Jana Kahánková,1 Roman Pantucek,1*Christiane Goerke,3 Vladislava Ružicková,1
Pavla Holochová2 and Jirí Doškar1
1Department of Genetics and Molecular Biology, 2CzechCollection of Microorganisms, Institute of ExperimentalBiology, Faculty of Science, Masaryk University, Brno,Czech Republic.3Institut für Medical, Mikrobiologie und Hygiene,Universitätsklinikum Tübingen, Tübingen, Germany.
Summary
Given the great biological importance and high diver-sity of temperate Staphylococcus aureus bacterioph-ages, a method is needed for the description of theirgenomic structure. Here we have updated a multiplexPCR strategy for the complex characterization of S.aureus phages of the family Siphoviridae. Based onthe comparative genomic analysis of the availablephage sequences, a multilocus PCR strategy fortyping the major modules of the phage genome wasdesigned. The genomic modules were classified onthe basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaCand dnaD (four types), dUTPase (four types), portalprotein (eight types), tail appendices (four types) andendolysin (four types) corresponding to the integraselocus, lysogeny control region, and modules for DNAreplication, transcription regulation, packaging, tailappendices and lysis respectively. The nine PCRassays designed for the above sequences wereshown to be capable to identify the bacteriophagegene pool present both in the phage and bacterialgenomes and their extensive mosaic structure. Theestablished multiplex PCR-based multilocus diagnos-tic scheme is convenient for rapid and reliable phageand prophage classification and for the study of bac-teriophage evolution.
Introduction
Staphylococcus aureus is an important opportunistichuman and veterinary pathogen with the capability ofcausing a wide range of local and systemic infection orfood poisoning. Much of the dissimilarity between patho-genic S. aureus strains is dependent on the presence ofvirulence factors encoded mainly by mobile genetic ele-ments. Microarray studies have shown that prophagesintegrated in the bacterial chromosomes are the mostwidespread mobile genetic elements in S. aureus strains(Witney et al., 2005), with most of them carrying betweenone and four prophages (Pantucek et al., 2004; Goerkeet al., 2009). The acquisition or loss of temperate bacte-riophages during the course of infection increases thegenome plasticity in host strains, thus facilitating theadaptation of the pathogen to various host conditions(Moore and Lindsay, 2001; Resch et al., 2005; Goerkeet al., 2006a,b). The bacteriophages that contain in theirgenome additional virulence genes located upstream ofthe right attachment site mediate positive lysogenic con-version of different virulence factors such as enterotoxin A(Betley and Mekalanos, 1985), Panton-Valentine leukoci-din (Kaneko et al., 1998), exfoliative toxin A (Yamaguchiet al., 2000) and immune evasion factors (van Wamelet al., 2006), or negative conversion of chromosomal viru-lence genes for b-haemolysin or lipase by interruptingupon insertion (Lee and Iandolo, 1986; Carroll et al.,1993) in S. aureus genome. Moreover, it is becomingincreasingly apparent that generalized transduction bybacteriophages is responsible for horizontal transfer ofgenes, but the distribution and frequency of such phagesare unknown.
Variation in prophage content of a lysogenic S. aureusstrain is apparent in two ways: (i) the prophage genomecan integrate into the host chromosome at least at 9different insertion sites (attB) that are recognized by thetype of the bacteriophage integrase (Lindsay, 2008;Rashel et al., 2008) and (ii) a remarkable feature of theseelements is their modular structure and mosaicism result-ing from recombination, horizontal transfer of sequencesand the selective pressure imposed by antiviral systems.The phage genes are usually organized into functionalmodules coding for lysogeny, DNA replication, regulation
of transcription, packaging, head and tail, tail appendicesand lysis (Iandolo et al., 2002). A genomic module foundin a phage can be replaced in another phage by asequence-unrelated module that frequently fulfils thesame or a related function (Botstein, 1980; Canchayaet al., 2003). Understanding the bacteriophage carriage istherefore of great importance not only to the determina-tion of the high genomic variability in S. aureus but also,from a global genomics perspective, to the elucidation ofthe evolution and virulence of bacterial strains.
The taxonomy of S. aureus siphophages is still beingupdated. Ackermann and DuBow (1987) recognized sixtentative phage species of S. aureus (3A, 11, 107, 77, 187and 2848A) classified into Siphoviridae based on capsidmorphology, serological properties (serogroups A, B, C, Fand L) and DNA homology. However, this taxonomic clas-sification scheme adopted also by ICTV is outdated andthe proper methodology for classification of bacterioph-ages exhibiting extensive genome mosaicism is dis-cussed (Nelson, 2004; Morgan and Pitts, 2008). Recently,a genome-based taxonomy for phages based on thephage proteomic tree using a complete amino acidsequence has been proposed (Rohwer and Edwards,2002). The proteomic tree for the Staphylococcussiphophage group correlates with the phage serogroups,probably because of the large proportion of morphogen-esis genes in the genome (Daniel et al., 2007). Anothercomparative genomics approach using dot-plot align-ments of whole phage genomes has classified the staphy-lococcal siphoviruses into two phage genera, Sfi21-likeand Sfi11-like, with several distinct structural head-tailgene modular types (Canchaya et al., 2003).
The nomenclature of S. aureus siphophages is incon-sistent. Phage and bacterial genome sequencing (Naritaet al., 2001; Iandolo et al., 2002; Matsuzaki et al., 2003;Kwan et al., 2005; Bae et al., 2006; García et al., 2009)and microarray studies of S. aureus strains (Lindsayet al., 2006) revealed a high diversity of prophages thatsomewhat confuses the classification. In the post-genomic age, Lindsay and Holden (2004) suggested clas-sifying bacteriophages into integrase gene families on thebasis of integrase gene homology. Recently, we havereported the first PCR-based molecular assays for thecharacterization of a subgroup of S. aureus phages andprophages that are members of the Siphoviridae familybased on morphogenesis (Pantucek et al., 2004) andintegrase genes (Goerke et al., 2009). However, theseassays did not cover mosaicism of the whole phagegenome.
