonging to the phenetic cluster 'S. fulvissimus' were investigated by chemical and NMR spectroscopic methods. A teichoic acid from the cell wall of S. spectabilis INA 00606 was studied in more detail, and this was shown to represent 1,3-poly(glycerol phosphate) substituted with glucosamine (alpha-D-GlcNAc) and L-glutamic acid (non-stoichiometric substitution). For the first time, glutamic acid is identified as an acyl substituent in teichoic acids of streptomycetes. The polymer chain is built of the following fragments: Cell walls of other streptomycetes of the phenocluster under study contain 1,3-poly(glycerol phosphates) with glucosamine as a glycosyl substituent at O-2 of the glycerol phosphate units and L-glutamic acid and lysine as O-2 acyl substituents. Not all amino sugar residues in the polymers of these strains are N-acetylated, and the content of the glucosamine and lysine residues in the polymers of different strains is not the same. Despite certain quantitative differences in the structures of the polymers, one may consider streptomycetes of the phenocluster 'S. fulvissimus' as closely related microorganisms, the details of the structures serving as additional criteria for the determination of the species status of a strain under study.     

Taxonomic study on nonpathogenic streptomycetes isolated from common scab lesions on potato tubers

Numerical analysis was carried out to compare sixteen nonpathogenic actinomycetes isolated from common scab lesions on potato tubers with Streptomyces scabiei type strain as well as with other streptomycete groups. These isolates were divided into two classes according to their level of similarity with S. scabiei. Isolates resembling S. scabiei were associated with S. griseoruber or with S. violaceusniger while isolates exhibiting less than 61% of similarity with S. scabiei were phenotypically related to S. albidoflavus or to S. atroolivaceus. Sequence of the 16S rRNA gene of each isolate was obtained and compared against the GenBank nucleotide database. No significant match could be established between the sequences of two potato isolates and the ones available in the GenBank database. The other isolates were closely related with S. setonii (S. griseus), S. mirabilis, S. fimbriatus, S. violaceoruber, S. melanosporofaciens and S. thermocarboxydus.     

Characterization of Streptomyces plasmid-phage pFP4 and its evolutionary implications

Autonomous-replicating plasmid pFP4 of Streptomyces sp. FR1 isolated from a heavy metal-contaminated land was cloned and sequenced. Surprisingly, the 40,949-bp pFP4 contains a cluster of 20 genes, resembling these chromosome-integrated prophages of Streptomyces sp. SPB78 and Streptomyces scabiei 87.22. Plasmid pFP4 could transfer by conjugation and a replication locus, iteron/repA/repB, was identified. The filtered FR1 culture could infect both FR1 and FR1 cured of pFP4 to form plaques, and also six out of 13 strains from the same land, but failed to form plaques on other seven strains from same source and all ten Streptomyces species from different sources. pFP4 phage particles were observed by transmission electron microscopy. Major structural proteins (capsid, portal and tail, etc.) of pFP4 virions were encoded by twelve pFP4 genes. pFP4 phage DNA contained 3' protruding cohesive ends of 9-nt. Streptomyces pFP4 represents a novel plasmid-phage.     

<Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis> sp. nov., isolated from soil in Karnataka,India

During the course of screening for industrially important microorganisms, an alkali-tolerant and thermotolerant actinomycete, strain DAS 131T, was isolated from a soil sample collected from the Gulbarga region, Karnataka province, India. The strain was characterized by a polyphasic approach that showed that it belonged to the genus Streptomyces. Growth was observed over a wide pH range (pH 6-12) and at 45 degrees C. The 16S rRNA gene sequence of strain DAS 131T was deposited in the GenBank database under the accession number DQ317411. 16S rRNA gene sequence analysis revealed that strain DAS 131T was most closely related to Streptomyces venezuelae ISP 5230T (AY999739) with a sequence similarity of 99.5% (8 nucleotide differences out of 1,477). Despite this very high sequence similarity, strain DAS 131T was phenetically distinct from S. venezuelae. The DNA relatedness between these strains was 54%, indicating that strain DAS 131T is a distinct genomic species. On the basis of phenetic and genetic analyses, strain DAS 131T is classified as a new species in the genus Streptomyces, for which we propose the name Streptomyces gulbargensis sp. nov.     

