genomic and proteomic characterization

7/31/2019 Genomic and Proteomic Characterization

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Btriog 1:3, 152-164; My/Jun 2011; 2011 Lnd Bioin

ReseaRch papeR

152 Btriog Volum 1 Iu 3

*Correspondence to: Masatada Tamakoshi; Email: [email protected]

Submitted: 04/13/11; Revised: 07/21/11; Accepted: 07/22/11

http://dx.doi.org/10.4161/bact.1.3.16712

Introduction

Bacteria-inecting phages are ubiquitous in our world, especiallyin the ocean, and are probably the most abundant biologicalentity on earth.1 It is believed that they drive microbial evolu-tion via lateral gene transer and also inuence biogeochemicalcycles.2 Among these phages, tailed phages are the largest, mostwidespread and most diverse group o phages.3 Phages in oneo the amilies, known as Myoviridae, have a contractile tails.They show the most complex structure, where the phage par-ticle is ormed in a complicated and sophisticated manner. T4,the archetype o the T-even phages, is one o the most inten-sively studied large myoviruses.4 Phages related to T4 are wide-spread in the biosphere, and probably they have diverged rom

Genomic and proteomic characterizationof the large Myoviridae bacteriophage TMA

of the extreme thermophile

Thermus thermophilusMtd Tmkoi,1,2,* ay Murkmi,1 Motoki sugiw,1 Knji Tunizumi,1 sigki Tkd,3 Toiiko ski,3

Tki Izumi,4 Toiiko akib,5 Koru Mituok,5 hidiro To,6 atui Ymit,7 Fumio arik,8 Mir httori,9

Tiro Oim10 nd akiiko Ymgii1

1Drtmnt o Molulr Biology; Tokyo Univrity o prmy nd Li sin; hioji, Tokyo; 2RIKeN spring-8 cntr; hrim Intitut; syo, hyogo; 3Drtmnt o

cmitry nd cmil Biology; Grdut sool o enginring; Gunm Univrity; Kiryu; 4Drtmnt o Biomitry; Gunm Univrity Grdut sool o Mdiin;

Mbi, Gunm; 5Biomdiinl Inormtion Rr cntr (BIRc); Ntionl Intitut o advnd Indutril sin nd Tnology (aIsT); Koto-ku, Tokyo; 6advnd

sin Intitut; RIKeN; Yokom; 7Kitto Intitut or Li sin; Kitto Univrity; sgmir; 8Drtmnt o Li sin; Grdut sool o Bioin nd

Biotnology; Tokyo Intitut o Tnology; Yokom, Kngw; 9Grdut sool o Frontir sin; Univrity o Tokyo; Kiw, cib; 10Intitut o environmntl

Mirobiology; Kyow-kko co.; Mid, Tokyo Jn

Ke wo:Thermus thermophilus, myovirus, genomics, antagonistic coevolution, proteomics

a common ancestor by acquiring the ability to inect dierenthost bacteria.5

Phages ound in the hot water environment also show largediversity.6 For example, thermophilic archaeal viruses display anexceptional degree o diversity with regard to both morphotypeand genome.7 In addition to evolutionary interest, the gene prod-ucts o the thermophilic phages that are involved in nucleotidemodifcation are also o great interest or various molecular biolog-ical applications.8 So ar, more than one hundred Thermus inect-ing phages, the largest number or any thermophilic bacteria, havebeen isolated. These phages are morphologically diverse, as seenin mesophilic phages, including the Myoviridae, Siphoviridae,Tectiviridae and Inoviridae amilies, and more than hal o the iso-lates are tail-less.9 The complete genome sequences o a myovirus,

a lyti g, digntd TMa, w ioltd rom Jn ot ring uing Thermus thermophilus hB27 n

inditor trin. eltron mirooi xmintion owd tt TMa d n iodrl d nd ontrtil til. T

irulr doubl-trndd DNa qun oTMa w 151,483 b in lngt, nd it orgniztion w ntilly m

tt oYs40 xt tt t TMa gnom ontind gn or ir o trno nd rolv, nd gn or

rin to rgin ubtitutd ortolog o t rotin involvd in t initition o t Ys40 gnomi DNa ynti.

T dirnt ot ifiti oTMa nd Ys40 ould b xlind by t qun dirn in t c-trminl

rgion o tir ditl til fbr rotin. T pilA knokout trin o T. thermophilus owd imultnou lo o

nitivity to tir ognt g, ilu trutur, twiting motility nd omtn or nturl trnormtion, tu

uggting tt t g intion rquird t intt ot ili. puld-fld gl ltroori nlyi o t TMa

nd Ys40 gnom rvld tt t lngt o tir DNa xdd 200 kb, inditing tt t trminl rdundny

i mor tn 30% o t lod irulr orm. protomi nlyi o t TMa vir ion uing ombintion o N-trminl

quning nd m tromtri nlyi o tid rgmnt uggtd tt t mturtion o vrl rotin

involvd in t g mbly ro w mditd by tryin-lik rot. T gn ordr o t g truturlrotin w lo diud.


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maturation process o the protein components involved in phagehead ormation.

