Coevolution of antibiotic production andcounter-resistance in soil bacteriaemi_2125 1..15

Paris Laskaris,1 Sahar Tolba,1,2 Leo Calvo-Bado1

and Liz Wellington1*1Department of Biological Sciences, University ofWarwick, Coventry, UK.2Microbiology department, Ain Shams University, ••, UK.

Summary

We present evidence for the coexistence and coevo-lution of antibiotic resistance and biosynthesis genesin soil bacteria. The distribution of the streptomycin(strA) and viomycin (vph) resistance genes wasexamined in Streptomyces isolates. strA and vphwere found either within a biosynthetic gene clusteror independently. Streptomyces griseus strainspossessing the streptomycin cluster formed part ofa clonal complex. All S. griseus strains possessingsolely strA belonged to two clades; both were closelyrelated to the streptomycin producers. Other moredistantly related S. griseus strains did not containstrA. S. griseus strains with only vph also formed twoclades, but they were more distantly related to theproducers and to one another. The expression of thestrA gene was constitutive in a resistance-only strainwhereas streptomycin producers showed peak strAexpression in late log phase that correlates with theswitch on of streptomycin biosynthesis. While thereis evidence that antibiotics have diverse roles innature, our data clearly support the coevolution ofresistance in the presence of antibiotic biosyntheticcapability within closely related soil dwelling bacteria.This reinforces the view that, for some antibiotics atleast, the primary role is one of antibiosis during com-petition in soil for resources.

Introduction

Antibiotic biosynthetic gene clusters are widespread insoil bacteria and Streptomyces species in particular(Watve et al., 2001). However, the detection of naturallyproduced antibiotics within soil has met with limitedsuccess (Morningstar et al., 2006). This is due both tonutrient limitations placing constraints on bacterial growth

and secondary metabolite production (Anukool et al.,2004) and because many antibiotics strongly adsorb ontosoil or clay particles making them difficult to extract anddetect (Sarmah et al., 2006). A study following the fate oftetracycline and tylosin added to two soil types provedthat these antibiotics remain biologically active even whentightly bound to soil particles (Chander et al., 2005). Anti-biotics can therefore retain their bactericidal propertieswhether free or adsorbed and are thus biologically rel-evant in soil antagonism even when they cannot bedetected. This has important implications for effluent con-taminated with high levels of antibiotics (Larsson et al.,2007). Antibiotic production by biocontrol bacteria haslong been implicated as an important characteristic in thecontrol of soil-borne plant pathogens. Haas and Keel(2003) reviewed the evidence for production in situ basedon chemical extraction and expression of antibiotic bio-synthesis genes. There was convincing evidence for theproduction by pseudomonads of antifungal antibioticsincluding phenazines, 2,4-diacetylphloroglucinol and pyo-luteorin (Haas and Keel, 2003). A number of Streptomy-ces species are also used for biocontrol (Raaijmakerset al., 2002) and can reduce the extent of infection causedby the potato pathogen Streptomyces scabies. This is dueat least in part to the antibiotics produced by the suppres-sive strains, as S. scabies mutants resistant to some ofthese antibiotics are able to readily infect potato tubers(Neeno-Eckwall et al., 2001). In another study, RT-PCRprovided indirect evidence for the production of strepto-thricin in soil by Streptomyces rochei although no antibi-otic could be extracted (Anukool et al., 2004).

An important indicator of antibiotic production in soil isthe widespread occurrence of antibiotic resistance in soilbacteria; a screen of 480 Streptomyces isolates (collectedfrom various environments) against 21 antibiotics showedthat all had resistance to one or more antibiotics (D’Costaet al., 2006). The organisms were on average resistant toseven to eight antibiotics and resistance to every com-pound used in the trial was demonstrated. It was recentlyreported that soil bacteria were able to subsist on antibi-otics and strains had levels of resistance ranging from 20to 1000 mg l-1 of antibiotic (Dantas et al., 2008). Thesestudies prove that soil bacteria can exhibit a resistancephenotype exceeding the levels of antibiotics found inboth serum of antibiotic-treated patients (MacGowan andWise, 2001) and in antibiotic-polluted soils (Sarmah et al.,

Received 10 August, 2009; accepted 29 October, 2009. *Forcorrespondence. E-mail e.m.h.wellington@warwick.ac.uk; Tel. (+44)(24) 7652 3184; Fax (+44) (24) 7652 3701.

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2006). Large numbers of bacteria resistant to gentamicin,kanamycin, streptomycin, chloramphenicol or tetracyclinehave been isolated from multiple Siberian permafrostsediments dating back three million years (Mindlin et al.,2008).