In this study, we focused on the development, validationand application of a PCR-based assay for detailed char-acterization and identification of S. aureus bacterioph-ages based on the sequences of seven loci covering themain phage genome modules. This analysis enabled us to
identify several phage functional modular types and genevariants representative of each module. The establishedmultiplex PCR-based multilocus diagnostic scheme isconvenient for rapid and reliable classification of S.aureus siphophages including prophages.
Results
Classification of phage genomic modules and multiplexPCR assay design
To investigate bacteriophage variability and lysogeny of S.aureus in detail, we focused on designing a novel multi-plex PCR strategy for the characterization of the modularstructure of staphylococcal phages classified into thefamily Siphoviridae. Comparative genomic analysis ofmore than 66 complete phage or prophage genomes cur-rently available in the databases (Fig. 1) allowed the iden-tification of nucleotide sequences conserved in allgenome-sequenced S. aureus siphoviruses. Recognizingthe protein domain functions in predicted open readingframes (ORFs) enabled us to define the borders of thefunctional module types or their parts where recombina-tion events may frequently occur. Seven functionalgenomic modules covering the entire phage genome andcorresponding to the phage integrase locus, lysogenycontrol region, DNA replication module, transcriptionregulation module, packaging and head-tail modules, tailappendices module, and lysis module were used for thedescription of the genome modular structure and then indesigning a multilocus PCR strategy for bacteriophageidentification. The comparison of nucleotide sequences ofthe above-mentioned modules was performed using aprobabilistic global alignment algorithm and the trees indi-cating the major modular types were reconstructed(Fig. 2). Sequences for each modular type were alignedand grouped into clades and one representative genevariant from each functional module was chosen for oli-gonucleotide primer design so that the primers werecapable of differentiating between variants of each targetgene.
In addition to the 10 S. aureus bacteriophage integrasegene classes analysed previously (Goerke et al., 2009),we specified five types of the lysogeny control region, fourtypes of the DNA replication module, four types of thetranscription regulation module, eight types of the pack-aging module, four types of the tail appendices moduleand four types of the lysis module according to theirrelatedness. Although the sequence similarity betweensome target genes was low, it was still compatible withtheir structural and functional analogy. The proposed mul-tilocus typing system has been designed to compare thegenetic variation in staphylococcal phages efficientlywithout the need for sequencing the entire phage
Sa6(TW20) FN433596 Sa6 ant1d dnaD1a dut3 Bc B ami1
ROSA NC_007058 Sa6 ant4b dnaC1 dut1 Bd B ami2
77 NC_005356 Sa6 ant4b+1e dnaC1 dut1 Fa F ami2
53 NC_007049 Sa7 ant1a dnaC1 dut1 Ba B ami2
NM2 DQ530360 Sa7 ant1a dnaC1 dut3 Ba B ami1
85 NC_007050 Sa7 NT dnaC1 dut1 Ba B ami1
92 NC_007064 Sa7 ant2 dnaC1 dut4 Be B ami1
X2 NC_007065 Sa7 ant3 dnaD2c dut4 Be B ami1
P954 NC_013195 Sa7 ant4a dnaD2b dut1 Fa F ami3
RF122 NC_007622 Sa8 ant4a+1a dnaD2b dut2 Ba B ami1
IPLA88 NC_011614 Sa8 ant4a dnaD2b dut2 Ba B ami1
96 NC_007057 Sa9 ant2 dnaD2b dut2 Bb B ami2
MR11 NC_010147 Sa12 ant5 dnaD2c dut1 Bc B ami1
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Fig. 1. In silico classification of the complete S. aureus phage genomes into genomic module types based on int, ant, replication proteins(polA, polB, dnaC or dnaD), dut, por, serogroup specifying and ami genes. Identical types are colour coded (see online version); IEC, immuneevasion cluster type; NT, non-typeable.
Differentiation scheme for S. aureus siphoviruses 2529
Fig. 2. Circle trees illustrating the nucleotide sequence similarity of six phage genomic modules: (A) lysogeny control module; (B) DNAreplication module; (C) regulation of transcription module; (D) packaging module; (E) tail appendices region of morphogenesis module; and (F)lytic module. The phage names are written next to each branch and the clusters corresponding to bacteriophage genomic module types areindicated by brackets and named according to representative gene types. The trees A and C–F were reconstructed by FSA and the tree B byMAVID.
genomes. When disregarding the identical prophages inclonally related strain variants (JH1 and JH9, variants ofUSA300, Mu3 and Mu50), the designed system makes itpossible, together with the detection of the genes forphage-borne virulence factors, to differentiate betweenmost phages and prophages with the as yet sequencedgenomes except fPVL-CN125 that cannot be distin-guished from fPVL108, fSa3mw that is indistinguishablefrom fSa3ms and f52A that is not distinct from f80(Fig. 1).
Integrase locus. The phage integrases mediate site-specific integration of the phage genome into the bacterialchromosome. In the previous work, we categorizedexperimentally the known genome-sequenced S. aureusbacteriophages of the family Siphoviridae according tothis locus into integrase types Sa1int–Sa7int (Goerkeet al., 2009). In this work, multiplex PCR assay 1 wasupdated to include Sa8int (bovine prophage fRF122) andSa9int (f96) integrase types and the staphylococcal DNApositive control was added (Fig. 3A). As integrase typeSa12int (fMR11) was not detected in a collection of 100clinical isolates of different origins (data not shown), theprimers targeting this rarely occurring integrase type werenot included in the multiplex PCR assay 1. Phages f37(NC_007055) and fEW (NC_007056) were excluded fromthe typing system design as related to phages fPH15(NC_008723) and fCNPH82 (NC_008722) of the speciesStaphylococcus epidermidis (Daniel et al., 2007) andnone of these phages lyses coagulase-positive staphylo-cocci (Rippon, 1956; Dean et al., 1973).