Results

iolo o ge eou o T. thermophilus hB27.In order to isolate a novel myovirus, we screened hot water oAtagawa hot spring with HB27 as an indicator strain. Severalplaques were ound and one o them was cloned by two serialpassages o phage dilutions. The newly isolated phage, designatedas TMA, ormed clear plaques on the lawn o HB27 cells spreadon double agar layer plates. Clear plaques were also ormed withsimilar efciency on the lawns o HB8 and T. favusAT-62 cells,but not on T. aquaticusYT-1 cells.

Electron micrograph o negatively stained TMA showedthat its morphology was very similar to that oYS40 (g. 1).Accordingly, TMA had an isometric, rather than prolate, head,

a contractile tail and a thick baseplate with tail fbers. Like a typi-cal myovirus, such as T4, the tail tube oTMA protrudes romthe bottom o the baseplate when the tail sheath contracts. Therewere several cases where lipid vesicles appeared to be bound to thebottom o the baseplate o the phage particles (g. 1).

Geoe eue o tma ys40. The genomes oTMA and YS40 consisted respectively o 151,483 and 152,372base pairs (bp) long double-stranded DNAs (g. 2), and werepredicted to contain 168 and 170 protein-coding genes, respec-tively (tble s1). The GC content (32.63%) o the TMAgenome was very similar to that (32.59%) o the YS40 genome.One hundred and fty-nine o the protein-coding genes werecommon to both genomes. Just as in YS40, approximately 25%o the predicted proteins in TMA showed sequence similarityto proteins o known unction, including nucleotide metabolism(nine genes), DNA replication/recombination (nine genes), com-ponents o phage particles (nine genes) and others (13 genes).The amino acid sequence identity between the orthologs in thetwo genomes ranged rom 43% to 100% and the average was94%. Three tRNA genes were ound in the TMA genome, andthe positions and nucleotide sequences o the three tRNA genesoTMA were identical to those oYS40. The gene order wasalso conserved between these two genomes (g. 2 tble s1).

YS40,10 two siphoviruses with the longest known tails, P23-45and P74-26,11 two tail-less tectiviruses, P23-77,12 and T. aquaticusphage IN93,13 have already been reported. Notably, o all thepredicted genes, only approximately 25% have been assigned any

putative unction, indicating the novelty o their gene products.Purifcation and subsequent N-terminal amino acid sequencingo the lysozyme encoded in the genome oIN93 demonstratedthat its primary sequence was very dierent rom the knownlysozymes.14 The membrane-containing, icosahedral phage P23-77 oT. thermophilus is evolutionarily related to the viruses andplasmids o the halophilic archaea, all o which share two majorcapsid proteins and a putative packaging ATPase.12 Among theThermus phages, YS40 is the only one whose genome is largerthan 100 kb, and has been extensively characterized at the molec-ular level that includes determining the transcriptional profle15and host gene response ater the inection.16

In general, the role o lytic phages in cellular evolution is

not as clear as that o temperate phages, which are thought tobe important vehicles or lateral gene transer via transduction.Lytic phages o Thermus species, however, seem to contribute tothe evolution o their host cells because DNA released rom thelysed cells ollowing inection could serve as good substrates oruptake by other Thermus cells. In this regard, it is noteworthythat because o their natural competence,17T. thermophiluscantake up DNA without any sequence specifcity rom all domainso lie,18 regardless o the growth phase.19

To gain evolutionary insights into the Thermus large myovi-ruses through comparat ive genomic analysis, we isolated aT. ther-mophilusphage, TMA, whose morphology and genome lengthwere similar to those o the well-characterized phage YS40,except that it showed broader host cell specifcity. Subsequently,we determined and compared the complete genome sequenceso TMA and YS40. Analysis o spontaneously occurringphage-resistant strains and gene-knockout strains revealed thatthe phage inection was dependent on the presence o type IVpili on the host cell surace. The linear size o the genome othe phage particle, estimated by pulsed-feld gel electrophore-sis, revealed an exceptionally large redundancy or the circularpermutation. Proteomic analysis o the TMA particle was per-ormed to identiy the putative gene products and determine the

Figure 1. eltron mirogr o t two T. thermophilus g: (a) TMa nd (B)Ys40. T br indit 100 nm.


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gene encoding the long tail fber protein is highly likely to beresponsible or discriminating the host-phage recognition speci-

fcity. Consistent with this idea, comparison o amino acidsequences between the TMA_001 and YSP_001 proteins showeda striking dierence, especially in the C-terminal region, includ-ing a deletion o 30 amino acid residues in the TMA_001 proteinand low amino acid identity o the sequence ollowing the dele-tion (66%) (g. 4).

Both TMA and YS40 are lytic phages. Although lysozymeand holin are generally involved in the lysis process in double-stranded lytic phages,20 we have not ound the genes or lysozymeand holin in TMA and YS40.

reequeg o e ys40 geoe. YS40 was origi-nally isolated by our group,21 and we determined the completegenome sequence oYS40. However, during the preparationo this manuscript, the genome sequence (152,372 bp, 170 pro-tein-coding genes) oYS40 has also been reported by anothergroup (accession no. DQ997624).10 Genome sequence align-ment analysis showed that eight nucleotides were dierent in theprotein-coding regions o these two reported YS40 sequences,resulting the indicated amino acid replacements in the ollow-ing genes: YSP_004 (I88L, ATTCTT), YSP_011 (I102V,ATAGTA), YSP_031 (E202D, GAGGAT), YSP_032(S30L, TCGTTG), YSP_068 (T153K, ACAAA A), YSP_082(H243Q, CATCAG), YSP_152 (I278S, AATAAG), and

One o the noticeable dierences between these twogenomes is the presence o the genes or the putative transposase

(TMA_131) and resolvase (TMA_132) in the TMA genome(see Materials and Methods or the locus_tag prefxes). The geneproduct o TMA_131 showed 57% amino acid identity to thetransposase (accession no. ACN98377) o the hyperthermophileSulurihydrogenibium azorenseAz-Fu1, a member o the Aquifcalesamily. The gene product o TMA_132 showed 59% amino acididentity to the resolvase-like protein (accession no. BAG02822) othe cyanobacteriaMicrocystis aeruginosaNIES-843.