There is strong evidence that antibiotic use in agricul-ture has led to increased levels of resistance in terrestrialand aquatic environments, contributing to native resis-tance gene reservoirs (Vrints et al., 2007; Gaze et al.,2008; Byrne-Bailey et al., 2009). Soil bacterial antibioticresistance genes are selected for by indigenous produc-ers and external antibiotic inputs, and such genes can betransferred to potential pathogens (Riesenfeld et al.,2004). A number of opportunistic human pathogens areknown to reside in the rhizosphere, such as Burkholderia,Enterobacter, Pseudomonas and Staphylococcus (Berget al., 2005). Due to the ease with which horizontal genetransfer (HGT) can occur in the soil (Davison, 1999), andthe fact that resistance genes can be transferred betweenphylogenetically distant bacterial groups, it seems likelythat some of the resistance genes found in pathogenshave originated from antibiotic producers (Benveniste andDavies, 1973), whose resistance is necessary to avoidsuicide or in order to provide resistance against nichecompetitors. There are a number of homologous genesfound in pathogens and antibiotic producers. Homologuesto the otrA and otrB tetracycline resistance genes presentin tetracycline producer Streptomyces rimosus have beenfound in Mycobacterium fortuitum (Pang et al., 1994). Asstreptomycete plasmids can be acquired by Mycobacte-rium species via natural transformation (Bhatt et al.,2002), it is likely that they were transferred from the pro-ducer. The vanH, A and X resistance genes found inEnterococcus species have orthologues in vancomycinproducers Streptomyces toyocaensis and Amycolatopsisorientalis (Marshall et al., 1998).

Members of the genus Streptomyces are prolific pro-ducers of antibiotics (Paradkar et al., 2003). While thishas been exploited commercially, few studies havefocused on the ecological importance of antibiotic produc-tion. Our aim was therefore to elucidate the relationshipbetween speciation and evolution of antibiotic biosynthe-sis. We targeted the Streptomyces griseus species thathas been the subject of previous studies (Huddlestonet al., 1997; Wiener et al., 1998; Egan et al., 2001; Tolbaet al., 2002) and asked the question ‘do all membersof this species have the capacity to biosynthesize strep-tomycin, have some lost it or have some just retainedresistance?’

Streptomycin resistance is ubiquitous in soil and widelydistributed globally (Huddleston et al., 1997; Sevenoet al., 2002), due to the widespread presence of strepto-mycin in the environment; it is estimated that 1% of soilactinobacteria can synthesize streptomycin (Sm) (Baltz,

2006). The streptomycin resistance gene (strA) has alsobeen mobilized between streptomycetes in soil (Tolbaet al., 2002). We hypothesize that closely related S.griseus strains are likely to be niche competitors andwould either be Sm producers or solely resistant strains.We report here a detailed study of the distribution anddiversity of strA, coding for an aminoglycoside phospho-transferase [APH(6′)], and the biosynthetic gene strW inS. griseus with multi-locus sequence typing to establishstrain phylogenies. The chromosomal location of strA wasinvestigated in two soil isolates for evidence of genemobility, and preliminary expression analysis was done.The distribution within S. griseus of a further gene clusterfor viomycin (Vm) biosynthesis, producing an RNA-binding peptide antibiotic that inhibits prokaryotic proteinsynthesis and group I intron self-splicing, and its resis-tance gene (vph) was studied to compare their evolution-ary history with that of the str cluster and strA resistancegene.

Results

Delimitation of species and species groups

A tree was constructed using the partial concatenatedsequences of eight housekeeping genes (Fig. 1). Themulti-locus sequence analysis (MLSA) provided well-defined clades identified as species for S. griseus, S.violaceoruber and S. limosus, with improved phylogeneticresolution compared with the 16S tree (Fig. S1). Isolatesrecovered in the S. limosus clade had a very similarphenotype to S. griseus and were included in this study onthis basis but were clearly members of the S. limosusspecies. Pairwise analysis of the housekeeper sequencesindicated they shared a similar evolutionary history, asevidenced by high correlation coefficients (Fig. S2). TheS. griseus clade was more diverse in comparison withthe S. violaceoruber and S. limosus clades. The MLSA,however, did not resolve interspecies relationships andindividual housekeeper gene phylogenies differed atthe species group level (Figs. S3–10). There was oneinstance of recombination among the housekeeper genesexamined, where S. griseus DSM 40932 clustered with S.bacillaris DSM 40598 in all trees except for rplC, where itwas recovered with the S. griseus type strain (DSM40236) (Fig. S7). The GARD algorithm identified onerecombination breakpoint in atpD, rplC and rpoB and twoin 16S, indicating that parts of these genes have under-gone recombination (P < 0.01).

Strain DSM 40931 is listed as belonging to the S.griseus group; however, the 16S sequence fromGenBank, and the sequence we obtained, showed only a95% sequence identity to that of S. griseus DSM 40236. Ithas a 99% sequence identity to S. albus NBRC 3711,indicating that this strain was misidentified.

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Fig. 1. Phylogeny of strains derived using the concatenation of seven partial housekeeper gene sequences. Clades containing the strA andvph resistance genes are marked. The CW, DW, RB and E strains were isolated from UK soils, CB from Cuban soils, AR and CR fromGerman soils, Z34 from Zambian soils and 650 and 666 from Brazilian soils.