Lysogeny control module. The lysogeny control regioncontains lysogeny module sequences, excluding the inte-grase gene. It is bounded by a conserved stem-loopsequence between the integrase and excisionase genes(Iandolo et al., 2002; Pantucek et al., 2004) at the 3′ endand an ORF upstream of the fPVL orf38 or fPVL orf39homologues at the 5′ end. A characteristic feature of thelysogeny control region is the alternation of regions withhigh DNA sequence similarity and DNA segmentsshowing low or no sequence similarity resulting fromnumerous short-range modular exchanges. For the deter-mination of the lysogeny control region types, we chosethe anti-repressor gene that contributes to lysogeny main-tenance, although the cI or cro genes are more relevant tolysogeny control (Ganguly et al., 2009). The anti-repressor gene is suitable for the differentiation ofmodular types because of its length and the presence ofboth conservative and variable regions. In spite of thehigh sequence diversity of the lysogeny control module,the anti-repressor genes are highly conserved at the DNAlevel as well as the integrase gene or part of the repressorgene (Lucchini et al., 1999). Moreover, the anti-repressor
types correlate with the lysogeny control types based onthe module sequences and allow rapid recognition of thisregion (Fig. 2A).
Anti-repressors are usually composed of two domainsand recombination between the regions coding for theseprotein domains is possible. Several domain types havebeen identified in S. aureus phages. The first domain typeis Bro-N (baculovirus repeat ORFs proteins), a DNA-binding domain almost always located at the N-terminus,and on many occasions, the Bro-N domain is fused to theC-terminal domain. Other identified domain types are theDNA binding KilA-C domain (involved in killing of hostcell), AntA domain (anti-repressor), ORF6C domain andRha domain (Iyer et al., 2002).
Sequence alignment of the anti-repressor genes of S.aureus bacteriophages revealed five major types reflect-ing domain structure with several subtypes based on thesequence variation. Within the major types, the nucleotidesequence similarity was 79–100%, with the exception ofsubtypes ant1e and ant1f that clustered separately; thesimilarity between types was 34–70%. Based on aminoacid sequence homology and domain composition, theanti-repressor variants corresponding to particular lysog-eny control module types were designated ant1 withBro-N and KilA-C domains containing six subtypes, ant2and ant3 both with Bro-N and ORF6C domains, ant4 withAntA and KilA-C domains containing two subtypes, andant5 (Rha protein). The bacteriophage genomes wherethe anti-repressor gene homologue had not been found,similarly as in some temperate phages from other low GCcontent Gram-positive genera (Lucchini et al., 1999),were assigned as non-typeable. Multiplex PCR assay 2(Fig. 3B) was developed to amplify the DNA fragmentscorresponding to the 10 determined ant gene types orsubtypes using 14 primers.
DNA replication module. In this region that is delimited byfPVL orf38 homologue at the 3′ end and by fPVL orf50homologue at the 5′ end, DNA replication-related proteinsare encoded. The analysis identified four types of the DNAreplication module which differ from one another in genecomposition and replication initiation mechanism, as wasshown previously (Weigel and Seitz, 2006). We chosethree different representative genes for inclusion in thediagnostic scheme (Fig. 2B): the phage DNA polymeraseA gene (polA) for phages probably using DNA replicationinitiation by transcription factors (tDR), the dnaC gene forthe modules encoding the replication initiator with thednaC helicase loader (IL-type replication), and the dnaDgene for phages possessing a (putative) initiator gene butwithout detectable helicase loader or helicase genes(dnaD1 ~ I-solo type) or an initiator gene followed byhelicase gene (dnaD2 ~ IH-type). In addition, a novel typeof replication module with putative DNA polymerase
Differentiation scheme for S. aureus siphoviruses 2531
Sa int1Sa int3Sa int5Sa int4Sa int9Sa int7Sa int8Sa int6
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BbBaBcBeSAUBd
Fig. 3. Agarose gel electrophoresis showing multiplex PCR patterns for the bacteriophages in genome sequenced and control S. aureusstrains: (A) multiplex PCR assay 1 for the integrase locus; (B) multiplex PCR assay 2 for lysogeny control region; (C and D) multiplex PCRassays 3 and 4 for DNA replication module; (E) multiplex PCR assay 5 for transcription regulation module; (F) multiplex PCR assay 6 for DNApackaging module of serogroup B phages; and (G) multiplex PCR assay 9 for lytic module.
B-type and unknown mechanism of replication has beendetected in the recently sequenced prophage fAvb.
Multiple alignment of the polymerase A genesequences revealed one conserved type with the nucle-otide sequence similarity from 98% to 100%. Sequencealignment of the dnaC genes revealed two different types.Within groups, the nucleotide sequence similarity wasfrom 86% to 100%, while the similarity between groupswas 56–58%. Based on amino acid sequence homology,DnaCs belong to the AAA+ superfamily of ATPase whichis an ancient group of ATPases. The DnaC1 and DnaC2differ in motifs content. Sequence alignment of the dnaDgenes revealed two types with several subtypes (two sub-types in dnaD1 and three subtypes in dnaD2). Within thednaD subtypes, the nucleotide sequence similarity was96–100%, while the similarity between groups was35–80%. Based on amino acid sequence homology, allDnaD genes contain a DnaD-like domain. DnaD is a com-ponent of the PriA primosome that recruits the replicationfork helicase onto the DNA. Moreover, DnaD2a subtypealso contains an orgN domain. This domain belongs to thePhage_rep_org_N superfamily of the N-terminal phagereplisome organiser.
Multiplex PCR assays 3 and 4 (Fig. 3C and D) weredeveloped to identify distinct genes located in the replica-tion module: multiplex PCR 3 for the dnaD gene andmultiplex PCR 4 for the DNA polymerase A and dnaCgenes.
Transcription regulation module. This module associatedmostly with transcription regulation contains sequencesdownstream of fPVL orf50 homologue to the rinB geneand it includes numerous short ORFs of unknown func-tion. For the differentiation of this module, we chose thedUTPase gene (dut) (Fig. 2C). The dUTPase hydrolysesdUTP to dUMP and pyrophosphate. Sequence alignmentof the dut gene revealed four major types which were inaccordance with those identified based on amino acidsequence homology. Within groups, the nucleotidesequence similarity was 76–100%, while the similaritybetween groups was 42–70%. Dut1 is the dUTPase whichcontains the all-beta fold common to the majority of organ-isms; dut2, dut3 and dut4 are members of dUTPase_2superfamily. Multiplex PCR assay 5 was developed toamplify the DNA sequences corresponding to four majortypes of the dUTPase gene using six primers (Fig. 3E).