It has been reported that the YS40_065 protein oYS40 haslow sequence similarity to a portion o the terminal protein orDNA replication oBacillus subtilisphage 29 and that the ser-ine residue essential or the protein-primed replication o lineardsDNA genome o29 is conserved in the YS40_065 proteinoYS40.10 Thus, YS40_065 was annotated as a gene encodingthe terminal protein or DNA replication. However, its ortholog(TMA_064) protein in TMA showed only 65% amino acididentity to the YS40_065 protein, and contained an asparagineresidue in place o the serine residue that was essential or theDNA priming (g. 3), suggesting that the TMA_064 proteinis unlikely to unction as a terminal protein or DNA replicationin TMA.TMA ormed clear plaques on both HB8 and HB27 ef-

ciently, whereas YS40 ormed clear plaques only on HB8. The

Figure 2. Linr rrnttion oTMa nd Ys40 gnom. Gn r own uing olord rrow inditing t trnrition dirtion: blu,

rotin-oding gn on t oitiv trnd; orng, rotin-oding gn on t ngtiv trnd; grn, rotin-oding gn in on gnom

wo ortolog r miing rom t otr gnom; nd blk, tRNa gn.


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were selected in the presence o excess TMA as described inMaterials and Methods. These mutants arose at a requency oapproximately 10-5. A well-isolated colony was cloned by serialpassage o diluted cells, and was designated as 27R1. Similarly,a mutant strain resistant to YS40 was isolated rom HB8, andwas designated as 8R. The resistance o 27R1 toTMA and thato 8R to YS40 were urther confrmed using the plaque-orm-ing assay.

The wild-type HB8 and HB27 grown on agar plates showedspreading zones at the edges o their growing areas, which isa characteristic o the twitching motility (results not shown).Visual inspect ion showed that the extent o spreading o HB27was smaller than that o HB8, whereas the phage-resistant

YSP_153 (E71K, GAAAA A). Furthermore, a deletion o onenucleotide was ound between the genes YSP_066 and YSP_067and an insertion o one nucleotide was ound between thegenes YSP_124 and YSP_125. Another dierence was that thegene YSP_096 (our annotation, this study), which is encodedin the same strand as the anking genes (g. 2), was notound in the latter study reported by Naryshkina et al. who,instead, assigned YS40_096 (accession no. ABJ91490) to agene encoded in the strand opposite to the strand encoding theanking genes. Thus, the genes YSP_096 and YS40_096 arenot the same genes.

te iv l-eee eo o tma ys40.Several spontaneous mutant strains o HB27 resistant to TMA

Figure 3. amino id qun lignmnt o t uttiv trminl rotin oYs40 (Ysp_065) nd it orronding ortolog in TMa (TMa_064).

T rin ridu wo ydroxyl grou bn rumd to orm ootr bond wit t 5'-trminl daMp o t Ys40 gnom or DNa

rlition i igligtd in blk. Gry ding indit idntil mino id ridu.

Figure 4. amino id qun lignmnt o t til fbr rotin oTMa (TMa_0 01) nd Ys40 (Ysp_001). Gry ding indit idntil mino

id ridu.


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apilA deletion mutant strain, KA271, was constructed. We sub-sequently confrmed that thispilA mutant strain lacked both thepilus fber on the cell surace (g. 6c) and competence or natu-ral t ransormation. ThepilA mutant strain showed resistance toinection byTMA and non-motility on agar plate (g. 5c).The HB8 genome also contained the pilin gene (TTHA1221,

accession no. NC_006461) and the pilin gene in HB8 showedonly 39% amino acid sequence identity to its counterpart inHB27 (g. 7), which is much lower than those o many otherorthologs o HB8 and HB27. Notably, the location o the cys-teine residues, supposed to be involved in the disulfde-bondedloop ormation, and which are conserved in many type IV pili,was very dierent. To confrm that the pilin gene o HB8 isinvolved in the phage inection process, a mutant strain AU81, inwhich this gene was disrupted, was constructed. Electron micro-scopic analysis showed that AU81 lacked the pilus flament ( g.6). The mutant strain AU81 was also resistant to both YS40and TMA, non-motile (g. 5) and noncompetent or DNAuptake as was seen with 8R. These results demonstrated that thepilin gene o HB8 is one o the requirements or inection withYS40 and TMA phages.

pule-fel gel eleooe. To determine the topologyand sizes o the packaged DNAs oTMA andYS40, the capsidDNA was digested separately with a number o dierent restric-tion enzymes, each enzyme recognizing its own unique restrictionsite, and subsequently the intact and restriction enzyme-digestedcapsid DNAs were subjected to pulsed-feld gel electrophoresis(PFGE). As shown in gue 8, the intact genomes o the ther-mophilic phages were larger than 200 kb (average DNA length,

mutant strains 8R and 27R1 showed dome-shaped colonieswith sharp edges (results not shown). Micromorphology othe twitching or non-twitching zone edge was also examinedby light microscopy. The wild-type strains, HB27 and HB8,showed the characteristic motile rats o cells at the leading edgeo the moving zone (g. 5a d). The motility o HB27

was less intense than HB8, which is consistent with the resultsobtained by visual inspection. In the phage-resistant mutants27R1 and 8R, the motility was completely abolished (g. 5B E).

