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

Authors


  • Present address: College of Environmental Science and Forestry, State University of New York, Syracuse, NY 13210, USA; Bauer Center for Genomics Research, Harvard University, Cambridge, MA 02138, USA; §Animal and Plant Health Inspection Service, United States Department of Agriculture, Riverdale, MD, 20737, USA.

Summary

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.

Introduction

Horizontal gene transfer (HGT) has played a major role in bacterial evolution, enabling rapid acquisition of new traits that improve fitness in specific environmental niches, including pathogenic associations with eukaryotic hosts. Pathogenicity and virulence genes affect host range or the phenotype of the pathogenic interaction and may be mobilized among bacteria (Ochman et al., 2000; Hacker and Carniel, 2001; Ochman and Moran, 2001; Schmidt and Hensel, 2004). Pathogenicity islands (PAIs) are discrete clusters of such genes, sometimes organized into modules (Alfano et al., 2000), which possess, or have possessed, the ability to mobilize into other genetic backgrounds. Because HGT often occurs across bacterial genera, coding regions within PAIs frequently have a G+C content different from the core genome. PAIs often contain mobile genetic elements, have repeated sequences at their borders and are inserted into tRNA or other highly conserved loci. A bacterial strain may contain one or more PAIs and closely related bacteria may have a different PAI complement or lack PAIs entirely (Hacker and Kaper, 2000). PAIs have been known to be important in Gram-negative bacteria for some time (Hacker and Kaper, 2000; Kim and Alfano, 2002). Data on the role of PAIs in the evolution of pathogenicity in Gram-positive bacteria has lagged, but PAIs have now been described in many Gram-positive animal pathogens, including Enterococcus species (Shankar et al., 2002; Leavis et al., 2004), Staphylococcus aureus (Lindsay et al., 1998; Fitzgerald et al., 2001; Yamaguchi et al., 2002; Yarwood et al., 2002), Mycobacterium avium (Tizard et al., 1998; Stratmann et al., 2004) Streptococcus pneumoniae (Brown et al., 2001), Bacillus anthracis (Read et al., 2003) and Rhodococcus equi (Jain et al., 2003). However, there has been a lack of evidence for PAIs in Gram-positive plant pathogenic bacteria.

Streptomyces are filamentous, high G+C, Gram-positive bacteria with a complex morphology. This genus is best known for its ability to produce a diverse array of biologically active secondary metabolites. Streptomyces coelicolor is a model system for the study of secondary metabolism and morphogenesis. Its 8.7 Mb linear chromosome has been sequenced and is organized into a central core region of essential genes and chromosome arms containing gene clusters for secondary metabolism, mobile genetic elements and other presumably dispensable genes (Bentley et al., 2002).

Although hundreds of Streptomyces species have been described, only a few are known to cause plant diseases. Potato scab is an economically important plant disease caused by a polyphyletic group of Streptomyces species. Streptomyces scabies (syn. Streptomyces scabiei) was first described in 1891 and has a global distribution (Lambert and Loria, 1989a). During the last 75 years, two additional species, Streptomyces acidiscabies (Lambert and Loria, 1989b) and Streptomyces turgidiscabies (Miyajima et al., 1998), have independently emerged as pathogens in the USA and Japan respectively. The emergence of new phytopathogenic species has led us to postulate that the genetic determinants for plant pathogenesis are being shared between Streptomyces species that are adapted to specific ecological niches. Genetic analysis of strains of S. scabies, S. acidiscabies and S. turgidiscabies strongly suggests that HGT from S. scabies to saprophytic species in agricultural systems is the basis for the emergence of S. acidiscabies and S. turgidiscabies (Healy et al., 1999; Bukhalid et al., 2002).

