Research Article Analysis of Complete Genomes of ...pagk 2348 pagk 2352 pagk 2355 pagk 2346 pagk 2351 pagk 2354 ... 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374 2375 2376

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 918320 11 pageshttpdxdoiorg1011552013918320

Research ArticleAnalysis of Complete Genomes of Propionibacterium acnesReveals a Novel Plasmid and Increased Pseudogenes in an AcneAssociated Strain

Gabriela Kasimatis1 Sorel Fitz-Gibbon1 Shuta Tomida1

Marthew Wong1 and Huiying Li12

1 Department of Molecular and Medical Pharmacology Crump Institute for Molecular Imaging University of CaliforniaLos Angeles CA 90095 USA

2UCLA-DOE Institute for Genomics and Proteomics Los Angeles CA 90095 USA

Correspondence should be addressed to Huiying Li huiyingmednetuclaedu

Received 6 February 2013 Accepted 9 April 2013

Academic Editor Andrew McDowell

Copyright copy 2013 Gabriela Kasimatis et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The human skin harbors a diverse community of bacteria including the Gram-positive anaerobic bacterium Propionibacteriumacnes P acnes has historically been linked to the pathogenesis of acne vulgaris a common skin disease affecting over 80 of alladolescents in theUS To gain insight into potentialP acnes pathogenicmechanisms we previously sequenced the complete genomeof a P acnes strain HL096PA1 that is highly associated with acne In this study we compared its genome to the first publishedcomplete genome KPA171202 HL096PA1 harbors a linear plasmid pIMPLE-HL096PA1This is the first described P acnes plasmidWe also observed a five-fold increase of pseudogenes in HL096PA1 several of which encode proteins in carbohydrate transport andmetabolism In addition our analysis revealed a few island-like genomic regions that are unique to HL096PA1 and a large genomicinversion spanning the ribosomal operons Together these findings offer a basis for understanding P acnes virulent propertieshost adaptation mechanisms and its potential role in acne pathogenesis at the strain level Furthermore the plasmid identifiedin HL096PA1 may potentially provide a new opportunity for P acnes genetic manipulation and targeted therapy against specificdisease-associated strains

1 Introduction

Acne vulgaris commonly called acne is a disease of thepilosebaceous unit of the skin It affects over 80 of alladolescents in the US [1] and persists into adulthood in50 of the cases [2 3] While the etiology of the disease isundefined four pathogenicmechanisms have been proposedincreased sebum production changes in the follicle hor-mone and the activity of the follicular microflora [4ndash8]Antibiotic treatment is one of the main acne therapies target-ing the microbes living in the follicle

Propionibacterium acnes a Gram-positive anaerobic bac-terium has been associated with acne pathogenesis largelydue to the fact that it is commonly isolated from acnelesions [9 10] and that it can cause inflammation in thehost skin Contrarily P acnes is accepted as a commensal

bacterium and in some cases has been shown to play aprotective role against invading pathogenic colonization [11]Our study of the human skin microbiome associated withacne demonstrated that P acnes dominated the pilosebaceousunit in both healthy individuals and acne patients [12] How-ever at the strain level P acnes distributions were signifi-cantly different in the two cohorts suggesting that differentP acnes strains may contribute differently to skin healthand diseaseTherefore understanding the genetic differencesbetween acne-associated strains and other strains is essentialto understanding the phenotypic differences of the strainsand their different roles in acne

Complete genome sequences provide detailed insightsinto genetic variations among strains which may explaintheir phenotypic differences We previously sequenced acompleteP acnes genomeHL096PA1 [12]This strain belongs

2 BioMed Research International

to recA type IA and ribotype (RT) 5 which is highly associ-ated with acne It is resistant tomultiple antibiotics includingtetracycline clindamycin and erythromycin with resistance-conferring mutations in the 16S ribosomal RNA (rRNA)gene (G1058C) and the 23S rRNA gene (A2058G) To dateHL096PA1 is the only available complete genome of acne-associated strains [12]The first sequenced P acnes strain witha complete genome is KPA171202 [13] This strain belongs torecA type IB and RT1 which was not specifically associatedwith acne [12] The KPA171202 genome is 256Mbp longwith 60 GC content 2333 open reading frames (ORFs) areencoded To investigate whether genomic variations amongP acnes strains can explain their differences in virulentproperties in this study we performed a detailed genomecomparison of the genome of HL096PA1 to KPA171202

2 Materials and Methods

21 P acnes HL096PA1 Genome Sequencing Assembly andAnnotation HL096PA1 was sequenced using Roche454FLX as previously described [12]The genomewas finished bymultiple long-range PCRs combinedwith Sanger sequencingGenome assembly and annotation were previously described[12] Extensive manual inspection and editing of the genomeannotation were performed The GenBank accession num-bers for HL096PA1 chromosome and plasmid pIMPLE-HL096PA1 are CP003293 and CP003294 respectively