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Discovery of two distinct strA and twodistinct vph phylogroups

All strA sequences fell into two distinct clades (Fig. 2);Group A were from Sm producers and Group B were fromSm resistance-only strains. Strains representing GroupsA and B were screened for Sm production via a bioassaywith isogenic strains of Escherichia coli and a competitiveenzyme immunoassay. The vph sequences likewiseformed Group 1 from Vm producers and Group 2 fromresistance-only strains (Fig. 3). The Sm producers formeda clonal complex (Fig. S11) and also had invariant strAgenes, although strain Z34 had one SNP difference instrA. The Vm producers were more diverse and their vphsequences had multiple SNPs. The only Sm producer thatdid not form part of a clonal complex was DSM 40654,although it was still part of the same clade as the otherproducers and had an identical strA sequence. One addi-tional strain, CR50 identified as a S. platensis species,contained a more diverse str cluster with an 84%sequence homology to the S. griseus DSM 40236 cluster,but provided a clear case of HGT. The phylogeny of thisstrain, based on eight housekeeper genes and the 16Sgene (Fig. 1 and Fig. S1) was incongruent with both strA

and strW. Group B and Group 2 contained two subgroups,with sequences considerably more diverse than Group Aand Group 1. Group B sequences had 73.0–77.0% nucle-otide sequence identity to their Group A homologues,while Group 2 strains had a 76.5–80.5% sequence iden-tity to Group 1. These subgroups were also recovered inthe housekeeper tree (Fig. 1), indicating vertical ratherthan horizontal descent being the norm for both strA andvph (the concatenated housekeeper gene sequences hada congruence of r = 0.913 with the strA sequences andr = 0.991 with the vph sequences). Within S. griseus, onlymembers of the clade comprising Groups A and B pos-sessed strA and this gene was found in all isolatesidentifying with this clade. Conversely vph had a discon-tinuous distribution, being present in three separateclades of the S. griseus species group. The strW biosyn-thetic gene tree (Fig. S12) showed significant similarity tothe strA tree (pairwise analysis r = 0.997) demonstratingthat the evolutionary history of at least one of the biosyn-thetic genes is the same as for the resistance gene inproducers. The phylogenetic distances of the vioB andvioD biosynthetic genes and the vph resistance genewere more divergent (pairwise analysis r = 0.738 vioBto vioD, r = 0.740 vioB to vph, r = 0.652 vioD to vph).

Fig. 2. The phylogeny of the Sm resistance gene strA, revealing two major clades; one associated with production (A) and one withresistance (B).

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GARD identified one recombination breakpoint in strA(P < 0.01) and strW (P < 0.05) and none in vph, vioBand vioD.

The ratio of resistant to non-resistant strains based onthe groupings from the concatenated tree revealed thatmembers of Group A and Group 1 uniformly showed highlevels of resistance (growth at 50 mg ml-1) (Fig. S13). Incontrast, Group B had mixed resistance phenotypes,including some sensitive strains and Group 2 has mostlyhighly resistant strains and two with low-level resistance(growth at 10 mg ml-1). All the remaining S. griseus strainsthat lacked strA were recovered in Group C and thoselacking vph in Group 3. A small proportion of Group C(23.5%) and Group 3 (37.5%) had predominantly low-level resistance of unknown origin (the Sm resistance wasnot ribosomal or due to aphE). The majority of S. limosusstrains were sensitive (68.4%) to streptomycin and allwere sensitive to viomycin, although some strains dem-onstrated high resistance (31.6%) to streptomycin. All S.

violaceoruber strains were sensitive to both streptomycinand viomycin, with the exception of M110, which had beendeliberately engineered to be highly resistant to strepto-mycin via a ribosomal point mutation. The number ofstrains resistant to streptomycin belonging to Group Bwas significantly higher (c2 test P = 0.0316) than thatfound in Group C, as was that between Group 2 andGroup 3 (c2 test P < 0.0001). There was no significantdifference between Group B and S. limosus strains(P = 0.0880) or between Group C and the S. limosusstrains (P = 0.8658). Of the other strains possessing strAhomologues S. humidus DSM 40263, S. galbus DSM40480 and S. cinnamoneus DSM 40005 showed no resis-tance to Sm, unlike S. glaucescens DSM 40155 and S.mashuensis DSM 40221 that had low-level resistanceand S. netropsis DSM 40093 that had high S. platensisDSM 40041, which lacks an strA gene, was sensitive toSm, but S. platensis CR50, which possesses an strcluster, had low-level resistance.

Fig. 3. Vm resistance gene vph phylogeny, revealing two major clades separating resistance-only strains from producers.1

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Rate of strA evolution compared withhousekeeper genes

The non-synonymous (Ks) to synonymous (Ka) nucle-otide substitution ratios of housekeeper genes and hopB,a gene outside of the core region of the chromosome(Bentley et al., 2002), from Group A and Group B, werecompared (Fig. 4). All the genes in Group A gave a ratio ofzero as none of them showed any non-synonymous sub-stitutions. In contrast, the majority of genes from Group Bhad substitution ratios greater than zero due to the moreancient divergence of their members and strA was clearlythe least conserved gene in the group. The vph gene wasalso significantly more diverged than the housekeepergenes in both Group 1 and Group 2 strains, with theGroup 2 gene appearing slightly but not significantly morediverse (data not shown). The two Vm biosynthetic genesalso had a slightly but not significantly higher divergenceto the housekeeper genes.