Electron micrographs o the wild-type cells are shown ingue 6. As shown previously in reerence 22, we confrmed thepilus flament at the poles o the HB27 cells (g. 6a). The pilusflaments with uniorm width were also ound on the surace oHB8, radiating especially rom the poles (g. 6d). Both phageresistant strains 27R1 and 8R lacked such pilus (g. 6B E).

It was previously shown that the uptake o DNA by T.thermophilusHB27 is dependent on the presence o the pili.22Because both 8R and 27R1 strains lacked their pili, we testedtheir competence or natural transormation using a gene knock-out vector that conerred kanamycin resistance as described inMaterials and Methods. We consequently ound that neither othese two mutant strains produced any transormed colonies,whereas the wild-type strains HB8 and HB27 were transormedwith efciencies o 2.0 x 104 and 1.3 x 104 CFU/mg plasmidDNA, respectively.

ThepilA gene o HB27 encoding the pilus structural proteinpilin has been identifed previously in reerence 23. In order toconfrm that the presence o the pili is required or competency,

Figure 5. Twiting motility o wild-ty nd mutnt T. thermophilus. (a) hB27 (wild-t y), (B) 27R1 (TMa-ritnt), (c) Ka271 (pilA), (D) hB8 (wild-

ty), (e) 8R (TMa- nd Ys40-r itnt), (F) aU81 (T Tha1221-). T br indit 50 m.


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amino acid sequence o the 24 kDa proteinby Edman degradation even though thisprotein band was prominent on the SDS-PAGE, suggesting that the N-terminus othe 24 kDa protein might be blocked.

We also identifed fve phage subunits

by mass spectrometric analysis o peptideragments derived rom in-gel trypsindigestion. The mass spectrometric resultsshowed that the trypsin digested peptideragments rom the 71 kDa protein bandwere derived rom the product o theTMA_068 gene. In the similar manner,the peptide ragments obtained rom in-geltrypsin digested 47 kDa, 45 kDa, 26 kDaand 24 kDa proteins (N-terminal sequenceo the last one was ound to be blocked byEdman degradation analysis as describedabove) were also analyzed by mass spec-trometry. Based on these analyses, we

ound that the 47 kDa, 45 kDa, 26 kDaand 24 kDa proteins were derived romthe TMA_072, TMA_019, TMA_066and TMA_067 genes, respectively. These

results were consistent with the N-terminal sequencing results othe TMA_072 and TMA_019 proteins.

Discussion

Ater tectivirus, myovirus is the second most ubiquitous amilyo Thermus phages isolated so ar. However, all o the collectedThermus myoviruses, except or YS40 (which has a genome sizeo >100 kb), resemble coliphage Mu or P2 with a genome size o

2834 kb.9

This report describes the isolation o another myovi-rus, TMA, with large genome size and morphology similar tothose o the YS40, but with dierent host specifcity.

The closed circular map o both TMA and YS40 genomesconsisted o approximately 152 kb long DNA. This va lue is, how-ever, inconsistent with the previous report in which the molecu-lar weight o the YS40 virion DNA, as determined by sucrosedensity gradient centriugation, was estimated to be 1.36 x 108(approximately 206 kb, assuming that the average molecularweight o a single DNA base pair is 660) using the T4 DNAas an internal marker (1.3 x 108, approximately 197 kb),21 sug-gesting that the YS40 genome is longer than the T4 genome.In this study, the length o the genomic DNA purifed rom thephage particles was determined more precisely using PFGE, andwas ound to be more than 200 kb. In the T4-related phages,DNA packaging starts rom any ree end o a long concatemerduplicated in the host cell and terminates when the head is ull,giving rise to a linear DNA with various terminally redundantends within their capsids.24 The redundancy accounts or the cir-cular permutation o the genome and is usually several percento the circular map, which, or example, is approximately 3% inT4. In contrast, the length o the DNA within the phage par-ticles oTMA and YS40 is more than 50 kb longer than the

as determined rom three independent experiments, was 220 4kb or YS40 and 203 2 kb or TMA) and the digested prod-uct appeared as smeared bands (g. s1), which suggests the exis-tence o variable DNA termini in phage particles. These resultsindicate that the genomic DNA is linear and the terminal redun-dancy is 68 4 kb or YS40 and 52 2 kb or TMA, whichcorresponded to 44% and 34%, respectively, o their closed cir-cular orms.