Morphogenesis module. The late-expressed generegions coding for structural virion proteins share commonorganization features: DNA packaging – head and tail. Assuggested from the dot-plot comparisons, all the threeregions are closely linked and ordinarily horizontally trans-ferred together in S. aureus siphophages. Therefore, onlythe most conserved DNA packaging region was chosen to
be included in the diagnostic scheme (Fig. 2D). The pack-aging region is likely to start with a terminase smallsubunit gene and ends with a prohead- or Clp-proteasegene in serogroup A and F phages respectively, or withphage head morphogenesis protein (Mu-F) in serogroupB phages. Based on the packaging module, we can dis-tinguish serogroup F and B phages more precisely, whileserogroup A phages have a single morphogenesis typeand are identified by means of PCR assay 8 describedbelow. Previously reported multiplex PCR assay 6 target-ing the portal protein gene types of serogroup F phagesdesignated Fa and Fb (Pantucek et al., 2004) was supple-mented with multiplex PCR assay 7 in this work in order toinclude the detection of five different portal protein genetypes of serogroup B phages designated Ba–Be. Thenucleotide sequences are highly conserved within A, Fa,Fb or Ba–Be portal gene types (94–100%), while there isno significant similarity between groups. Multiplex PCRassay 7 (Fig. 3F) distinguishing the portal gene types ofserogroup B phages uses primer pairs specific for each ofthe types.
Tail appendices. The tail fibre morphogenesis modulecontains the genes localized downstream of the tapemeasure protein gene to the conserved sequence TCGGYACTGRCTTTTTATTT upstream of the holin gene. Thenucleotide sequence specificity in this module is consis-tent with serogroups A, B, F and L in phages of theInternational Typing Set and experimental phages (Pan-tucek et al., 2004). Previously described multiplex PCRassay 8 for the detection of A-, B-, F- and L-bacteriophagetypes (Pantucek et al., 2004) has been updated in thiswork using a degenerate primer pair (SGB-D) in order toinclude the detection of B phages fRF122 and fX2. Bac-teriophage fX2, initially classified into serogroup Cphages which are antigenically closely related to sero-group B phages (Rountree, 1949), belongs to B phagesaccording to the nucleotide sequence of this module(Fig. 2E).
Lysis module. This module covers two genes coding forholin and endolysin that contribute to the final step of thebacteriophage lytic life cycle – lysis of the host cell. Theclassification of the holin gene classes of S. aureus bac-teriophages of the Siphoviridae family has been recentlyproposed on the basis of the gene length polymorphism(Goerke et al., 2009). For PCR detection of the lysismodule, we propose an endolysin-based classificationthat correlates well with the holin gene types (Fig. 2F).The majority of endolysins have a modular organization,composed of at least two distinctly separate functionaldomains: a C-terminal cell-wall binding domain (SH3b)which directs the enzyme to its target and an N-terminalcatalytic domain (Young, 1992).
Differentiation scheme for S. aureus siphoviruses 2533
Endolysins of all S. aureus siphoviruses contain aCHAP (cysteine, histidine-dependent amidohydrolases/peptidase) domain corresponding to the peptidase func-tion. Multiple alignment of the endolysin gene sequencesrevealed four types with nucleotide sequence similarityfrom 89% to 100% within groups and only 26% to 58%similarity between groups. On the basis of amino acidsequence homology and domain organization, the endol-ysin types were designated ami1, containing a CHAPdomain, amidase_2 domain (N-acetylmuramoyl-L-alanineamidase activity) and SH3b domain; ami2, containing aCHAP domain, amidase_3 domain (N-acetylmuramoyl-L-alanine amidase activity) and SH3b domain; ami3, with aCHAP domain only; and ami4, containing a CHAP domainand amidase_3 domain. Multiplex PCR assay 9 wasdeveloped for assigning the lysis module variants. It com-prises four primer pairs for identifying four endolysin typesand gave rise to four products (Fig. 3G).
Classification system for S. aureus siphoviruses
The analysis of modular types of S. aureus phages andprophages illustrated high genomic mosaicism and in thegenome-sequenced phages, it revealed the prevalence ofthe genes proposed to be representative of the genomicmodule types and some links between these types. Theproposed extended typing scheme covering seven majorgenomic modules enabled us not only to differentiatephages but also to design a reliable classification system.
For the basic description and rapid differentiation of aphage strain, the integrase gene class, serological grouptype corresponding to the morphogenesis module, andendolysin type are the most relevant (Fig. 1). Using oursimplified scheme, e.g. phage f11 can also be labelled asSa5int-Ba-ami1 or phage f13 as Sa3int-Fb-ami3. More-over, it is evident that some integrase and amidase typesare closely linked with particular virulence factors. Sa1int-ami4 phages contain the eta and ear genes, while thegenes for PVL are only harboured by Sa2int-ami2 phages,and all Sa3int-ami3 phages described to date but f42Ecarry some genes of the immune evasion cluster. Inter-estingly, ami1 type is never associated with any knownvirulence factor gene. Despite the multiple mosaic vari-ants, there are some additional close links between par-ticular modular types in the sequenced genomes, e.g. allphages and prophages coding for DNA polymerase are ofserogroup A.
For a complete description of S. aureus siphophageswe designed a novel typing scheme based on the moduletypes that are listed in the order in which they appear inthe genome. For example, phage f11 can also bedescribed as Sa5int-ant1a-dnaD2a-dut1-Ba-ami1 whilephage f13 can be characterized as Sa3int-ant4a-dnaC1-dut3-Fb-ami3. A similar approach to the general classifi-
cation of bacteriophages based on reticulation has beenproposed previously (Lawrence et al., 2002; Lima-Mendez et al., 2008).