Analysis of strA divergence

The amino acid sequences of strA from Group B strainshad an average of 70.0% homology compared with thatfrom Group A. However, the two of the three active sitespresent in the translated protein sequence obtained in thisstudy showed relative conservation. The APH universalcatalytic site (HWDLHYEN in Group A) only had oneamino acid difference; the glutamic acid (E) has beenreplaced either by aspartic acid (D) or by glycine (G).Aspartic acid has very similar physico-chemical charac-teristics to glutamic acid; however, glycine is non-polarand has a neutral rather than positive side-chain charge.The second conserved motif is a glycine-rich flexible loop,

which is involved with Mg2+ binding (GDPGFDLWP inproducers) and was completely conserved in all non-producing strains except for AR23, where the secondproline (P) had been replaced with a serine (S). Thephysico-chemical characteristics of these two residuesdiffer; while both have neutral side-chains, serine is polarand proline is non-polar.

Chromosomal location of strA and the Sm cluster

The str cluster of S. griseus NBRC 13350 (Ohnishi et al.,2008) had a different chromosomal location from the strAgene in S. griseus AR23. There was no evidence forretention of str vestigial or pseudogenes (Fig. 5A and B).The flanking regions of the str cluster in S. platensis CR50showed no similarity to those in either the S. griseus typestrain DSM 40236 or AR23.

The strA gene in AR23 was highly divergent (75%sequence homology to Group A strA). The non-sequentialposition of neighbouring ORFs SGR_172 and SGR_400compared with the genome sequence of S. griseus indi-cated that there had been multiple recombination eventsin the flanking regions of strA. The strA gene in AR23 wasfound 1.85 Mb away from the location of the Sm cluster inthe S. griseus NBRC 13350 genome. BPROM identifiedthree putative promoter regions, two of which werelocated within SGR_4326 and SGR_400, indicating thatthey are unlikely to be functional; however, the promoterlocated at the start of the SGR_4326 homologue may beactive. No homologues to CDS3 were found. In the S.griseus type strain expression of strA commences mainlyat the transition between exponential and stationaryphases, once vegetative growth has ceased. Conversely,

Fig. 4. Comparison of substitution rate inhousekeeper and secondary metabolitegenes in Group B strains (S. griseus strainspossessing only strA). The ratio was 0 for allGroup A genes (S. griseus strains containingthe entire str cluster).

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strA expression in AR23 was constitutive with transcriptdetected in the exponential phase (Table 1).

In CR50 the str cluster genes were found in the sameorder and had homologous intergenic regions to those ofthe S. griseus type strain, indicating that the promoterregions were also conserved (Fig. 5C). The averagesequence homology for the str genes was 85% andranged from 80% (strR) to 91% (strF). The CR50 strA hadan 81% sequence homology to the S. griseus type strainstrA; however, the strB2 gene (encoding a scyllo-inosamine-4-phosphate amidinotransferase) was partiallydeleted. Only a 204 bp fragment from the 3′ end of thegene (out of a total of 1053 bp) was present where strB2was located in S. griseus. Only one of the genes(SGR_5941) flanking the str cluster in S. griseus NBRC

13350 was present in CR50. SGR_5941 was previouslytermed strZ but has no known function. There was 76%sequence homology between the two homologues and inS. griseus SGR_5941 is located on the 5′ end of strT,whereas in CR50 a gene has been inserted between strTand the SGR_5941 homologue. This inserted gene hadlow homology (68%) to a family 39 glycosyl transferasefrom Saccharopolyspora erythraea NRRL2338. Otherthan these two genes, no further significant homologieswere found in the flanking regions. A region homologousto a sequence flanking a putative truncated transposasewas on the 5′ end of the streptomycin cluster and aregion homologous to a putative IS110 transposase/integrase was on the 3′ end of the CR50 cluster. Thetransposase/integrase on the 3′ end has been partiallydeleted and the transposase on the 5′ end has beencompletely lost leaving only its flanking region behind;however, these remnants strongly suggest that thecluster arrived at that location via transposition. Therewere a number of terminal inverted repeats and plasmidsequences in this region, suggesting that there havebeen many additional instances of mobilization. It was notpossible to map the str cluster location on the chromo-some of CR50, as these flanking regions were radicallydifferent from all sequenced genomes. The str cluster inS. griseus NBRC 13350 is located in the core region

Fig. 5. The chromosomal location and structure of cloned str clusters.A. Comparison of positions of str cluster (striped), strA (black) and their flanking regions (dotted and grey respectively) on the genomes ofS. coelicolor A3(2), S. griseus NBRC 13350 and S. griseus AR23. Not drawn to scale; Mb positions based on the NBRC 13350 genome.B. Position of strA and its flanking regions in non-Sm producer strain S. griseus AR23.C. Comparison of str cluster in S. platensis CR50 (top) and S. griseus NBRC 13350 (bottom).

Table 1. Approximate transcript levels of strA in the AR23 and DSM40236 S. griseus strains.

Hours AR23 strA DSM 40236 strA

8 + -10 + -14 + ++20 ++ ++24 + ++++36 + -48 + -

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close to boundary with the right arm (Ohnishi et al.,2008). If the SGR_4330 and SGR_4331 homologues inAR23 have the same position as in the S. griseus typestrain, then the AR23 strA will also be located in the coreregion of the chromosome. Other diverse type speciesincluded in the strA tree (Fig. 2) were reported to bestreptomycin producers (DSMZ, 2004) but clearly showedsignificant divergence.