<Emphasis Type="Italic">Streptomyces roseoalbus</Emphasis> sp. nov., an actinomycete isolated from soil in Yunnan,China

A strain YIM 33098T (= CCTCC AA001027T = DSM 41831T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098T was clustered in the same subclade with Streptomyces tendae ATCC19812T and Streptomyces eurythermus ATCC14975T. Similarities of strain YIM 33098T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

Biosynthesis of epsilon-rhodomycinone from glucose by Streptomyces C5 and comparison with intermediary metabolism of other polyketide-producing streptomycetes

The catabolism of glucose by Streptomyces C5, a producer of anthracycline antibiotics, was investigated to determine the pathways that supply precursors for anthracycline biosynthesis. Carbons for the biosynthesis of epsilon-rhodomycinone, an anthracycline aglycone, from radiolabelled glucose were derived primarily from the Embden-Meyerhof-Parnas pathway, with a minor contribution from the pentose phosphate pathway. Furthermore, the anthracycline-producing strain, Streptomyces C5, as well as Streptomyces aureofaciens and Streptomyces lividans, strains that produce nonanthracycline polyketide antibiotics, displayed enzyme activities indicative of the Embden-Meyerhof-Parnas and pentose phosphate glycolytic pathways. As determined from labelling patterns, Streptomyces C5 apparently has a complete tricarboxylic acid cycle, but does not have a glyoxylate bypass pathway.     

Anionic carbohydrate-containing polymers of cell walls in two streptoverticille genospecies.

The cell walls of two streptoverticille genospecies which belong to a historically isolated group of the genus Streptomyces contain anionic polymers of different structure. Streptomyces hachijoensis VKM Ac-191T and Streptomyces cinnamoneus subsp. azacoluta VKM Ac-606T assigned to one genospecies on the basis of DNA--DNA hybridization [5] contain 37% of an identical sugar-1-phosphate polymer. The repeating disaccharide units of the polymer, 2-amino-2-deoxy-alpha-D-glucopyranosyl-(1-->6)-2-acetamido-2-deoxy-al pha-D-glucopyranose, are linked at C-1 and C-6' by phosphodiester bonds. The cell walls of Streptomyces biverticillatus VKM Ac-891T and Streptomyces baldaccii VKM Ac-821T, members of another genospecies, contain about 30% 1,3-poly(glycerol phosphate) completely substituted by 2-amino-2-deoxy-alpha-D-glucopyranosyl residues at C-2. Due to the presence of an amino sugar with a free amino group in the repeating unit, the polymers exhibit neutral properties. Polymer structures were determined by chemical methods and NMR spectroscopy. The data indicate taxonomic specificity of anionic polymers in streptoverticille cell walls.     

Streptomyces manipurensis sp. nov., a novel actinomycete isolated from a limestone deposit site in Manipur, India