We are characterizing virulence loci in phytopathogenic Streptomyces species. The primary pathogenicity determinant is the phytotoxin thaxtomin (Lawrence et al., 1990; Healy et al., 2000), a nitrated dipeptide toxin. The biosynthesis of thaxtomin A occurs through a highly specialized nitration event involving a bacterial nitric oxide synthase, encoded by nos, as well as a non-ribosomal peptide synthetase and associated modifying enzymes, encoded by txtABC (Healy et al., 2000; 2002; Kers et al., 2004). Thaxtomin has a novel mechanism of action: it inhibits cellulose biosynthesis and thus is able to induce plant cell hypertrophy in expanding plant tissues (Fry and Loria, 2002; Scheible et al., 2003). It is likely that, by compromising the integrity of the cell wall, thaxtomin-producing pathogens facilitate their intracellular penetration. The low G+C (54%) gene nec1 encodes a small, secreted, necrogenic protein with an uncharacterized plant cell target (Bukhalid et al., 1998). The Nec1 protein is not required for thaxtomin production and therefore represents an independent virulence factor. Through physical mapping and DNA sequencing of regions flanking nos and nec1 in S. turgidiscabies, we discovered a very large mobilizable PAI that appears to be responsible for the emergence of new pathogenic species. This article describes the first PAI in a Gram-positive plant pathogenic bacterium. We demonstrate PAI conservation in several polyphyletic plant pathogenic Streptomyces species, PAI transfer and site-specific integration into the S. coelicolor chromosome, and the ability of the PAI to confer a pathogenic phenotype in Streptomyces diastatochromogenes.

Results

DNA sequencing and analysis

A cosmid walking and DNA sequencing effort was initiated in S. turgidiscabies to explore regions of DNA flanking the nec1 and nos virulence loci (Fig. 1A). PFGE was used to map cosmids containing either nec1 or nos to a single 425 kb AseI restriction fragment in S. turgidiscabies. The nec1 gene mapped to a position approximately 70 kb upstream from the 5′AseI site and the nos gene mapped to a location approximately 30 kb downstream from the middle 5′AseI site. Thus, these two virulence determinants lie on a DNA fragment that spans 325 kb. A total of 154 kb of unique DNA sequence was obtained from cosmids flanking nos and nec1. Annotation of this sequence uncovered additional genes with homologues known to be involved in plant pathogenicity, gene regulation, transport, genes of unknown function, pseudogenes and mobile genetic elements (Fig. 1A; Table 1, GenBank Accession No. AY707079AY707081). The low G+C content of many of the open reading frames (ORFs), compared with the average 71% G+C content of the genome (Miyajima et al., 1998), suggests that these ORFs were acquired by HGT. The existence of multiple pathogenicity loci, ORFs with G+C content divergent from the bacterial genome and multiple mobile genetic elements supports the existence of a PAI in S. turgidiscabies.

Figure 1.

A. The genetic organization of virulence loci in S. turgidiscabies. DNA sequencing of cosmids flanking the nos and nec1 loci revealed the plant fasciation (fas) and tomatinase (tom) loci. Cosmid walking from virulence loci to AseI endonuclease sites demonstrate these four loci span approximately 325 kb of DNA.
B. Southern blot hybridization demonstrating conservation of the tomatinase (i), necrosis (ii), plant fasciation locus (iii) and thaxtomin biosynthetic pathway (iv) in three polyphyletic plant pathogenic Streptomyces species. Lane assignments: 1, S. coelicolor M145; 2, S. turgidiscabies Car8; 3, S. scabies 87.22; 4, S. acidiscabies 84.104. Southern hybridization of PstI digests of genomic DNA was performed using a 673 bp α-32P-labelled nos probe, 463 bp fas4 probe, 367 bp nec1 probe or 765 bp tom probe.
C. A single DNA fragment containing virulence loci is mobilizable from the pathogen S. turgidiscabies to the non-pathogen S. coelicolor. (i) PFGE of AseI digests of S. turgidiscabies Car811 (lane 1), S. coelicolor M145 (lane 2) and three transconjugant PAI S. coelicolor recipients (lanes 3–5). Site-specific integration of the transferred element is demonstrated by the absence of a 511 kb AseI fragment in S. coelicolor transconjugants strains (compared with S. coelicolor WT) and the presence of polymorphic 815 kb and 375 kb AseI fragments in the transconjugant strains. The 815 kb and 375 kb polymorphic bands present in transconjugant strains are contiguous, as demonstrated by hybridization of α-32P-labelled cosT44 (which contains an AseI site) to both fragments (ii). The 815 kb AseI fragment contains the nos (iii) and tomatinase virulence loci (iv), as demonstrated by Southern blot hybridization of the PFGE gel using a 673 bp α-32P-labelled nos probe and 765 bp tom probe.
D. A single DNA fragment containing virulence loci is mobilizable from the pathogen S. turgidiscabies to the non-pathogen S. diastatochromogenes. (i) PFGE of AseI digests of S. turgidiscabies Car811 (lane 1), S. diastatochromogenes (lane 2) and three transconjugant S. diastatochromogenes recipients (lanes 3–5). A 770 kb polymorphic band is present in S. diastatochromogenes transconjugants. An additional 375 kb polymorphic band is contiguous with the 875 kb polymorphic band as demonstrated by hybridization of α-32P-labelled cosT44 (which contains an AseI site) to both fragments (ii). The 770 kb AseI fragment contains the nos (iii) and tomatinase virulence loci (iv), as demonstrated by Southern blot hybridization of the PFGE gel using a 673 bp α-32P-labelled nos probe and 765 bp tom probe.