Alternative streptomycin resistance mechanisms

A large portion of the rpsL gene encoding the S12 ribo-somal protein was amplified and sequenced for all thestrains used in this study. The nucleotide sequence wastranslated into amino acids and examined for mutationsthat might confer streptomycin resistance. Lys-43 wasreplaced with an Asn in M110 and a Thr in CB148, Lys-88was replaced with an Arg in CB141 and an additional Argwas inserted between Gly-85 and Arg-86 in CB158. Themutations at positions 43 and 88 have previously beenfound to confer Sm resistance (Musser, 1995), whichexplains why M110, CB148 and CB141 all showed high-level resistance to Sm. An insertion between positions 85and 86 has not been described before and is not known toconfer Sm resistance, but CB158 also possesses a copyof strA and has high-level Sm resistance. The 530 loopregion on the 16S rRNA gene was also examined formutations, as most 16S mutations resulting in Sm resis-tance occur at that site (Springer et al., 2001); however,none were found in any of the strains included in thisstudy. The distribution of aphE, a 3′ aminoglycoside phos-photranspherase conferring resistance to streptomycin,was also determined via amplification and sequencing. Itwas detected in all strains belonging to Group A andGroup B1, with the exception of the DW15 strain fromGroup B1, which lacked the gene, and S. olivoviridis DSM40211, which possessed an aphE homologue that failedto grant Sm resistance.

Discussion

The MLSA analysis provided clear evidence of the con-servation and stability of the genes found within the corechromosome as defined by Bentley et al. (2002). Selectedgenes associated with secondary metabolism such asstrA have incongruent phylogenetic history due to HGT,although highly stable conserved genes such as hopB(this study) and chiF (Ul-Hassan, 2007) also occur in thearm regions. The arm regions showed a high level ofrearrangement and deletions in synteny comparisons, inaddition to the presence of transposases and insertionsequence elements (Ikeda et al., 2003; Choulet et al.,2006). The evidence for recombination in four out of ninehousekeeper genes confirms that streptomycetes fre-

quently undergo recombination in soil (Wellington et al.,1992). The presence of two breakpoints in the 16Ssequence supports the hypothesis that it can undergorecombination and can therefore potentially generate mis-leading phylogenetic trees (Acinas et al., 2004). Concat-enated gene trees buffer distortions on a phylogenygenerated by recombination (Hanage et al., 2005). Weobserved only one clear instance of housekeeper geneHGT (rplC) indicating that while core genes can undergorecombination, as noted in other studies (Daubin andLerat, 2006), it is generally rare. Based on the soil isolatesused in this study, we conclude that strains in the Strep-tomyces genus form clusters with high bootstrap valuesthat match the definition of a species cluster (Hanageet al., 2005) rather than a continuous spectrum of geno-typic variation (Gevers et al., 2005). The uncertain statusof interspecies relationships found in this study was alsoevident in other MLSA studies of streptomycetes (Antony-Babu et al., 2008; Guo et al., 2008). The HGT betweenthe ancestors of the species groups may have resulted inthe phylogenetic signal being lost. This, however, runscounter with our findings from the pairwise analysis, whichindicates that all the housekeeper genes have similarphylogenetic histories. Another possible explanation isthat the ancestors of all these species groups diverged ina very brief period and thus a phylogenetic tree cannotresolve the exact pattern of divergence. The S. griseusformed a robust species containing many synonyms andclearly had a global representation.

Within the S. griseus species we obtained evidenceproving that the str resistance gene shares a commonancestor with the same gene in the str cluster and thatthese have clearly evolved in parallel in soil, demonstrat-ing the importance of antibiotic production for antagonismin nature. There are a number of possible hypotheses forhow coevolution of resistance and production occurredwithin closely related S. griseus strains. The ancestralstr-like gene cluster must have spread and diversifiedearly on in its evolution, as evidenced by the presence ofrelated clusters in the different species S. galbus, S.glaucescens, S. humidus, S. cinnamoneus, S. mashuen-sis and S. netropsis. The str cluster is estimated to haveoriginated approximately 610 million years ago (Baltz,2005) and HGT is implicated in its evolution, as S. griseusNBRC 13350 appears to have acquired it from anotherstrain (Egan et al., 1998; Tomono et al., 2005). Our find-ings of sequences associated with transposases flankingthe str cluster in CR50 reinforce that hypothesis andsuggest that the absence of these sequences in S.griseus NBRC 13350 is because they have been com-pletely lost. The different location of the str cluster in thechromosome of S. platensis CR50 also indicates that itarrived there through a different gene transfer event com-pared with S. griseus producers. The biosynthetic clusters

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in S. cinnamoneus, S. mashuensis and S. galbus, report-edly Sm producers, were highly divergent indicating par-allel evolution towards production of the same compound,further substantiating the adaptive importance of strepto-mycin production.