Application of the multiplex PCR typing scheme forthe characterization of phages from the InternationalTyping Set
A set of multiplex PCR assays were applied to character-ize 23 S. aureus bacteriophages of the InternationalTyping Set and phages f11, f12, f80a, f42D, f187 andfX2 (Table S1). The results obtained for 17 of these bac-teriophages, i.e. genome-sequenced phages, were com-pared to the nucleotide sequence database. Fourteen ofthe tested genome-sequenced phages corresponded tothe genomes annotated in the database. However, theobtained data revealed three discrepancies. First, ourf42E strain was not carrying the ant1a gene as genome-sequenced f42E (Kwan et al., 2005) and furthermore, itwas of a different endolysin type, ami2, in contrast to ami3determined in the sequenced phage strain. Second, ourf80 variant carried dut2 gene type in contrast to dut1 ingenome-sequenced f80 (DQ908929) and third, our f80avariant carried ami1 endolysin type versus ami2 ingenome-sequenced f80a (Tallent et al., 2007). However,an older sequence of f80a harbouring the amidase geneis of ami1 type (Bon et al., 1997). Similar discrepancieshave been reported recently for the S. aureus bacterioph-age f11 amidase gene (Donovan et al., 2008).
Discussion
In the present report, we provide a systematic approach tothe experimental study of genome structure diversity instaphylococcal siphoviruses. We have developed a reli-able classification scheme using a multilocus PCR typingtool based on a comparative sequence analysis thatshould shed some light on the phylogenetic relationshipsbetween phages. It has been established for the molecu-lar typing of bacteriophages and prophage profiling oflysogenic S. aureus strains through the generation of theirmodular genomic patterns. The well-known modulartheory of phage evolution (Botstein, 1980; Canchayaet al., 2003; Kwan et al., 2005) states that the individualphage genomes evolve through exchanges of groups offunctionally related genes (modules) which can be rela-tively freely substituted between phages otherwise differ-ing in many respects. Therefore, it is possible to identifythe phylogenetic relationships from the individual modulesrather than from the entire phage genomes.
The designed multilocus characterization scheme haspotential not only to identify phages and/or phagesinduced from lysogenic strains but also to profile proph-ages within the host genomes. Such data would improve
understanding of the impact of phages on bacterial fitnessand virulence. The usefulness of detecting phage derivedORFs in the typing of MRSA strains has been reportedrecently (Suzuki et al., 2006). Ma and colleagues (2008)designed several PCR assays targeting phage genes todistinguish PVL-phage lineages. However, their workscover only a limited set of phage strains. On the otherhand, our diagnostic approach reflects the phage modulegenome organization and its dynamics. As new phagesand gene variants are expected to be described, thismultilocus typing scheme will be easily extendable toinclude newly generated genotypic data on staphylococ-cal phages and their epidemiological significance.Detailed phage characterization will be helpful not only inthe classification of newly isolated phages, laboratoryexperiments or epidemiological studies, but will also berelevant to the study of phage genome stability duringpropagation and long-term preservation.
The multiplex PCR assays targeting integrase, portalprotein or serogroup-specific and amidase genes enabledto infer the prophage content in polylysogenic strains. Themethod reliably identified the prophage content of theprototypic genome-sequenced S. aureus strains NCTC8325, COL, Newman, USA300, MW2, Mu50, N315,MRSA252, MSSA476 and RF122. The sizes of the ampli-fied DNA fragments matched those expected for therespective module variants except the dnaD1a subtypePCR product obtained with strain MRSA252 which wasshorter than that in strain Newman (phage fNM3), inagreement with the fact that dnaD1a of fSa3(252B) has a11 bp deletion. Although 10 sequenced strains provide areasonable coverage of human clinical S. aureus strainsand include a wide range of mobile genetic elements, theirprophages do not contain all module variants occurring inS. aureus phages that are currently available in the data-bases. Therefore, we added more validation controls. Therarely occurring module variants were validated in S.aureus strain 1039 lysogenized with selected Interna-tional Typing Set phages and strain CCM 7725, orgenome-sequenced phages fX2 and f187 for which labo-ratory lysogenization was not obtained. Bacteriophagesf83A and f3C were used as controls for anti-repressortypes ant1c and ant5 instead of phages fSLT and f88respectively, and their nucleotide sequences were esti-mated. No PCR product was obtained in non-lysogenicstrain S. aureus 1039.
Various degrees of genome mosaicism have been welldescribed in Siphoviridae (Canchaya et al., 2003) andcould also be observed in staphylococcal siphophages.The early- and middle-expressed gene segments, from intto rinB are more dynamic both among and withinmodules. There are three characteristic features of thisregion contributing to the great variability of modules andmaking the phylogenetic analysis difficult. The first one is
mosaicism of the genes that specify module function, e.g.the lysogeny control region of phage f92 is clustered toant1a complex but harbours ant2 gene type. The secondfeature is the presence of ORFs with unknown functioninserted between the genes that determine module func-tion, e.g. the lysogeny control region of fPVL108 (Maet al., 2006). The third characteristic feature is that somephages either have modules with a reduced gene contentor some modules are missing in them. Quite common isthe absence of the int or ant genes, therefore virulentphages such as f3A with deletions in the lysogeny modulecannot be classified using these two loci. Almost all ORFsof the transcription regulation module are missing infSa3ms and fSa3mw phages.
The late-expressed genes encoding structural proteinsinteracting during capsid morphogenesis and the corre-sponding module sequences typically show low alterna-tion of DNA segments and are relatively homogenouswithin the module boundaries. These stable regions gen-erally diversify mainly by point mutations, although, rarely,some of the situations reported for the early- and middle-expressed genes can take place.
In conclusion, in this work we report the establishmentand application of a novel multilocus PCR strategy forthe complex characterization of the modular structure ofS. aureus siphoviruses. It allows rapid and specific iden-tification of the phage strains and at the same time, itenables the description of the modular composition ofprophages isolated from clinically relevant strains andcan contribute to the specification of S. aureus phagespecies of the family Siphoviridae. This tool will be usedprimarily to investigate the evolution of bacteriophagesin particular S. aureus clonal lineages, to determinewhether recombination only occurs within the lineageboundaries or is evolutionarily more ancient than thelineage branching and to track phage dynamics duringclonal outbreaks.