Table 1. ORFs found on the S. turgidiscabies PAI.
Gene locusGene nameG+C contentORF length (bp)Putative function
 1 67.81223β-Glucosidase, pseudogene (partial CDS)
 2 tomA 64.21602Tomatinase
 3 70.81023LacI family transcriptional regulator
 4 69.61191Transposase
 5 66.2 438Conserved hypothetical protein
 6IS162970.11296Transposase
 7 nec1 54.2 666Necrosis protein
 8 ORFtnp 68.51238Transposase, pseudogene
 9 66.7 771Resolvase
10 70.31236Transposase
11 fas5 66.01338Cytokinin oxidase
12 fas4 62.7 756Isopentyl transferase
13 fas3 65.0 933Transketolase
14 fas2 66.2 891Ferredoxin transketolase
15 fas1 69.31203Cytochrome P450
16 65.6 843Methyltransferase
17 65.4 855Methyltransferase
18 68.61470Transmembrane transport protein
19 fas6 67.0 576Hypothetical protein
20 69.6 827Transposase
21 nos 68.71203Nitric oxide synthase
22 67.21302Cytochrome P450
23 64.5 234Transposase (partial CDS)
24 67.4 765Transposase (partial CDS)
25 73.2 220Transposase (partial CDS)
26 69.61224Transposase
27 55.7 639AraC family regulatory protein
28 68.01205Transposase, pseudogene
29 txtA 69.54359Thaxtomin synthetase A
30 txtB 69.74518Thaxtomin synthetase B
31 txtC 64.91188Cytochrome P450

Two loci on the S. turgidiscabies PAI are homologous to known plant pathogenicity determinants in other Gram-positive and fungal plant pathogens. Homologues of the six genes in the plant fasciation (fas) operon in Rhodococcus fascians were identified from the S. turgidiscabies PAI. In R. fascians these genes are located on a linear plasmid and comprise a cytokinin biosynthetic pathway that is required for leafy gall formation on plants (Crespi et al., 1992; Goethals et al., 2001). The S. turgidiscabies fas operon is colinear with the fas operon of R. fascians, with the exception of fas6, which appears to have undergone a rearrangement on the S. turgidiscabies chromosome. We also identified a gene (tomA) encoding a homologue to tomatinase, a well-characterized enzyme from plant pathogenic fungi. Tomatinase detoxifies the anti-microbial saponin, tomatin (Lairini and Ruiz-Rubio, 1998; Quidde et al., 1998; Roldan-Arjona et al., 1999), which can lead to the suppression of plant defence responses to pathogen attack (Osbourn, 1996; Bouarab et al., 2002). The S. turgidiscabies tomatinase is a member of the glycosyl hydrolase family 10, which contains characterized tomatinase proteins. S. acidiscabies and S. scabies were analysed for the presence of fas and tom, using Southern hybridization (Fig. 1B). Like the nec1 and txt loci, tom was conserved in S. acidiscabies, S. scabies and S. turgidiscabies but absent in the non-pathogen S. coelicolor (and 10 additional non-pathogenic species; data not shown). In contrast, the fas operon was only present in the newly emerged pathogen S. turgidiscabies.