poe ooo o e tma o. To identiy the

structural proteins oTMA, the TMA virions were purifedby cesium chloride density gradient ultracentriugation. Basedon the sequence analysis (by Edman degradation) o the band-orming proteins isolated rom the SDS-PAGE (g. 9), we iden-tifed three N-terminal sequences: ALN AAG QVA E or the47 kDa protein, MTS QGY SLK Y or the 45 kDa protein andSVV DVT VEG or the 30 kDa protein. As described below,peptides identical to each o these three N-terminal sequenceswere ound respectively in three separate proteins encoded by theputative ORFs o the TMA genome, and none o them showedany signifcant homology to proteins encoded by the other ORFsoTMA. The peptide sequence ALN AAG QVA E was oundin the protein encoded by the TMA_072 gene and started atposition 16 (A16) o the encoded protein. This result indicatedthat the TMA_072 protein, the major head protein, is processedbetween residues K15 and A16. The peptide sequence MTSQGY SLK Y was ound in the N-terminal region o the proteinencoded by the TMA_019 gene. The peptide sequence SVVDVT VEG was ound in the protein encoded by the TMA_166gene and started at position 110 (S110) o the encoded protein.This result also suggested that, similar to the TMA_072 protein,the TMA_166 protein was processed between the residues K109and S110. We, however, were unable to determine the N-terminal

Figure 6. eltron mirogr o ty IV ili o wild-ty nd mutntT. thermophilus.

(a) hB27(wild-ty), (B) 27R1 (TMa-ritnt), (c) Ka271 (pilA), (D) hB8 (wild-ty), (e) 8R (TMa- nd

Ys40-ritnt), (F) aU81 (T Tha1221-). T br indit 100 nm.


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mechanism that involves interaction between the core and shellproteins.26 The putative major head proteins o TMA andYS40, encoded by TMA_072 and YSP_073 genes, respectively,could be responsible or the dierences in space within the cap-sid. However, other capsid proteins could also be responsible ordetermining the length and shape o the capsid.27

T4 has approximately 290 probable protein-coding genespacked into its 169 kb long genome whereas TMA and YS40have 168 and 170 protein-coding genes, respectively, in their 152kb long genomes. The average length o ORFs in the TMAand YS40 genomes are 837 bp and 836 bp, respectively, whichare longer than the average length o ORFs (588 bp) in the T4genome, suggesting that the protein-coding genes in the thermo-philic phages are longer than those in the T4 phage. The pres-ence o smaller number o protein-coding genes in TMA andYS40 compared with those in the counterpart mesophilic largevirulent phages suggests that the thermophilic phages might havea simpler lie cycle than the mesophilic ones. Alternatively, it canbe argued that the thermophilic phages might require longer pro-teins to attain stability at higher temperature; however, compara-tive analysis o complete genomes o mesophilic, thermophilicand hyperthermophilic organisms indicated a trend towardshortened thermophilic proteins relative to their mesophilic

circular map, and thus, the redundancy is more than 30%. Thelow mobility o the YS40 genomic DNA in the PFGE assaycould not be explained on the basis o its covalent binding to aprotein, such as the YSP_065 protein that was predicted as theprotein primer in DNA replication, because the DNA sampleused in the PFGE analysis was pre-treated with proteinase K.

The large redundancy may acilitate homologous recombination,contributing to the repair o requently occurring mutations at ahigh temperature. Consistent with this idea, several genes sup-posedly involved in the DNA repair, such as the putative recAand recBgenes, were ound in the TMA and YS40 genomes.It is, however, unclear whether, among all the Thermus myovi-ruses, the large redundancy is specifc to YS40 and TMA,because we currently do not have appropriate inormation on theprecise genome sizes and complete genome sequences o otherThermus myoviruses. Another possible explanation or such along-terminal repeat is that when the thermophilic phages arepropagated in environments other than their native environment(i.e., in the laboratory), many genes are no longer needed and arelost while the terminal repeat is increased in length to physicallycompensate or the lost genes in their capsids and to maintain theefciency o DNA packaging and injection. Results o the PFGEassay also suggest a larger dierence in length between the DNAsoTMA and YS40 genomes than their nucleotide sequencesuggest. The dierence might reect the dierence in capsidlengths, one o the determinants o DNA length in T4.25 It hasbeen shown that the head length o the T4 capsids could be con-trolled by mutations in certain amino acids o the major struc-tural protein, and the head length is determined by a vernier-type

Figure 7. amino id qun lignmnt o t r-ilin o hB8 nd hB27. Gry ding indit idntil mino id ridu. T ytin ri-du involvd in t ormtion o uttiv c-trminl diulfd-bondd loo r igligtd in blk.

Figure 8. pFGe o t gnomi DNa oYs40 nd TMa. ln 1 nd 5,

lmbd DNa lddr; ln 2, T4; ln 3, Ys40; ln 4, TMa.

Figure 9. sDs-paGe nlyi o t TMa virion rotin. T cBB-

tind sDs/olyrylmid gl owing rotin bnd o urifd

TMa virion. protin (bnd inditd uing rrow) wr idntifd

by edmn dgrdtion nd/or m tromtry. Rltiv migrtion

o MW mrkr rotin r inditd on t lt.