Experimental procedures
Origin of bacteriophages and bacterial strains
Twenty-three S. aureus bacteriophages of the InternationalTyping Set, phages f42D and f187 and their propagationstrains were kindly provided by Professor V. Hájek (PalackýUniversity, Olomouc, Czech Republic). Bacteriophages f11and f12 were induced from RN1HG strain (Pohl et al., 2009).Bacteriophage fX2 (NCTC 9858) and its propagation strainNCTC 9317 as well as S. aureus strain NCTC 8325 wereobtained from the National Collection of Type Cultures(Health Protection Agency, London, UK). Bacteriophagef80a was obtained from Professor A. Cheung (DartmouthMedical School, Hanover NH, USA). S. aureus strains Mu50,N315, MW2, MSSA476, and MRSA252 were suppliedthrough the Network on Antimicrobial Resistance in Staphy-lococcus aureus (NARSA) program. The S. aureus strain
Differentiation scheme for S. aureus siphoviruses 2535
COL was obtained from Dr A. Tomasz and Dr H. de Lencastre(The Rockefeller University, New York). Strain S. aureusRF122 was provided by courtesy of Dr J. R. Fitzgerald (Uni-versity of Dublin, Trinity College, Dublin, Ireland). The S.aureus strains USA300 and Newman were obtained fromProf F. Götz (University of Tübingen, Tübingen, Germany). Aquadruple lysogenic strain of S. aureus CCM 7097 was pre-pared previously (Pantucek et al., 2004). The prophagelessindicator strain of S. aureus 1039 (Yoshizawa, 1985) wasobtained from Dr Y. Yoshizawa (Jikei University, School ofMedicine, Tokyo, Japan). A set of laboratory lysogenizedstrains of S. aureus 1039 with phages f3C, f47, f55, f71,f83A and f96 (CCM 7730 – CCM 7735), and monolysogenicstrain of S. aureus CCM 7725 harbouring an exfoliative toxinA converting prophage fB122 were prepared as describedpreviously (Borecká et al., 1996) and deposited in the CzechCollection of Microorganisms. Prophage identification wasdone on a set of clinical strains collected from several hos-pitals in the Czech Republic and obtained from the NationalInstitute of Public Health (Prague).
Prophage induction
The phage particles were isolated from bacterial cells culti-vated in Nutrient Broth CM1 (Oxoid, Basingstoke, UK) at37°C with aeration after they reached the logarithmic growthphase. Twice washed cells resuspended in 10 ml of salinesolution (0.85% NaCl) to optical density OD600 = 0.15 wereirradiated with a 15 W UV lamp (254 nm) at a distance of60 cm, for 30 s. The next cultivation steps were performedaccording to Duval-Iflah (1972).
Phage propagation
The phage lysates were plated on the respective propagationstrain, then one plaque was picked up and propagated in500 ml of a bacterial culture of the S. aureus 1039 straingrown to OD600 = 0.22 for 2 h at 37°C. The remaining bacteriaand bacterial cell debris in lysates were removed by centrifu-gation at 5000 g for 15 min and by subsequent filtration with0.45 mm L.E. nylon syringe filters (OlimPeak, Teknokroma).
Phage DNA extraction
Small volumes (250 ml) of phage lysates were used forsample preparation and DNA extraction. Bacterial DNA andRNA were removed by DNase I (1 mg ml-1), and RNase A(5 mg ml-1) in the presence of 100 mM MgCl2 for 30 min at37°C. Then the phage DNA was extracted using a QIAEX IIkit (Qiagen, Hilden, Germany). The extracted DNA was usedfor PCR amplification.
Phage and prophage typing by a multilocusPCR strategy
Bacterial DNA for PCR was isolated using a PCR TemplatePreparation Kit (Roche Diagnostics, Germany) according tothe manufacturer’s protocol modified by prolonged lysis usinglysostaphin (Dr Petry Genmedics, Reutlingen, Germany)
added to a final concentration of 30 mg ml-1. The types ofbacteriophage genomic modules or lysogenic types of bac-terial strains were classified on the basis of nine multiplexPCR assays targeting the genes for integrase (int), anti-repressor (ant), replication proteins (polA, dnaD or dnaC),dUTPase (dut), portal protein (por), structural tail proteinsand amidase (ami). The multiplex PCR assays included aninternal amplification control targeting the conservedsequence of the S. aureus species (Štepán et al., 2001).Three primer pairs (SAU1–SAU6) corresponding to threerespective amplification products: 217, 470 or 851 bp, wereused (Table S2).
The primer design was performed after multiple alignmentsof the bacteriophage genomic sequences using Oligo PrimerAnalysis Software v6 (Molecular Biology Insights, USA). Theprimers for the PCR assays and their concentrations aregiven in Table S2. Reactions were performed in a finalvolume of 25 ml, which contained 50 ng of template chromo-somal DNA or 5 ng of phage DNA. Multiplex PCR-1, PCR-2,PCR-3, PCR-4 and PCR-5 were carried out with a MultiplexPCR kit (Qiagen, Hilden, Germany). The reaction conditionsfor multiplex PCR-6, and PCR-8 were described previously(Pantucek et al., 2004); the reaction conditions for multiplexPCR-7 and PCR-9 using Taq DNA polymerase (Invitrogen,Carlsbad, USA) are summarized in the Table S2 footnote.The PCR assays were performed using a T-Gradient thermalcycler (Biometra, Goettingen, Germany).
Validation of the multiplex PCR assays was carried out with10 genome-sequenced S. aureus strains, prophageless S.aureus 1039, S. aureus 1039 lysogenized with differentphage types and genome-sequenced phages f187 and fX2.In addition, triple lysogenic S. aureus NCTC 8325 lysog-enized with f77 (CCM 7097) was used. The positive controlstrains for PCR assays are listed in Table S2.
Sequence analysis
Sequences of 66 complete S. aureus bacteriophagegenomes were retrieved from the GenBank database orextracted from the complete bacterial genomes (http://www.ncbi.nlm.nih.gov/Genbank/index.html). The phagegenomic module borders were identified after global align-ments with rearrangements performed by mVista Server(Shuffle-LAGAN program) at http://www-gsd.lbl.gov/vista/.Multiple sequence alignments and database searches wereperformed with the programs ClustalW2 – web-based serviceat EBI (http://www.ebi.ac.uk/Tools/clustalw2/) and BLAST –web-based service at NCBI (http://blast.ncbi.nlm.nih.gov/).The ORFs were deduced from the whole phage or prophagegenomes by Vector NTI Advance Software v11 (Invitrogen,Carlsbad, USA). The conserved domains in ORFs were iden-tified using the CDD web-based service at NCBI (Marchler-Bauer et al., 2009). The relatedness of the phage genomicmodules was estimated and, subsequently, the tree wasreconstructed by the MAVID and Fast Statistical Alignment(FSA) programs via a web interface (http://baboon.math.berkeley.edu/mavid/download/ and http://orangutan.math.berkeley.edu/fsa/).