Mobilization of the pathogenicity island

Antibiotic resistance markers were used to detect transfer of the PAI from the donor S. turgidiscabies Car811 [camS, strS, thioR (nec1::tsr)] to recipient strains S. coelicolor M145 (camR, thioS) and S. diastatochromogenes (strR, thioS). The maximum frequency of PAI transfer observed in our experiments, as estimated by the acquisition of thioR by S. coelicolor, is 1.3 × 10−3 per recipient colony-forming unit (cfu). Putative thioR transconjugants were subsequently tested using PFGE in order to determine whether a large fragment of DNA had been transferred. No plasmids were observed in S. turgidiscabies or S. coelicolor wild-type (WT) and transconjugant strains following PFGE of undigested agarose plugs. S. diastatochromogenes appears to possess a 700 kb plasmid in both WT and transconjugant strains. Digestion of S. coelicolor transconjugant DNA with AseI revealed that a 511 kb AseI fragment present in S. coelicolor WT was absent in all transconjugant strains (Fig. 1C, i). Nine of 15 transconjugants tested contained 815 kb and 375 kb AseI fragments that were absent in the S. coelicolor and S. turgidiscabies WTs. The continuity between the 815 kb and 375 kb fragments was established by hybridization of a cosmid that overlaps the AseI site flanking the nos locus. Hybridization of cosT44, which contains an AseI site, to both the 815 kb and 375 kb fragments confirms that a single DNA element integrated into the S. coelicolor 511 kb AseI fragment (Fig. 1C, ii). Hybridization of both nos (Fig. 1C, iii) and tom (Fig. 1C, iv) probes demonstrate that all known pathogenicity loci were mobilized during the conjugation events. From these data, we can infer that a DNA element of approximately 660 kb has integrated into the S. coelicolor chromosome. The polymorphic DNA bands present in the transconjugant strains are consistent with mobilization of the single internal AseI site but not the terminal AseI sites (Fig. 1A). Five of 15 S. coelicolor transconjugants possess insertions of a 100 kb PAI fragment containing nec1 into the 511 kb AseI fragment and may represent specific transfer of a smaller fragment of the PAI (data not shown). Conjugal transfer of the PAI from S. coelicolor transconjugants to S. lividans demonstrates that the PAI is self-mobilizable and as such the genetic determinants for mobilization exist on the PAI (data not shown).

Site-specific integration of the PAI

The availability of the S. coelicolor genome sequence greatly facilitated mapping of the PAI integration point in the transconjugants. The PAI integration point was mapped using comparative restriction fragment length polymorphism analyses of S. coelicolor WT and transconjugant genomic DNA. The PAI integrated into SCO1326, an 876 bp ORF encoding a putative integral membrane lipid kinase. Homologues of SCO1326 have also been described as undecaprenol kinase or bacitracin resistance protein (BacA) (Cain et al., 1993; Chalker et al., 2000) in other bacteria. DNA sequencing of the PAI integration point in three independent S. coelicolor transconjugants revealed that the PAI is integrating site specifically into an 11 bp region of SCO1326 that contains an 8 bp palindrome (Fig. 2A). Although five copies of this 8 bp palindrome are present in the S. coelicolor genome, the PAI integrated exclusively into SCO1326. An 876 bp bacA homologue with 85% nucleotide identity to SCO1326 is found on the 5′-terminus of the PAI in S. turgidiscabies (Fig. 2B). Integration of the PAI in SCO1326 results in an in-frame insertion event and the formation of a hybrid lipid kinase. In the S. coelicolor transconjugants the 3′ PAI terminus contains at least 126 bp of putatively non-coding DNA flanking the 3′ truncated portion of SCO1326 (GenBank Accession No. AY707082 and AY707083).