There are two possible explanations for the origin of theresistance gene in the non-Sm producing S. griseusstrains. One is that the common ancestor of the producersand resistance-only S. griseus strains lacked a biosyn-thetic cluster and the non-producers acquired their resis-tance gene from another species with a related cluster viaHGT and the common ancestor of the producers similarlyacquired the entire str cluster independently. This transfercould have consisted solely of the resistance gene or ofan entire or partial cluster that decayed, leaving onlystrA present. The independent acquisition of strA bythe resistance-only strains is supported by the highersequence identity of the resistance-only strA to the resis-tance genes of the spectinomycin (79–83%) and hydrox-ystreptomycin (81–83%) clusters compared with that ofthe streptomycin cluster (73–77%). The strA phylogenetictree also places the spectinomycin and hydroxystrepto-mycin resistance genes in a single clade alongside theresistance-only strA sequences; however, their bootstrapvalues are low, which indicates that this may not be a realclade. The alternative and possibly more parsimonioushypothesis for the origin of these genes is that thecommon ancestor of the resistant strains and the Smproducers possessed an str cluster but that the biosyn-thetic and regulatory genes were lost in the resistantstrains and strA arrived at a different location in the chro-mosome via recombination. The advantage of this hypoth-esis is that it only needs to posit one HGT event, that ofthe original cluster to the ancestor of all the S. griseusstrains possessing strA, rather than one for the bio-synthetic cluster and one or two such events for theresistance gene. The two Sm resistance-only cladessuccessively branch out from the clade containing theproducers; however, as the relevant bootstrap values arelow they may form part of a single clade that underwent aHGT event to acquire strA and then rapidly diverged intotwo subgroups. However, the concatenated housekeepergenes displayed a 97% sequence identity between S.griseus NBRC 13350 and AR23, while strA had only 75%.Such a level of divergence in one gene only pointstowards the independent acquisition of strA in Groups Aand B. Regarding the vph homologue found in Group 2strains, it is more parsimonious to posit that it arrived tothe clades containing it via HGT, as at least four indepen-dent deletions of the gene would have been required for itto be absent from the remaining S. griseus clades if it hadbeen present in their common ancestor. We cannot becertain whether that transfer included an entire or partialVm cluster; however, the vph homologue (SGR_421) in

S. griseus NBRC 13350 is not flanked by sequenceshomologous to the Vm cluster, indicating that if they weretransferred alongside the resistance gene these geneshave since been lost.

Both Sm producers and resistance-only strains arefound in similar locations worldwide. The fact that almostall the Sm producers form a clonal group demonstratesthat the streptomycin production phenotype is highlyadvantageous, as it has enabled the global radiation ofthis clonal group marking them as highly successful soilcolonists. The predominance of the S. griseus clonalgroup among streptomycin producers may be because itsporulates very readily and in large amounts, doing soeven in liquid culture (Kendrick and Ensign, 1983). TheSm resistance-only group is also widespread; however, itis more diverse than producers, being split into two sub-groups, which in turn are more distantly related to oneanother. The resistance gene in Sm producers is highlyconserved, as it is necessary for the cell to avoid suicide.In non-producers it is more divergent, as a high level ofSm resistance is not essential for these organisms,enabling them to tolerate a decrease in the efficiency ofthe enzyme as Sm levels in soil are probably low. Cur-rently no particular pattern of amino acid changes couldbe identified as having a definitive correlation with resis-tance phenotype. This will require cloning and expressionof the genes in a different host without the promoters. Asimilar pattern of coevolution of resistance and productionwas identified for viomycin within S. griseus, where diver-sity of the resistance gene, vph, was greater among non-producers. The diversification of the resistance gene mayalso be driven by the organism’s exposure to other relatedantibiotics, which have selected for an enzyme that canprovide resistance at a lower efficiency but against awider spectrum of antibiotics. The strA and vph genes innon-producers may have unknown substrates other thanantibiotics that provide evolutionary pressure for retentionof the genes; however, the statistically significant correla-tion between possession of the resistance genes and Smor Vm resistance suggests that they do play a role inproviding resistance. There was also some evidence forSm resistance in the absence of strA, which was not dueto ribosomal mutations and may indicate the presence ofother more diverse APH enzymes or of mutations to thersmG gene, encoding a 16S rRNA methyltransferase,which can result in low-level streptomycin resistance(Nishimura et al., 2007). Strains resistant to viomycin butlacking vph homologues were also isolated, highlightingthe fact that bacteria can develop multiple methods ofneutralizing antibiotics present in their environment.Clearly the constitutive expression of strA in non-producers also supports the presence of Sm in the soiland its role in competition. Nine strains of S. griseus inGroup B that had strA but were sensitive may express the

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gene under different conditions. Further studies areneeded to fully elucidate the key promoter for strA inGroup B strains.

In S. griseus NBRC 13350, Sm production is regulatedby the A factor cascade (Onaka et al., 1995). AfsA syn-thesizes A-factor in a growth-dependent manner. WhenA-factor reaches a critical level during the transitionphase, it binds to the repressor ArpA and dissociates itfrom the promoter of adpA. AdpA triggers the transcriptionof strR and strA, with StrR acting as a transcriptionalactivator for the biosynthetic genes (Horinouchi, 2007).The completely different expression pattern in AR23(Table 1) and absence of strR indicates a different regu-latory mechanism. As strA is mainly transcribed due toreadthrough from the strR gene that is located upstreamof it in producers (Tomono et al., 2005), strA in AR23 hasto be transcribed in a different manner.

