Characterization of an insertion sequence element associated with genetically diverse plant pathogenic Streptomyces spp

Streptomycetes are common soil inhabitants, yet few described species are plant pathogens. While the pathogenicity mechanisms remain unclear, previous work identified a gene, nec1, which encodes a putative pathogenicity or virulence factor. nec1 and a neighboring transposase pseudogene, ORFtnp, are conserved among unrelated plant pathogens and absent from nonpathogens. The atypical GC content of nec1 suggests that it was acquired through horizontal transfer events. Our investigation of the genetic organization of regions adjacent to the 3' end of nec1 in Streptomyces scabies 84.34 identified a new insertion sequence (IS) element, IS1629, with homology to other IS elements from prokaryotic animal pathogens. IS1629 is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase. Transposition of IS1629 generates a 10-bp target site duplication. A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis. A functional copy of IS1629 from S. turgidiscabies 94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the lambda cI857 repressor. IS1629 is present in multiple copies in some S. scabies strains and is present in all S. acidiscabies and S. turgidiscabies strains examined. A second copy of IS1629 was identified between ORFtnp and nec1 in S. acidiscabies strains. The diversity of IS1629 hybridization profiles was greatest within S. scabies. IS1629 was absent from the 27 nonpathogenic Streptomyces strains tested. The genetic organization and nucleotide sequence of the nec1-IS1629 region was conserved and identical among representatives of S. acidiscabies and S. turgidiscabies. These findings support our current model for the unidirectional transfer of the ORFtnp-nec1-IS1629 locus from IS1629-containing S. scabies (type II) to S. acidiscabies and S. turgidiscabies.     

Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24.

A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.     

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) possesses a centrally located replication origin and shows significant homology to the transposon Tn4811.

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325-660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non-pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site-specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram-positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.     

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S. scabies growth and repress thaxtomin production represents an attractive alternative to classical control strategies. Pseudomonas sp. LBUM 223 produces phenazine-1-carboxylic acid (PCA), an antibiotic that inhibits the growth of plant pathogens and contributes to the biological control of plant diseases. In this study, the involvement of LBUM 223's PCA-producing ability in the growth inhibition of S. scabies, repression of thaxtomin biosynthesis genes (txtA and txtC) and the biological control of common scab of potato was investigated using a mutant defective in PCA production (LBUM 223phzC(-) ). Streptomyces scabies growth was inhibited to a significantly lesser degree by LBUM 223phzC(-) than by the wild type. LBUM 223 also significantly repressed txtA and txtC expression in S. scabies and protected potato against disease, whereas LBUM 223phzC(-) did not. These results suggest that PCA production is central to the ability of LBUM 223 to limit pathogen growth, repress the expression of key pathogenicity genes and control common scab of potato.     

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends.

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.     

Cloning and sequence of a gene encoding macrotetrolide antibiotic resistance from Streptomyces griseus.

A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S. lividans TK64. The sequence (3326 bp) of the cloned DNA revealed three complete open reading frames (ORFs) and one incomplete ORF encoded on one strand of the DNA. The nonR gene (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases. A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring. The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyl-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.     

Lack of plasmids in Streptomyces hydrogenans

Abstract Wild-type cells of Streptomyces hydrogenans ATCC 19631, strain HY A₁, show a remarkable degree of genetic instability with regard to the biosynthesis of 17β-hydroxysteroid dehydrogenase. As plasmids might be responsible for this phenomenon we tried to detect plasmids in lysates of this microorganism. Streptomyces lividans , strain TK64 (pIJ916), was used as reference strain, containing a 19-kb plasmid with low abundancy. Whereas plasmid DNA could be shown in lysates of S. lividans TK64, no plasmid DNA was detectable in lysates of S. hydrogenans .     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

Primary structure analysis of a duplicated region in the amplifiable AUD6 locus of Streptomyces ambofaciens DSM40697

Abstract In Streptomyces ambofaciens , an amplifiable unit of DNA ( AUD6 ) contains two homologous sequences, one located on the right extremity of the AUD ( S1R ), the other being internal ( IHS ). This paper presents the molecular analysis of this duplication. The nucleotide sequences are almost identical (95%) and each contains an ORF of about 330 codons, the two ORFs being nearly identical. The two hypothetical proteins, deduced from these sequences, show about 30% identity with different bacterial repressors. They also show a particularly strong similarity (90% identity between the full-length sequences) with hypothetical proteins of Streptomyces lividans 66 encoded by sequences also present on an amplifiable DNA region ( AUD1 ).     

Production of thaxtomin a by two species of Streptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five out of them were pathogenic on potato minitubers and four of the pathogenic isolates produced thaxtomin A in infected tubers tissues. The lesion surface areas induced by thaxtomin A were highest in treatment of the minitubers with extract of OMB inoculated with S-6 and S-7, intermediate with that inoculated with S-4 and lowest with S-3. The pathogenic isolates were identified by their colour of aerial mycelia, melanin pigment productivity (+ or -), the type of spore chains morphology and carbon utilization as either S. scabies strains S-3, S-4 and S-8, or S. acidiscabies strains S-6 and S-7. S-3 and S-4 produced 0.65 and 1.60 micrograms thaxtomin A per milliliter of OMB, respectively, whereas S-6 and S-7 produced similar amounts of thaxtomin A, 2.36 and 2.10 micrograms per ml of OMB, respectively. The optimal temperature for production of thaxtomin A by S. scabies and S. acidiscabies was 28 degrees C. Production of thaxtomin A by S. scabies strain S-4 and S. acidiscabies strain S-6 was suppressed at least 50-fold at 0.5 and 0.3% of glucose, respectively. Fructose enhanced the production of thaxtomin A by both S. scabies and S. acidiscabies.     

The Linear Plasmid SCP1 of Streptomyces coelicolor A3(2) Possesses a Centrally Located Replication Origin and Shows Significant Homology to the Transposon Tn4811

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.     

Characterization of Streptomyces scabies mutants deficient in melanin biosynthesis

Streptomyces scabies, a causal agent of common scab, produces both melanin and a secondary metabolite called thaxtomin A. To establish a possible relation between melanin and thaxtomin A production in S. scabies, we carried out N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and isolated 11 melanin-negative mutants of S. scabies EF-35. These mutants were characterized for thaxtomin A production, pathogenicity, sporulation, and stress resistance. Nine of these mutants showed a significant reduction in thaxtomin A production when compared with the wild strain. However, only a few mutants exhibited a reduced level of virulence or a loss in their ability to induce common scab symptoms on potato tubers. Other pleiotrophic effects, such as higher sensitivity to heavy metals and incapacity to sporulate under certain stress conditions, were also associated with a deficiency in melanin production.