Figure 2.

Integration point of the PAI in the S. coelicolor chromosome.
A. The PAI is integrating site-specifically into an 11 bp region that contains an 8 bp palindrome.
B. Cartoon illustrating integration of the PAI into the S. coelicolor chromosome in SCO1326, a putative integral membrane lipid kinase at chromosome position 1401207 bp. In S. coelicolor transconjugants, the left PAI junction consists of the 5′ region of SCO1326 and the 3′ region of a putative integral membrane lipid kinase from S. turgidiscabies as the result of a putative in-frame recombination event between the S. turgidiscabies PAI and the S. coelicolor chromosome. The right PAI junction consists of a putative non-coding region from S. turgidiscabies and the truncated 3′ region of S. turgidiscabies bacA.

Similar results were obtained when S. diastatochromogenes transconjugants were analysed using PFGE (Fig. 1D). Five of six transconjugants tested contained polymorphic 770 kb and 375 kb AseI fragments (Fig. 1D). As there was not a clear AseI fragment into which the PAI integrated, Southern hybridization using a bacA gene probe from S. diastatochromogenes genomic DNA was used to determine that the PAI is inserting into a 460 kb AseI fragment in S. diastatochromogenes (data not shown).

Plant pathogenicity assays of transconjugant strains

The S. coelicolor and S. diastatochromogenes transconjugants were assessed for the ability to produce thaxtomin in media and to necrotize potato tuber tissue, characteristics of S. turgidiscabies and other scab-causing species. The S. coelicolor transconjugants did not acquire a pathogenic phenotype; none produced thaxtomin or nectrotized potato tuber tissue (data not shown). In contrast, all S. diastatochromogenes transconjugants did produce thaxtomin. A representative transconjugant strain produced 40.80 ± 4.49 µg of thaxtomin A (standard deviation reported) under the same conditions that a S. turgidiscabies culture produced 70.64 ± 15.19 µg of thaxtomin A. S. diastatochromogenes transconjugants were able to colonize and cause necrotic symptoms on potato slices similar to S. turgidiscabies(Fig. 3). S. diastatochromogenes and S. coelicolor WT strains did not produce thaxtomin or necrotize potato tuber slices.

Figure 3.

Acquisition of the S. turgidiscabies PAI confers plant pathogenicity on S. diastatochromogenes. A representative S. diastatochromogenes transconjugant is able to induce necrosis on a potato slice while S. diastatochromogenes WT is not able to induce necrosis.

Discussion

Streptomyces turgidiscabies contains a mobile, integrative PAI. This is the first PAI to be described from a Gram-positive plant pathogenic bacterium and the largest PAI described in any bacterium to date. The PAI is at least 325 kb and is located on a DNA element approximately 660 kb in size. This PAI contains multiple virulence-associated genes, including the thaxtomin biosynthetic genes, nec1, cytokinin biosynthetic genes encoded by the fas operon, and a tomatinase homologue. The S. turgidiscabies PAI is typical of other PAIs in that it contains mobile genetic elements, pseudogenes and genes with varied G+C content, consistent with the introduction of DNA from other taxa (Table 1). The presence of the fas operon on the PAI of the newly emerged pathogen S. turgidiscabies and its absence from the S. scabies and S. acidiscabies strains analysed here suggests that the fas operon has been independently acquired following acquisition of the PAI by S. turgidiscabies. However, an assessment of multiple strains of each of these species for the presence of the fas operon should be performed to test this hypothesis.