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HB8 (this study). These results demonstrate that the myovirusesinect T. thermophilusvia the pilus o the host strain. It has beenshown that the phage PO4 binds to the type IV pili expressing onthe surace oPseudomonas aeruginosa32 and also to the pili heter-ologously expressed on the cell surace oNeisseria gonorrhoeae,33suggesting that the P. aeruginosapili are the primary receptors

or the phage PO4. Because inection o the T. thermophilusstrains byTMA and YS40 was dependent on the existenceo the host pili, they could also be the primary receptors or thethermophilic phages. The amino acid sequence o the pilA geneproducts o HB8 and HB27 diered rom each other, especiallyin the C-terminal region that included the putative disulfde-bonded loop region, which has been shown to be critical or thepilus assembly and twitching motility in P. aeruginosa.34 It hasbeen hypothesized that the sequence diversity o pilin ound inpathogenic bacteria reects an evolutionary compromise betweenthe retention o the unction as a retractile tether or twitchingmotility and antigenic variation against host immune system.34On the other hand, the signifcant dierence ound in the pilusstructural protein o the T. thermophiluslikely reects competi-

tion against the phages. The astonishing divergences in the pri-mary sequences o pilins o the thermophiles and putative tailfber proteins o the phages must have resulted partly rom thecompetition between the thermophiles and the phages in the hotsprings. In addition to the dierences in the type IV pili, com-parative genomics o HB8 and HB27 have shown striking di-erences in the cell surace determinants, including the S-layerproteins and cell envelope-modiying enzymes, such as the gly-cosyltranserases.35 These strain-specifc surace structures otherthan the pili might also act as countermeasures against phages inthe natural environment.

SDS-PAGE analysis o the TMA structural proteins sug-gested that the proteins in the TMA phage particles are very

stable. We ound that proper denaturation o proteins by boilingprior to the sample loading on the gel was very important orreproducibility. Short time boiling (


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In conclusion, we isolated a thermophilic myovirus, TMA,evolutionarily related to YS40. Their divergence seems to bepartly driven by coevolution with the host thermophile thatproceeds via interaction between the tail fber o the phages andthe pili o the host cells and involves a mobile element encod-ing a transposase. The presence o small number o the predictedORFs suggests a unique simple lie cycle or the thermophiliclarge myoviruses. The unexpectedly large terminal redundancyin their genomes suggests a role in the maintenance o geneticinormation through circular permutation and also implies novelsignifcance in processes such as in DNA repair. The gene orderand processing o the head proteins o these phages resemble

those o the mesophilic phages.

Materials and Methods

Bel , e, ge dna. The Thermusstrains used in the present study are described in tble 2. Therich medium used or growingT. thermophilusconsisted o 0.8%(w/v) polypepton, 0.4% (w/v) yeast extract, 0.2% (w/v) NaCl,0.35 mM CaCl

2and 0.4 mM MgCl

2, and the composition o the

synthetic medium used or its growth has been described previ-ously in reerence 40. Escherichia coliJM109 was used or plasmidpropagation. The T. thermophilusphages used in this study wereTMA and YS40.21TMA was isolated rom Atagawa hotspring, Japan, with HB27 as an indicator strain. For plaque or-mation experiment, the top and bottom agar medium contained0.75% and 1.5% agar, respectively. Phage T4 was propagated andpurifed as described previously in reerence 41, to prepare thegenomic DNA that was used as a reerence or the pulsed-feld gelelectrophoresis. Synthetic oligonucleotides used as PCR primersare summarized in tble 3.

Eleo oo. An aliquot o the sample was directlyapplied to a copper grid covered with a thin carbon flm. Aterblotting o the excess liquid, the phages were immediately stained

TMA_072 protein did not show any sequence homol-ogy with the subunits o the registered virus particlesavailable in the public databases, except or the ortho-log ound in YS40.

We could also speculate on the unction o some othe other capsid proteins. First, the primary sequence

o the protein encoded by the TMA_068 gene revealedlow but signifcant homology with the tail sheath pro-teins o other Myoviridae amily o phages having acontractile tail and a linear double-stranded DNA.Although the Myoviridae virion generally containshigh copies o the sheath protein, the TMA_68 geneproduct was not abundant in the SDS-PAGE analy-sis. The transmission electron microscopic analysis oTMA showed requent contracted phage particles,which could have caused by osmotic shock duringthe dialysis ollowing the cesium chloride gradientultracentriugation step. Another reason or contrac-tion might be the sensitivity o the phage particles tohigh concentration o CsCl used or the density gradi-

ent centriugation. It has been shown previously thatYS40 is easily inactivated at high salt concentration.21 A similarsensitivity to high salt concentration could be responsible or thecontraction oTMA tail, leading to aggregation o the sheathprotein. We speculate that the aggregate is resistant to denatur-ation by the standard SDS/PAGE sample buer, and is too largeto enter the separating gel (see g. 9), which could be a majorreason or the observed low abundance o the TMA_068 geneproduct. Second, the product o the TMA_067 gene could be atube protein because o its abundance and molecular weight (24kDa), which is close to the tube proteins o other phages (e.g., 18kDa and 15.9 kDa or the T4 phage and K phage, respectively).Finally, products o the TMA_073 gene and that o its counter-

part YSP_074 gene oYS40 showed partial similarity to theHK97 prohead protease. A strictly conserved pair o serine andhistidine residues ound in the prohead protease superamily38was also conserved in the proteins encoded by the TMA_073 andYSP_074 genes. This gene is located next to the one encoding thephage major head structural protein described above. We, there-ore, hypothesize that the TMA_073 protein oTMA (and alsothe YSP_074 protein oYS40) is the protease involved in thehead maturation. In this way, the order o the genes encoding thehead protease, major capsid protein tail-related sheath and tubeproteins are highly conserved among other Myoviridae (tble 1).It is hard to speculate on the unction o the TMA_066 proteinin TMA virion morphogenesis, because its primary sequencedid not share any homology with the sequences o proteinsinvolved in virion morphogenesis that are available in the pub-lic database. However, it showed a weak homology (22% aminoacid sequence identity) with the putative tube protein encodedby the TMA_067 gene, a probable tube protein-encoding gene(see above). The gp54 protein o T4, whose amino acid sequenceshows partial similarity to the tail tube structura l protein o T4,is believed to unction as a tail tube initiator.39 It is possible thatthe TMA_066 protein could help in the ormation o the tailtube o the thermophilic phage.