The accession numbers of partial anti-repressor genesequences of bacteriophages f83A and f3C were depositedin the GenBank database (GU230886–GU230887).
This work was supported by grants from the Czech ScienceFoundation (310/09/0459), the European Union (LSHM-CT-2006-019064) and the Ministry of Education, Youth andSports of the Czech Republic (MSM0021622415). We thankEva Kodytková for valuable help.
References
Ackermann, H.W., and DuBow, M.S. (1987) Natural groups ofbacteriophages. In Viruses of Prokaryotes. Ackermann,H.W., and DuBow, M.S. (eds). Boca Raton, FL, USA: CRCPress, pp. 101–105.
Bae, T., Baba, T., Hiramatsu, K., and Schneewind, O. (2006)Prophages of Staphylococcus aureus Newman and theircontribution to virulence. Mol Microbiol 62: 1035–1047.
Betley, M.J., and Mekalanos, J.J. (1985) Staphylococcalenterotoxin A is encoded by phage. Science 229: 185–187.
Bon, J., Mani, N., and Jayaswal, R.K. (1997) Molecular analy-sis of lytic genes of bacteriophage 80 alpha of Staphylo-coccus aureus. Can J Microbiol 43: 612–616.
Borecká, P., Rosypal, S., Pantucek, R., and Doškar, J. (1996)Localization of prophages of serological group B and F onrestriction fragments defined in the restriction map of Sta-phylococcus aureus NCTC 8325. FEMS Microbiol Lett143: 203–210.
Botstein, D. (1980) A theory of modular evolution for bacte-riophages. Ann N Y Acad Sci 354: 484–490.
Canchaya, C., Proux, C., Fournous, G., Bruttin, A., andBrussow, H. (2003) Prophage genomics. Microbiol Mol BiolRev 67: 238–276.
Carroll, J.D., Cafferkey, M.T., and Coleman, D.C. (1993)Serotype F double- and triple-converting phage insertion-ally inactivate the Staphylococcus aureus beta-toxin deter-minant by a common molecular mechanism. FEMSMicrobiol Lett 106: 147–155.
Daniel, A., Bonnen, P.E., and Fischetti, V.A. (2007) Firstcomplete genome sequence of two Staphylococcus epider-midis bacteriophages. J Bacteriol 189: 2086–2100.
Dean, B.A., Williams, R.E.O., Hall, F., and Corse, J. (1973)Phage typing of coagulase-negative staphylococci andmicrococci. J Hyg (Lond) 71: 261–270.
Donovan, D.M., Foster-Frey, J., Garrett, W.M., andBlomberg, L. (2008) Resolving the database sequencediscrepancies for the Staphylococcus aureus bacterioph-age phi11 amidase. J Basic Microbiol 48: 48–52.
Duval-Iflah, Y. (1972) Lysogenic conversion of the lipase inStaphylococcus pyogenes group 3 strains. Can J Microbiol18: 1491–1497.
Ganguly, T., Das, M., Bandhu, A., Chanda, P.K., Jana, B.,Mondal, R., and Sau, S. (2009) Physicochemical proper-ties and distinct DNA binding capacity of the repressor oftemperate Staphylococcus aureus phage phi11. FEBS J276: 1975–1985.
García, P., Martínez, B., Obeso, J.M., Lavigne, R., Lurz, R.,and Rodríguez, A. (2009) Functional genomic analysis oftwo Staphylococcus aureus phages isolated from the dairyenvironment. Appl Environ Microbiol 75: 7663–7673.
Goerke, C., Koller, J., and Wolz, C. (2006a) Ciprofloxacinand trimethoprim cause phage induction and virulence
modulation in Staphylococcus aureus. Antimicrob AgentsChemother 50: 171–177.
Goerke, C., Wirtz, C., Fluckiger, U., and Wolz, C. (2006b)Extensive phage dynamics in Staphylococcus aureus con-tributes to adaptation to the human host during infection.Mol Microbiol 61: 1673–1685.
Goerke, C., Pantucek, R., Holtfreter, S., Schulte, B., Zink, M.,Grumann, D., et al. (2009) Diversity of prophages in domi-nant Staphylococcus aureus clonal lineages. J Bacteriol191: 3462–3468.
Iandolo, J.J., Worrell, V., Groicher, K.H., Qian, Y., Tian, R.,Kenton, S., et al. (2002) Comparative analysis of thegenomes of the temperate bacteriophages phi11, phi12 andphi13 of Staphylococcus aureus 8325. Gene 289: 109–118.
Iyer, L.M., Koonin, E.V., and Aravind, L. (2002) Extensivedomain shuffling in transcription regulators of DNA virusesand implications for the origin of fungal APSES transcrip-tion factors. Genome Biol 3: 0012.0001–0012.0011.
Kaneko, J., Kimura, T., Narita, S., Tomita, T., and Kamio, Y.(1998) Complete nucleotide sequence and molecular char-acterization of the temperate staphylococcal bacterioph-age phiPVL carrying Panton-Valentine leukocidin genes.Gene 215: 57–67.
Kwan, T., Liu, J., DuBow, M., Gros, P., and Pelletier, J. (2005)The complete genomes and proteomes of 27 Staphylococ-cus aureus bacteriophages. Proc Natl Acad Sci USA 102:5174–5179.
Lawrence, J.G., Hatfull, G.F., and Hendrix, R.W. (2002)Imbroglios of viral taxonomy: genetic exchange and failingsof phenetic approaches. J Bacteriol 184: 4891–4905.
Lee, C.Y., and Iandolo, J.J. (1986) Lysogenic conversion ofstaphylococcal lipase is caused by insertion of the bacte-riophage L54a genome into the lipase structural gene.J Bacteriol 166: 385–391.