It appears that this PAI is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems. Previous work had demonstrated that the ability to produce thaxtomin, a novel phytotoxin, was shared by polyphyletic plant pathogenic Streptomyces species, as was nec1, a low G+C gene encoding a necrogenic protein. The distribution of a PAI-associated insertion sequence, IS1629, in strains of S. scabies, S. acidiscabies and S. turgidiscabies, is consistent with mobilization of the PAI from the earliest known pathogen, S. scabies, to the newly emerged pathogens S. acidiscabies and S. turgidiscabies (Healy et al., 1999). Here we demonstrate that the thaxtomin biosynthetic genes and nec1 lie on a mobilizable PAI and that S. diastatochromogenes acquires a pathogenic phenotype upon acquisition of the PAI. S. diastatochromogenes transconjugants produce thaxtomin A and colonize plant tissue, demonstrating the functionality of the PAI in those recipients. The fact that none of the S. coelicolor transconjugants produce thaxtomin suggests that the functionality of the PAI depends on the genetic background of the recipient strain. However, we cannot rule out small deletions, rearrangements or incomplete transfer of the PAI in some or all transconjugants.

The availability of the complete genome sequence of S. coelicolor facilitated mapping of the PAI insertion site in the chromosome of transconjugant strains. Integration occurred exclusively into a 511 kb AseI fragment located on the left arm of the S. coelicolor linear chromosome. This is consistent with the location of other genes encoding non-essential functions, such as antibiotic biosynthetic pathways, on the Streptomyces chromosome arms rather than in the core genome where integration events are more likely to be deleterious (Volff and Altenbuchner, 2000; Bentley et al., 2002; Chen et al., 2002). Although many PAIs integrate into tRNA loci, the S. turgidiscabies PAI does not. The PAI integrated into SCO1326, an ORF encoding a putative integral membrane lipid kinase, homologues of which have been described as undecaprenol kinase or bacitracin resistance proteins (bacA) (Cain et al., 1993; Chalker et al., 2000) in other bacteria. BacA homologues are thought to be involved in the rate of peptidoglycan biosynthesis via phosphorylation of undecaprenol and are required for full virulence in S. pneumoniae and S. aureus (Chalker et al., 2000). Although homologues to SCO1326 are found in both Gram-positive and Gram-negative bacteria, this is the first report of a lipid kinase being targeted as an integration site for foreign DNA. It is not known whether the in-frame recombination between SCO1326 and the S. turgidiscabies bacA alters the activity of the enzyme in any way. Exclusive PAI integration within the 11 bp region of SCO1326 (Fig. 2) indicates a highly specific integration mechanism that is probably mediated by a site-specific recombinase. Site-specific integration of a DNA fragment greater than 600 kb has not previously been reported in Streptomyces and the genetic mechanism of this transfer event is not known at this time.

The high homology and colinear arrangement of the fas operon in S. turgidiscabies and R. fascians suggests a common origin. The fas operon is required for the production of leafy galls by R. fascians on plants and evidently codes for a cytokinin biosynthetic pathway (Goethals et al., 2001). The presence of the fas operon on the S. turgidiscabies PAI was unexpected, as neither this pathogen nor the other species known to cause potato scab produce plant galls. Interestingly, there is one Streptomyces species that has been reported to produce root galls on melon in Japan (Yoshida and Kobayashi, 1991), although the genetic basis of this pathogenic phenotype is not known.

The tomA  gene  encodes  a  homologue  of  tomatinase, a well-characterized enzyme present in fungi that are pathogens of solanaceous crops. Some tomatinase homologues are bifunctional proteins involved in the detoxification of anti-microbial saponins secreted by plants, as well as in the suppression of plant defence responses in tomato (Bouarab et al., 2002). Saponins generally complex with sterols in eukaryotic membranes, resulting in pore formation and the loss of membrane integrity (Nes, 1974). Potato also produces saponins, which could be the substrate for the putative tomatinase on the PAI. However, the absence of sterol biosynthesis in the majority of bacteria suggests that saponin detoxification is not the role of the tomatinase in plant pathogenic Streptomyces. Interestingly, the genome of the Gram-positive soilborne bacterium Clavibacter michiganensis ssp. michiganensis, the causal agent of bacterial wilt and canker of tomato, contains a homologue of the S. turgidiscabies tomatinase (Gartemann et al., 2003).