Table 1. Gn ordr or t id rotin o Trmu, styloou

nd erii g


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accession numbers are as ollows: AP011617 (TMA)and AP011616 (YS40). The locus_tag prefxes oTMA and YS40 are TMA_ and YSP_, respec-tively, while the locus_tag prefx oYS40 reported byanother group10 (accession no. DQ997624) is YS40_.

cezo o wg ol. The T.

thermophiluscolony to be tested was grown overnighton 1.2% agar (w/v) containing nutrient medium at70C, ater which the edge morphology o the colonieswere examined with a dissecting microscope (BX60,Olympus, Japan).

too ue. The natural transorma-tion efciency oT. thermophiluswas obtained usinga knockout vector, pRMAHTK7, which containsthe T. thermophilus ribosomal L11 methyltranseraseencodingprmA gene interrupted by the thermosta-bilized kanamycin resistant gene as the marker. Toconstruct the plasmid, a DNA ragment containingthe upstream region o the prmA gene was amplifedby PCR using the primers prm1-Kpn and prm2-Hin.

Another DNA ragment containing the downstreamregion o theprmA gene was amplifed by PCR usingthe primers prm3-Eco and prm4-Xba. Two PCRamplifed ragments were purifed. The upstreamragment was digested with the restriction enzymesKpnI and HindIII and the downstream ragment wasdigested with the restriction enzymes EcoRI andXbaI,

and the digested ragments were then sequentially cloned into thecorresponding sites o the plasmid pBHTK1.44 The procedure ornatural transormation o T. thermophilus was described previ-ously in reerence 17. Transormants were selected on 100 g/mL kanamycin supplemented T. thermophilusnutrient mediumagar (1.2%) plate.

iolo o T. thermophilus u e otma ys40. An overnight culture o T. thermophilusHB27 or HB8 cells (about 1 x 107 cells) was mixed with 1 x 108TMA orYS40. Ater overnight incubation at 70C, a survivingcolony on the plate was purifed and isolated. The mutant strainsresistant toTMA andYS40 derived rom HB27 and HB8 weredesignated as 27R1 and 8R, respectively. Resistance to the respec-tive cognate phage was confrmed by plaque orming experiments.

couo oT. thermophilus pilA . AT. thermophi-luspyrEstrain, AM114, was constructed rom HB8 as describedpreviously in reerence 45, with a slight modifcation. In brie, thewild-type HB8 was naturally transormed with pKN605, whichcontains the pyrimidine biosynthetic operon except the entirepyrEgene, by putting a ew drops o the plasmid solution on thetop o the cells grown on a non-selective plate. Ater overnightincubation at 70C, the cells were streaked on a selective platecontaining the minimum medium supplemented with 100 g/ml uracil and 200 g/ml 5-uoroorotic acid, and transormantswere selected and purifed. The deletion o thepyrEgene in theselected cell was confrmed by DNA gel blot hybridization analy-sis (results not shown).

The pyrEgene, amplifed by PCR with the primers 3TSDNand P5 and using pT8L2P46 as a template, was cloned in the

Table 2.T. thermophilus trin ud in ti tudy

with 2% uranyl acetate and the bacterial cells were stained with1% phosphotungstenic acid. The stained samples were observedusing a JEM-1230 transmission electron microscope (JEOL)operated at 100 kV, and the images were recorded using a CCDcamera, Fastscan F114T (TVIPS) or BioScan model 792 (Gatan).

Geoe equeg o. The genome sequences

oTMA and YS40 were determined using a whole-genomeshotgun strategy. For this purpose, we constructed small-insert(~2 kb) genomic libraries, and generated nucleotide sequences o4,224 genomic clones oTMA (12-old coverage) and 9,024genomic clones o YS40 (14-old coverage) rom both endsusing the ABI 3700 Sequencer (Applied Biosystems). Sequencereads were assembled with the help o the Phred-Phrap-Consedprogram and gaps were closed by direct sequencing o clonesthat spanned the gaps or sequencing o PCR products amplifedwith oligonucleotide primers designed to anneal to each end othe neighboring contigs. The overall accuracy o the fnishedsequence was estimated to have an error rate o


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r = 1.29 (4:6), r = 1.36 (5:5), r = 1.46 (6:4) and r = 1.55 (7:3).Approximately 8 ml o each solution was layered in a SPR28SArotor tube (HITACHI) and phage suspension was placed on thetop o the gradient. Ultracentriugation (20,000 rpm) was per-

ormed in a HITACHI SCP70H or 20 min at 4C. The liquidcontent inside the tube was ractionated into 12 tubes and eachraction was examined by SDS-PAGE. The raction containingTMA phage was dialyzed overnight against phosphate-bueredsaline, pH 7.4.