A novel actinobacterium, designated MBRL 201(T), was isolated from a sample collected from a limestone quarry at Hundung, Manipur, India. The strain was characterized using polyphasic taxonomy. Comparison of the 16S rRNA gene sequence of strain MBRL 201(T) and other Streptomyces species showed sequence similarities ranging from 93.0 to 99.6 % and strain MBRL 201(T) showed closest similarities to Streptomyces virginiae NBRC 12827(T) (99.6 %) and Streptomyces cinnamonensis NBRC 15873(T) (99.6 %). The DNA relatedness between MBRL 201(T) and the type strains of S. virginiae NBRC 12827(T) and S. cinnamonensis NBRC 15873(T) were 44.5 and 35.6 % respectively. Strain MBRL 201(T) contained LL: -diaminopimelic acid (A(2)pm) as the diagnostic diamino acid, with glucose as the main sugar, while small amounts of galactose, glucose, mannose, rhamnose, ribose and xylose were also present in cell-wall hydrolysates. The major fatty acids identified were anteiso-C(15:0) (38.9 %), iso-C(15:0) (19.9 %) and anteiso-C(17:1) (14.7 %). The predominant menaquinones detected were MK-9(H(6)) and MK-9(H(8)), while the polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides, with other unknown phospholipids and lipids. The G+C content of the genomic DNA was 72.9 %. The phenotypic and genotypic data showed that strain MBRL 201(T) merits recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces manipurensis sp. nov. The type strain is MBRL 201(T) (=DSM 42029(T)?=?JCM 17351(T)).     

Structure of anionic carbohydrate-containing cell wall polymers in several representatives of the order actinomycetales

A new teichoic acid was identified in the cell walls of Streptomyces griseoviridis VKM Ac-622T, Streptomyces sp. VKM Ac-2091, and Actinoplanes campanulata VKM Ac-1319T. The polymer is poly(glycosylglycerol phosphate). The repeating units of the polymer, alpha-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-galactopyran+ ++ osyl-(1-->1)-glycerols, are in phosphodiester linkage at C-3 of glycerol and C-6 of galactose. The structures of cell wall teichoic acids in the strains Streptomyces chryseus VKM Ac-200T and "Streptomyces subflavus" VKM Ac-484 similar in morphology and growth characteristics are also identical: 1,5-poly(ribitol phosphate) substituted at C-4(2) by 2-acetamido-2-deoxy-beta-glucopyranosyl residues and 1,3-poly(glycerol phosphate). The taxonomic aspects of these results are discussed.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Alteration of serine/threonine protein kinase activity during growth of the wild type Streptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-type Streptomyces avermitilis strain and its chloramphenicol-resistant (Cmlr) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [gamma-32P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cmlr mutation activates the protein kinase signalling system of S. avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.     

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.     

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.     

Degradation of latex and of natural rubber by Streptomyces strain La 7

Streptomyces strain La 7 was isolated from the banquete of a city high way in Karlsruhe. According to partial 16S rRNA gene sequencing it was identical with Streptomyces albogriseolus and Streptomyces viridodiastaticus. DNA-DNA-similarity studies revealed 80.3-82.4% similarity between each of two of the three strains. Although phylogenetically closely related, Streptomyces strain La 7 differed from the two reference strains by morphological as well as physiological features and might represent a new species aside of S. albogriseolus and S. viridodiastaticus. The new Streptomyces strain La 7 was grown in a medium containing a latex emulsion or squares of natural rubber gloves as the only carbon source. On agar plates with a latex overlay agar, translucent halo formation around the colonies was observed. The unvulcanized latex was metabolized and the carbon from the isoprene units was apparently used for cell growth. In shake cultures with unlimited oxygen supply, during 60 days of incubation, 140 mg of the 175 mg totally emulgated latex were degraded exponentially. In sterile control flasks about 3% of the initial amount of latex could not be recovered after incubation on a shaker, presumably due to photochemical transformation. During static incubation of sterile medium, the latex formed a sticky layer at the surface of the medium and on the glass walls and recovery of the material was more difficult. Estimation of the protein content of cells from total nitrogen resulted in about 50% of the degraded latex being incorporated into cells, if a standard cell composition was assumed. Direct protein analysis according to Bradford (1976) gave much lower estimates, presumably due to a low content of aromatic amino acids. Stripes of natural rubber were degraded by Streptomyces strain La 7 during 70 days to an extent of about 30%. Scanning electron microscopy demonstrated, that hyphes of Streptomyces strain La 7 colonized and penetrated the latex surface with a concomitant deterioration of the latex material.     

Development of a genotyping method for potato scab pathogens based on multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S-23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.