We have demonstrated that a PAI from a Gram-positive bacterium contains virulence loci that have a common origin with those found in other Gram-positive plant pathogenic bacteria (fas, tomA) and plant pathogenic fungi (tomA). These data indicate that Gram-positive plant pathogenic bacteria acquire genetic determinants for pathogenicity through HGT, as do other animal and plant pathogens. The remaining DNA sequence of the PAI should reveal additional evidence for HGT. Functional analyses of this PAI will provide insight on the nature of plant pathogenicity in Streptomyces and other Gram-positive bacteria. For example, the fact that pathogenicity is a very rare phenotype among streptomycetes suggests that either mobilization of the PAI is an infrequent event in nature or that acquisition of the PAI confers a pathogenicity phenotype on only a small proportion of recipients. Analyses of the PAI mechanisms of transfer and chromosomal integration, as well as PAI gene expression, should greatly inform our understanding of the evolution of pathogenicity in microbes.

Experimental procedures

Bacterial strains, growth media and DNA manipulations

Streptomyces scabies 87.22, S. acidiscabies 84.104 and S. turgidiscabies Car8 are plant pathogenic strains that have been described previously (Lambert and Loria, 1989a,b; Miyajima et al., 1998). S. coelicolor M145, S. lividans TK24 (Kieser et al., 2000) and S. diastatochromogenes ATCC12309 are non-pathogenic strains. The International Streptomyces Project Medium 2 and 4 (BD Biosciences) as well as Mannitol Soya Flour Medium (Kieser et al., 2000) were used for the routine cultivation of Streptomyces strains. Spore suspensions of Streptomyces strains were prepared as previously described (Kieser et al., 2000). DNA was extracted from Streptomyces cultures grown in CRM medium (Pigac and Schrempf, 1995) or YEME medium (Kieser et al., 2000) for 24–48 h at 30°C. Total genomic DNA was extracted using a modification of the procedure of Rao et al. (1987).

Cosmid walking, DNA sequencing, contig assembly and ORF annotation

We constructed a S. turgidiscabies Car8 cosmid library in Escherichia coli DH5∝ using the cosmid vector pOJ446 (Bierman et al., 1992). Cosmids containing either nec1 or nos virulence loci were [α-32P]-dCTP-labelled and used to screen the cosmid library using colony hybridization (Sambrook et al., 1989). Cosmids were isolated using a Qiaprep® spin miniprep kit (Qiagen), digested using PstI and BamHI, resolved by horizontal agarose gel electrophoresis and physically mapped to the cosmid probe using Southern blot hybridization. The physical chromosomal location of each overlapping cosmid was confirmed by hybridization of [α-32P]-dCTP-labelled cosmids to a PFGE membrane containing an AseI digest of S. turgidiscabies. Overlapping cosmids were subsequently used to chromosome walk bidirectionally away from the nec1 and nos loci. Cosmids were mutagenized for DNA sequencing using the EZ::TN <Kan-2> Insertion kit (Epicentre) as described by the manufacturer. DNA sequencing was performed using the Applied Biosystems Automated 3700 DNA Analyzer (Bioresource Center, Cornell University). Sequence data were assembled into contigs using SeqMan II 5.03 (DNAstar, Madison, WI) and Vector NTI Suite 8.0 (InforMax, Frederick, MD). Putative ORFs were identified using Frameplot 2.3.2 (Ishikawa and Hotta, 1999) and protein-coding regions were identified by database similarity search using blast (National Center for Biotechnology Information). These sequence data have been submitted to the GenBank database under Accession No. AY7079AY7083.