22 Genome Comparison P acnes genome visualization andsequence comparison were performed using the ARTEMIScomparison tool (httpwwwsangeracuksoftwareACT)[14] Best-BLASTp matches with a cutoff E-value of 1119864 minus 10were used to identify HL096PA1 and KPA orthologous pro-teins

23 Identification and Verification of Pseudogenes Predictedpartial or truncated HL096PA1 protein-coding ORFs werealigned to homologs or truncated proteins in the nonredun-dant protein database to identify pseudogenes For pseu-dogene verification primers flanking the gene regions withframeshifts were designed for suspected HL096PA1 pseudo-genes DNA fragments of 500ndash1000 bp were generated usingPCR Sanger sequencing was used to sequence the full lengthof the amplicons to verify the frameshifts

24 Verification of Genomic Inversion PCR targeting thechromosomal inversion region of HL096PA1 was performedusing the primer sets described in Figure 3(b) Each 20120583L-reaction contained 133 120583L molecular grade H

2O 2 120583L 10X

PCR buffer 06 120583L 50mM MgSO4 04 120583L 10mM dNTPs

08120583L primers (final concentration of 04 120583M) 01 120583L Plat-inum Taq DNA Polymerase High Fidelity (Invitrogen)and 2120583L 30 nguL genomic DNA template Genomic DNAextracted fromHL096PA1 or KPA171202was amplifiedTher-mocycling conditions were as the following initial denatu-ration step of 10 minutes at 94∘C 35 cycles of 45 seconds at94∘C 45 seconds at 54∘C and 7 minutes at 72∘C and a finalelongation step of 20 minutes at 72∘C The primer sequences

Table 1 Comparison of the general features of HL096PA1 andKPA171202 genomes

Features HL096PA1 KPA171202Chromosome Plasmid Chromosome

recA phylotype IA IBRibotype (RT) 5 1Acne association Yes NoSize (bp) 2494191 56169 2560265GC content () 60 62 60Coding sequences 2254 74 2297Pseudogenes 79 0 14Coding density () 90 83 91tRNAs 45 0 45RNA operons 3 0 3Sequencing method 454Roche 454Roche SangerSequencing coverage 50X 87X

used are the following P1 (51015840-CGCCACAGCATCACT-TAAT-31015840) P2 (51015840-ACTTGTTACCACAAACCTATTCTT-31015840) P3 (51015840-CAGACCACTTCTCTAACACACA-31015840) P4 (51015840-GGCTTACCTCAACAATGTTAAA-31015840) P5 (51015840-CACGCA-TTGGAATTACAGAG-31015840) and P6 (51015840-CGAACCTACTCA-GATGGAATTAC-31015840)

3 Results and Discussion

31 General Genome Features of P acnes Strain HL096PA1Similar to other sequenced P acnes strains HL096PA1 hasa circular chromosome of 249 4191 bp (Figure 1) It encodesthree sets of 16S 23S and 5S rRNA operons 45 tRNA genesand 2254 protein coding genes A comparison of the generalgenome features of HL096PA1 and KAP171202 is shown inTable 1 Although HL096PA1 belongs to a different lineage itshares 94 of the sequence with the genome of KPA171202Among the proteins encoded on theHL096PA1 chromosome91 are orthologous to KPA171202 proteins (gt90 aminoacid identity in gt60 sequence length) This suggests thatsmall genomic variations or extrachromosomal elementsmaybe responsible for different phenotypes of the two strains andtheir different associations with acne pathogenesis

32 Plasmid in HL096PA1 pIMPLE-HL096PA1 Comparedto KPA171202 the most significant specific feature ofHL096PA1 is a plasmid HL096PA1 genome sequencing andassembly revealed a second large contig in addition to thechromosome which appears to be an extrachromosomalplasmid [12] The plasmid named pIMPLE-HL096PA1is 56169 bp long It encodes 74 ORFs (PAGK 2319ndashPAGK 2392) and one origin of replication (RepA) Severalplasmid related proteins including plasmid partition proteinParA (PAGK 2332) and plasmid stabilization system toxinand antitoxin proteins (PAGK 2321 and PAGK 2322) areencoded Based on the sequencing coverage and quantitativePCR results the copy number of the plasmid is three pergenome [12] While our attempts to isolate the plasmid inculture have been unsuccessful possibly due to the low copy

BioMed Research International 3

HL096PA1

Propionibacterium acnes

2400000 0

240000

480000

720000

960000

12000001440000

1680000

1920000

2160000

Figure 1 Circular representation of P acnesHL096PA1 chromosome From the outer circle to the inner circle are circle 1 ORFs on the positivestrand (blue) circle 2 ORFs on the negative strand (green) circle 3 pseudogenes (red) circle 4 tRNA (gold) and rRNA (purple) circle 5GC content (lightdark grey) circle 6 GC skew (lightdark grey) DNA Plotter [46] was used in making the figure

number and the large size of the plasmid we have sinceidentified homologous plasmids in multiple other P acnesstrains that belong to the same lineage as HL096PA1 basedon genome sequencing [12]