In conclusion, streptomycetes form a substantial res-ervoir of resistance genes in soil; clearly antibiosis is animportant driver for their evolution in addition to theavoidance of suicide in producers. This is illustrated byour own research on Sm, but the presence of a numberof other resistance-only genes in streptomycetes hasalso been demonstrated (Pang et al., 1994; Hong et al.,2004). Streptomycetes have never been subjected toselection for high-level resistance required for survivalduring exposure to clinical antibiotic chemotherapy.Therefore, the importance of this phenotype that is pre-served within this group must be due to competition withclosely related strains that are capable of producing theantibiotic in soil. The natural role for antibiotics andother bioactive secondary metabolites cannot fully beexplained by antagonism as such a diversity of biologicalactivity exists (Vining, 1990; Demain and Fang, 2000).Other roles may involve a contribution to survival such assensing the external environment and cell–cell signalling(Price-Whelan et al., 2006; Martinez, 2008). There issome preliminary evidence that Sm may play a dual rolein nature, as it has been detected bound to a cell wallprecursor unit (Szabo et al., 1989) where it activated lyticenzymes in the cell wall (Szabo et al., 1990). If strepto-mycin played a key role in regulating growth and pepti-doglycan formation, the widespread occurrence of thismetabolite would be expected. In addition, the discon-tinuous distribution of the biosynthetic gene cluster withineven the species S. griseus strongly indicates nichespecialization and adaptation. Our data prove thatsome close relatives of producers have retained theresistance gene. More distantly related S. griseus strainsprobably never acquired the str cluster. The most parsi-monious route of origin we believe is that a member ofthe species acquired the cluster by HGT and thendiverged into two groups, one of which retained only theresistance gene.

Experimental procedures

Selection of strains for study

A total of 57 strains of S. griseus [as defined in this study bythe possession of > 97% 16S rRNA sequence homology tothe S. griseus ssp. griseus (Krainsky 1914; Waksman andHenrici 1948) type strain, DSM 40236] were studied; 34 wereselected from culture collections and 23 were isolates fromcollections of strains (512 total) retained from previousstudies of soils from Brazil (Huddleston et al., 1997), Cuba(Heuer et al., 1997), Europe (Tolba et al., 2002) and Zambia(Bradbear et al., 2004). In addition, soil isolates werescreened for the S. griseus phenotype and a subsample ofthese (39) were selected and included in this study. The S.violaceoruber species group was included that containsStreptomyces coelicolor A3(2), a well-characterized speciesnot known to contain an str gene cluster nor Sm resistancephenotype. Other type strains included representativesknown to produce Sm and related compounds together withS. platensis and a related soil isolate that had acquired the strgene cluster (Tolba et al., 2002). Sm and related compoundsproducers were as follows: S. cinnamoneus, S. mashuensis,S. galbus: streptomycin; S. glaucescens, hydroxystreptomy-cin; S. humidus, dihydrostreptomycin; S. netropsis, strepto-thricin. A total of 106 strains were included in this study. Allstrains were maintained as spore suspensions at 20% (v/v)glycerol held at -20°C. Strains were screened for growth at30°C on nutrient agar containing 0, 10 and 50 mg ml-1 strep-tomycin, which was recorded at 24 and 72 h after inoculation.

Detection of Sm production

Strains were grown at 30°C for 5 days and 25 ml of culturesupernatant was loaded on 6-mm-diameter Whatman paperdiscs and air-dried for 20 min. The discs were then placed onthe surface of soft nutrient agar inoculated with an overnightculture of approximately 107 cfu of E. coli ATCC 29842(streptomycin-resistant strain) or ATCC 29839 (isogenic sen-sitive strain). Plates were kept at 4°C for 1 h then incubatedovernight at 37°C and examined for zones of clearance. Smproduction was confirmed via a competitive enzyme immu-noassay using the Streptomycin EIA kit (Adgen DiagnosticSystems, Scotland) as per the manufacturer’s instructions.

DNA extraction method

Cells were grown in 100 ml flasks (with a coil of stainlesssteel spring inside to disrupt bacterial clump formation andimprove aeration) containing 40 ml of Tryptic Soy Broth(Oxoid, Basingstoke, UK) for 3 days in a 30°C shaking incu-bator. Extraction was performed using the Kirby procedure(Kieser et al., 2000), modified by use of a Heavy Phase LockTube (Eppendorf, Westbury, NY, USA) for the second phenolextraction to ensure removal of all protein contamination.DNA quality was examined via spectrophotometry and gelelectrophoresis.

PCR and sequencing of str, vph, hopB andhousekeeping genes

Primers were designed by aligning all streptomycete versionsof a gene available in GenBank and then selecting two 20 bp

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regions that were conserved across all the sequences. If nosuitable sequences were found, then degenerate primerswere designed. The optimal annealing temperature wasdetermined via a temperature gradient PCR. Reaction mixeswere made with 25 ml PCR Master Mix (Promega, Madison,WI, USA), 2.5 ml DMSO, 100 pM of each primer in 50 mltotal volume. The cycling protocol used was the same forall primers with only the annealing temperature varying(Fig. S14): 10 min denaturing step followed by 35 cycles of60 s at 95°C, 45 s at temperature varying and 90 s at 72°Cfollowed by a final extension step for 10 min at 72°C. ThePCR products were run on a 1% agarose gel and the productbands were cut out and extracted using the QIAquick GelExtraction Kit (QIAGEN, Venlo, Netherlands) as per manu-facturer’s instructions. The product was dialysed for 15 minusing 0.025 mm VSWP nitrocellulose membranes (Millipore,Billerica, MA, USA) placed on sterile water. Sequencing wasperformed using 50 ng of PCR product, 5.5 pM of primer andthe BigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, Foster City, CA, USA) on an ABI PRISM 3130xlGenetic Analyzer as per manufacturer’s instructions. Both theforward and reverse PCR primers were used for sequencingto ensure that there were no sequencing errors.