Lima-Mendez, G., Van Helden, J., Toussaint, A., and Leplae,R. (2008) Reticulate representation of evolutionary andfunctional relationships between phage genomes. Mol BiolEvol 25: 762–777.
Lindsay, J.A., and Holden, M.T. (2004) Staphylococcusaureus: superbug, super genome? Trends Microbiol 12:378–385.
Lindsay, J.A., Moore, C.E., Day, N.P., Peacock, S.J., Witney,A.A., Stabler, R.A., et al. (2006) Microarrays reveal thateach of the ten dominant lineages of Staphylococcusaureus has a unique combination of surface-associatedand regulatory genes. J Bacteriol 188: 669–676.
Lucchini, S., Desiere, F., and Brussow, H. (1999) Similarlyorganized lysogeny modules in temperate Siphoviridaefrom low GC content gram-positive bacteria. Virology 263:427–435.
Ma, X.X., Ito, T., Chongtrakool, P., and Hiramatsu, K. (2006)Predominance of clones carrying Panton-Valentine leuko-cidin genes among methicillin-resistant Staphylococcusaureus strains isolated in Japanese hospitals from 1979 to1985. J Clin Microbiol 44: 4515–4527.
Ma, X.X., Ito, T., Kondo, Y., Cho, M., Yoshizawa, Y., Kaneko,J., et al. (2008) Two different Panton-Valentine leukocidinphage lineages predominate in Japan. J Clin Microbiol 46:3246–3258.
Differentiation scheme for S. aureus siphoviruses 2537
Moore, P.C., and Lindsay, J.A. (2001) Genetic variationamong hospital isolates of methicillin-sensitive Staphylo-coccus aureus: evidence for horizontal transfer of virulencegenes. J Clin Microbiol 39: 2760–2767.
Morgan, G.J., and Pitts, W.B. (2008) Evolution withoutspecies: the case of mosaic bacteriophages. Br J PhilosSci 59: 745–765.
Narita, S., Kaneko, J., Chiba, J., Piemont, Y., Jarraud, S.,Etienne, J., and Kamio, Y. (2001) Phage conversion ofPanton-Valentine leukocidin in Staphylococcus aureus:molecular analysis of a PVL-converting phage, phiSLT.Gene 268: 195–206.
Nelson, D. (2004) Phage taxonomy: we agree to disagree.J Bacteriol 186: 7029–7031.
Pantucek, R., Doškar, J., Ružicková, V., Kašpárek, P.,Orácova, E., Kvardová, V., and Rosypal, S. (2004) Identi-fication of bacteriophage types and their carriage in Sta-phylococcus aureus. Arch Virol 149: 1689–1703.
Pohl, K., Francois, P., Stenz, L., Schlink, F., Geiger, T.,Herbert, S., et al. (2009) CodY in Staphylococcus aureus: aregulatory link between metabolism and virulence geneexpression. J Bacteriol 191: 2953–2963.
Rashel, M., Uchiyama, J., Ujihara, T., Takemura, I., Hoshiba,H., and Matsuzaki, S. (2008) A novel site-specific recom-bination system derived from bacteriophage phiMR11.Biochem Biophys Res Commun 368: 192–198.
Resch, A., Fehrenbacher, B., Eisele, K., Schaller, M., andGotz, F. (2005) Phage release from biofilm and planktonicStaphylococcus aureus cells. FEMS Microbiol Lett 252:89–96.
Rippon, J.E. (1956) The classification of bacteriophageslysing staphylococci. J Hyg (Lond) 54: 213–226.
Rohwer, F., and Edwards, R. (2002) The Phage ProteomicTree: a genome-based taxonomy for phage. J Bacteriol184: 4529–4535.
Rountree, P.M. (1949) The serological differentiation of sta-phylococcal bacteriophages. J Gen Microbiol 3: 164–173.
Štepán, J., Pantucek, R., Ružicková, V., Rosypal, S., Hájek,V., and Doškar, J. (2001) Identification of Staphylococcusaureus based on PCR amplification of species specific
genomic 826 bp sequence derived from a common 44-kbSmaI restriction fragment. Mol Cell Probes 15: 249–257.
Suzuki, M., Tawada, Y., Kato, M., Hori, H., Mamiya, N.,Hayashi, Y., et al. (2006) Development of a rapid straindifferentiation method for methicillin-resistant Staphylococ-cus aureus isolated in Japan by detecting phage-derivedopen-reading frames. J Appl Microbiol 101: 938–947.
Tallent, S.M., Langston, T.B., Moran, R.G., and Christie, G.E.(2007) Transducing particles of Staphylococcus aureuspathogenicity island SaPI1 are comprised of helper phage-encoded proteins. J Bacteriol 189: 7520–7524.
van Wamel, W.J., Rooijakkers, S.H., Ruyken, M., van Kessel,K.P., and van Strijp, J.A. (2006) The innate immune modu-lators staphylococcal complement inhibitor and chemotaxisinhibitory protein of Staphylococcus aureus are located onbeta-hemolysin-converting bacteriophages. J Bacteriol188: 1310–1315.
Weigel, C., and Seitz, H. (2006) Bacteriophage replicationmodules. FEMS Microbiol Rev 30: 321–381.
Witney, A.A., Marsden, G.L., Holden, M.T., Stabler, R.A.,Husain, S.E., Vass, J.K., et al. (2005) Design, validation,and application of a seven-strain Staphylococcus aureusPCR product microarray for comparative genomics. ApplEnviron Microbiol 71: 7504–7514.
Yamaguchi, T., Hayashi, T., Takami, H., Nakasone, K.,Ohnishi, M., Nakayama, K., et al. (2000) Phage conversionof exfoliative toxin A production in Staphylococcus aureus.Mol Microbiol 38: 694–705.
Yoshizawa, Y. (1985) Isolation and characterization of restric-tion negative mutants of Staphylococcus aureus. JikeikaiMed J 32: 415–421.
Young, R. (1992) Bacteriophage lysis: mechanism and regu-lation. Microbiol Rev 56: 430–481.
Supporting information
Additional Supporting Information may be found in the onlineversion of this article:
Table S1. Experimental characterization of the InternationalTyping Set and additional six phages.Table S2. Primers used for PCR assays.
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