For protein sequencing, the phage solution was mixed withour times its volume o cold (-20C) acetone. Ater centriuga-tion at 10,000x g or 15 min, the phage pellet was dissolved in150 l o 1% SDS and boiled or 5 min. We added 200 l o 6 Murea, 1 M thiourea, 2% Triton X-100 and 1% 2-mercaptoetha-nol. Proteins were precipitated by chloroorm and then washedwith methanol. Ater centriugation at 12,000x g or 2 min, theprecipitate was dissolved in SDS sample buer and applied ontoa 12.5% SDS-PAGE. For Edman degradation, all proteins weretranserred to a PVDF membrane ollowing the electrophoresisand then the membrane was stained with 0.1% CBB in 50%methanol. Pieces o the blotted membrane, each containing aprotein band, were directly subjected to protein sequence analy-sis using a protein sequencer (Applied Biosystems, Procise). Formass spectrometric analysis, in-gel protease digestion was per-ormed as ollows. Ater SDS-PAGE, protein bands were excisedrom the gel and the pieces were washed with 2 l o 25 mMNH

4HCO

3in aqueous 50% acetonitrile. Tryptic digestion was

EcoRV site o pBluescript II SK+ (Agilent Technologies,21205) in the opposite direction o the lacZgene bya blunt-ended ligation reaction. The resultant plas-mid was designated as p3TSDN1. For replacement othe TTHA1221 gene with the pyrEgene in the HB8genome, a knockout vector was constructed as ol-

lows. A DNA ragment encoding the upstream regiono the TTHA1221 gene was amplifed by PCR usingthe primers TTHA1221Kpn and TTHA1221Nde.Another DNA ragment o the downstream regiono the TTHA1221 gene was amplifed by PCR usingthe primers TTHA1221Eco and TTHA1221Bam.Ater purifcation, the upstream ragment was digestedwith the restriction enzymes KpnI and NdeI and thedownstream ragment was digested with the restrictionenzymes EcoRI and BamHI. The digested ragmentswere then sequentially cloned into the correspondingsites o the plasmid p3TSDN1. The resultant plasmid(ppilA1-pyrE) was used to transorm AM114 (pyrE)and one o the transormant was designated as AU81.

Similarly, a knockout vector or replacing thepilA geneo HB27 with thepyrEgene was constructed using thetwo sets o PCR primers, pilA27Kpn/pilANde andpilA27Eco/pilA27Bam, and the plasmid p3TSDN1.The resultant plasmid (ppilA4-pyrE) was used to trans-orm MT111 (pyrE) and one o the transormant wasdesignated as KA271. Replacement o the pilA genewith thepyrEgene in AU81 and KA271 was confrmedby DNA gel blot analysis (results not shown).

pule-fel gel eleooe.YS40 and TMA particleswere purifed by sucrose gradient centriugation as described inreerence 21. The purifed phage particles were dialyzed against abuer (10 mM Tris-HCl, pH 7.5-10 mM MgCl

2) or more than

six hours, ollowing which they were mixed with liquefed 1% lowmelting agarose (BioRad) in 1.2 M Sorbitol-0.1 M EDTA (pH8.0), and then cooled to room temperature to solidiy. The solidi-fed agarose was then cut into adequate sized blocks. The blockswere treated with 0.5 M EDTA (pH 8.0)-1% N-lauroylsarcosin-1mg/ml protease K at 50C overnight and then washed with TEbuer. The phage DNA was then separated on 1% pulsed-feldcertifed agarose gel in 0.5x TBE buer (45 mM Tris, 45 mMboric acid, 1 mM EDTA, pH 8.0) at 4C or 22 h at an angleo 120 using CHEF MAPPER CHEF DR-II (BioRad). Switchtime and voltage used or running the gel were 60 sec and 4.5 V/cm, respectively. Lambda-DNA ladder (BioRad, 170-3635) wasused as the size marker.

poe equee l. Purifcation oTMA or pro-teomic analysis was perormed as ollows. Phage particles romone-liter culture were purifed by ultracentriugation using a six-step cesium chloride gradient. Tris-M buer (82 ml o 1 M HCl,12.1 g o Tris base, 5 g o NaCl, 1 g o NH

4Cl) was used or the

preparation o the cesium chloride stock solution (100 g o CsCl,7.5 ml o Tris-M buer, 66 ml o H

2O), which was diluted with

an appropriate ratio o water to prepare a gradient layer. Followingsix dierent cesium chloride solutions o various density (r) wereprepared: r = 1.13 (stock solution:water = 2:8), r = 1.22 (3:7),

Table 3. Oligonulotid ud in ti tudy


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akowlegee

We thank M. Takahashi (Tokyo University o Pharmacy andLie Sciences), K. Oshima, K. Furuya, C. Yoshino, H. Inaba, K.Motomura and Y. Hattori (University o Tokyo), A. Tamura andN. Itoh (Kitasato University) or technical assistance.

noe

Supplemental material can be ound at: www.landesbioscience.com/journals/bacteriophage/article/16712

initiated with the addition o 5 l o 5 g/ml modifed trypsin(Promega, V5113) in 25 mM NH

4HCO

3. Samples were incubated

overnight at 37C with gentle shaking. Ater centriugation, theresultant peptide-containing supernatants were subjected to ESI-IT-MS analysis (Esquire 3000 plus, Bruker Daltonik GmbH,Bremen, Germany).

dloue o poel co o iee

No potential conicts o interest were disclosed.

reeee

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