Cloning of the str genes and flanking regions inS. griseus isolate AR23 and S. platensis isolate CR50

High-molecular-weight genomic DNA was extracted using theCTAB protocol (William and Feil, 2004). A fosmid library wascreated using the CopyControl Fosmid Library Production Kit(Epicentre Biotechnologies, Madison, WI, USA) as per manu-facturer’s instructions. The E. coli cells containing fosmidwere screened by whole colony PCR using strA, strW andstrT primers. The FosmidMAX DNA Purification Kit (EpicentreBiotechnologies) was used to extract the fosmid. Two fosmidscontaining the str cluster and flanking regions from CR50were sequenced by the Sanger Institute (Wellcome TrustGenome Campus, Cambridge, UK). An strA-positive fosmidcloned from AR23 was partially sequenced via primer walkingand the accuracy of the sequence confirmed by designingPCR primers for the strA gene and flanking regions, followedby amplification and sequencing of these regions. Codingsequences (CDS) were identified using fgenesB (http://www.softberry.com), a Pattern/Markov chain-based bacterialoperon and gene prediction program and the BLASTN algo-rithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Putative pro-moters were identified using the promoter detection programBPROM (http://www.softberry.com).

RT-PCR for detection of strA transcripts

RNA was extracted using the RNeasy Mini kit (QIAGEN) afterapplication of RNeasy Protect solution (QIAGEN) in the bac-terial culture. The manufacturer’s procedure was modified bythe replacement of the vortexing step with a 30 s period on aribolyser and the addition of DNase to the eluted RNA solu-tion rather than in the extraction column. A two-step RT-PCRwas performed using the Omniscript kit (QIAGEN). Theamount of transcript was estimated by comparing the bandintensity to that of a DNA standard on a 1D gel image usingthe Total-Laboratory software.

Phylogenetic analysis and multi-locus sequence typing

The forward and reverse reads were aligned in SeqMan(DNASTAR, Madison, WI, USA) and checked for sequencingerrors. The resultant sequences were aligned using theMUSCLE algorithm in Jalview (Clamp et al., 2004). Nearestneighbour phylogenetic trees were then constructed usingthe Kimura-2-Parameter distance in PHYLIP (Felsenstein,2005). Bootstrap values were generated using 10 000repeats. An eBurst population snapshot was generated fromseven housekeeping genes (Feil et al., 2004) for detection ofclonal complexes. A tree based on concatenated partialhousekeeper gene sequences was derived from eight genes.The gene sequence from rpsL included the streptomycinresistance determining region for target mutations.

Statistical analysis to determine the correlation coefficient(pairwise analysis) between selected genes was done usingthe Kimura 2-parameter formula in PHYLIP and the correlationcoefficients were calculated using Microsoft Excel. The valueof omega non-synonymous (Ks) to synonymous (Ka) nucle-otide substitution ratio was obtained for each pair of strainsusing the yn00 application according to the method outlinedby Yang and Nielsen (2000) and the mean of each gene wasthen derived from those ratios. The GARD algorithm on theDatamonkey web server was used to detect recombinationbreakpoints on the housekeeper gene sequences (Pond andFrost, 2005).

Acknowledgements

We gratefully acknowledge financial support from the Bio-technology and Biological Sciences Research Council, UKand SysMO (Grant P-UK-01-11-3i), the Natural EnvironmentResearch Council (Grant NE/E004482/1) and the EgyptianGovernment (S.T.).

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. The phylogeny of the 16S gene rrn.Fig. S2. Pairwise comparison of housekeeper gene trees.Fig. S3. The phylogeny of the argininosuccinate lyase geneargH.Fig. S4. The phylogeny of the ATP synthase beta chain geneatpD.Fig. S5. The phylogeny of the elongation factor P gene efp.Fig. S6. The phylogeny of the phosphoenolpyruvate carboxy-lase gene ppc.Fig. S7. The phylogeny of the ribosomal protein L3 generplC.Fig. S8. The phylogeny of the DNA-directed RNA polymerasebeta subunit gene rpoB.Fig. S9. The phylogeny of the 30S ribosomal protein S12gene rpsL.Fig. S10. The phylogeny of the Fe-containing superoxidedismutase gene sodF.

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Fig. S11. Population snapshot of the S. griseus clade. GroupA strains are black, Group B grey and Group C white. Thenumbers next to some of the sites denotes the number ofstrains with the same allelic profile.Fig. S12. The phylogeny of the ABC-type transport genestrW.Fig. S13. Resistance levels of strains studied divided by phy-logroup. The pale grey column indicates high-level resistance

(50 mg ml-1), the deeper grey moderate level resistance(10 mg ml-1) and the dark grey sensitivity.Fig. S14. PCR primers used in this study.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.

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