Insertion of a thiostrepton resistance marker on the PAI

A 4.0 kb DNA fragment containing nec1 was polymerase chain reaction (PCR) amplified from S. turgidiscabies Car8 DNA using primers TnpF (5′-CTATGCGGTGAGTTCGGTGG-3′) and ISR (5′-GTGATGCCGTAGAGGCTGAGG-3′). The PCR product was cloned in the TA cloning vector pCRTOPO (Invitrogen), excised as an EcoRI fragment, and cloned in the EcoRI site of pUC19 resulting in pUC1901. A 1.7 kb BamHI DNA fragment containing the tsr gene, encoding thiostrepton resistance, was isolated from pKK2007 and inserted in the BsmI site of pUC1901. The EcoRI fragment with a disrupted nec1 was then excised from pUC1901 and ligated to EcoRI-linearized pOJ260, resulting in pOJ81, which was subsequently propagated in E. coli ET12567 (Macneil et al., 1992) before  transformation  into S.  turgidiscabies  protoplasts using standard techniques (Kieser et al., 2000). After 20 h incubation  at  30°C,  regeneration  plates  were  overlaid  with  15 µg ml−1 thiostrepton (final concentration). ThioR transformants were recovered and counterselected on apramycin to recover thioR apraS strains in which nec1 was replaced by nec1::tsr via homologous recombination. Disruption of the nec1 gene was confirmed by Southern analysis of PstI-digested total genomic DNA.

Selection of transconjugant strains

Conjugal matings were carried out using S. turgidiscabies Car811 (nec1::tsr) (thioR) as a PAI donor strain and either naturally chloramphenicol-resistant (camR) S. coelicolor or streptomycin-resistant (strR) S. diastatochromogenes as a recipient. Strains were co-cultured on R2YE (Kieser et al., 2000) for 2 days at 28°C. A soft nutrient agar overlay containing 15 µg ml−1 thiostrepton and 20 µg ml−1 chloramphenicol or 10 µg ml−1 streptomycin (final concentration) was used to select for thioRcamR or thioRstrR transconjugants. Frequency of PAI transfer was estimated by replicate plating (n = 3) ratios of donor:recipient spores from 1:100 1:10, 1:1, 10:1 and 100:1 (approximately 106−108 cfu per 80 mm Petri dish) on R2YE medium.

Pulsed-field gel electrophoresis

PFGE was used to separate large DNA fragments. Mycelial plugs of Streptomyces species were prepared following a standard protocol (Kieser et al., 1992). Restriction endonuclease digests were performed for 6 h using AseI (New England Biolabs). A CHEF DR® III system (Bio-Rad) was used to resolve plug digests using Hepes-based buffers (Evans and Dyson, 1993) using a 3.9 V cm−1 voltage gradient and switch times of 60–120 s for 24 h. Southern blot hybridization was performed by alkaline transfer of DNA to Hybond-N nylon membranes (Amersham) using the manufacturer's protocol. [α-32P]-dCTP-labelled DNA probes were generated using the Prime-It II® labelling kit (Stratagene). Pre-hybridization and hybridization were performed at 65°C in Hyb-9® hybridization solution (Gentra Systems). The most stringent wash was 0.2× SSC and 0.5% SDS for 20 min at 65°C. Autoradiography was performed with X-OMAT AR X-ray film (Eastman Kodak) at −80°C.

Phytotoxin and pathogenicity assays

Streptomyces strains were cultured in oat bran broth (OBB) (Goyer et al., 1998), a medium known to induce production of thaxtomin in plant pathogenic species. Thaxtomin was extracted from OBB and quantitatively evaluated using high-performance liquid chromatography (HPLC) (Healy et al., 2000). The pathogenic phenotype of strains was evaluated using a potato tuber slice assay (Loria et al., 1995). Strains were cultured on oat bran agar medium, after which agar plugs of inoculum were inverted onto potato slices (cultivar Chippewa) and incubated in the dark at room temperature for 10 days.

Acknowledgements

Streptomyces coelicolor M145 was kindly provided by the John Innes Centre (Norwich, UK). This work was funded by the National Science Foundation (Grant No. MCB-0080326) and by the United States Department of Agriculture National Research Initiative, Biology of Plant–Microbe Associations Program (Grant No. 2002-35319-12633).

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