The assembled sequence contig suggests that pIMPLE-HL096PA1 is a linear plasmid with hairpin ends usedfor bidirectional replication Linear plasmids with terminalinverted repeats (TIRs) and 51015840 terminally attached proteinshave been found in bacteria fungi and higher eukaryotes[15ndash19]Well-studied examples of human pathogenic bacteriawith such linear plasmids include Spirochaete Borrelia andFilamentous Streptomyces [16 20ndash24] TIRs have been shownto be essential for bidirectional linear plasmid replication [2022ndash24] Similar to the linear plasmids observed in Borreliaand Streptomyces pIMPLE-HL096PA1 contains a 20 bp shortTIR with a sequence of ACGACACCAGCACCCACAAC ateach end of the plasmid Due to the difficulty in assemblingthe repeat sequences at the ends the lengths of both endsof the plasmid are not fully defined but were assembled tothe same Based on the assembled plasmid sequence theleft arm TIR is located 743 bp from the start of the plasmidsequence spanning the first ORF (PAGK 2319 an unknownprotein) The right arm TIR is located 743 bp from the 31015840end of the plasmid sequence Analysis of the sequences atthe ends of the plasmid reveals potential hairpin structureswithin the repeat sequences The last ORF on the plasmidPAGK 2392 is located near the right arm TIR and encodesresolvase A (ResA) ResAmay facilitate fusion and resolutionof hairpin telomeres during plasmid replication It may cutthe circular-like plasmid after replication into two linearplasmids with hairpin ends using a mechanism similar totelomere resolvase (ResT) an enzyme commonly encoded bylinear plasmids and shown to facilitate resolution of hairpintelomeres during plasmid replication [25]

The genes encoded on pIMPLE-HL096PA1 have littleredundancy with the HL096PA1 chromosome The plasmidshares only two genes (PAGK 2383 and PAGK 2391) withthe chromosome However it encodes 12 genes (PAGK 2331PAGK 2332 and PAGK 2337ndashPAGK 2346) homologous tothe genes in KPA171202 which are absent on the HL096PA1chromosome and are mostly hypothetical proteins withunknown functions It also encodes an additional est-eraselipase (PAGK 2386) Lipases have been shown to con-tribute to growth advantage and possible pathogenicity of Pacnes in acne Lipases hydrolyze triacylglycerols to free fattyacids whichmay reduce the invasion of other skin pathogensbut may also lead to inflammation and the development ofacne when over-produced [26 27]

Significant horizontal gene transfer is evident withinpIMPLE-HL096PA1 Thirty-five of the 74 predicted ORFs(PAGK 2344ndashPAGK 2378) are highly homologous to ORFsCLOLEP 00122ndashCLOLEP 00166 encoded inClostridium lep-tum DSM 753 (119864-value = 1119864 minus 100 to 0) (Figure 2(a)) aGram-positive commensal bacterium found in the humangut The same region is also highly homologous to thegenomes of Propionibacterium humerusii strains HL037PA2HL037PA3 HL044PA1 and P08 [28] P humerusii is a bac-terium found on the human skin [12] and is closely relatedto P acnes It shares several additional ORFs with pIMPLE-HL096PA1 (Figure 2(a)) Based on our sequencing data ofstrains HL037PA2 HL037PA3 and HL044PA1 the homol-ogous genes in these P humerusii genomes had a three tofive folds higher sequencing coverage than other genes in thegenomes (Figure 2(c)) Although these genomes are not com-plete genomes the higher sequencing coverage of these genessuggests that they may also be located on an extrachrom-osomal mobile genetic element in P humerusii It is unclearwhether this set of ORFs was horizontally transferred among


PAGK 2360 PAGK 2363 PAGK 2365 PAGK 2371 PAGK 2375

PAGK 2358 PAGK 2362 PAGK 2369 PAGK 2370

PAGK 2

PAGK 2376


PAGK 2378

PAGK 2348 PAGK 2352 PAGK 2355


PAGK 2353

PAGK PAG

PAG PAGKPAGKPAGK 2344

PAGK 2356PAGK 2349 0PAGK 2347PAGK 2345

PAGK 23

ORF (PAGK) 2319

2320

2321

2322

2323

2324

2325

2326

2327

2328

2329

2330

2331

2332

2333

2334

2335

2336

2337

2338

2339

2340

2341

2342

2343

2344

2345

2346

2347

2348

2349

2350

2351

2352

2353

2354

2355

2356

2357

2358

2359

2360

2361

2362

2363

2364

2365

2366

2367

2368

2369

2370

2371

2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

2391

2392

P acnes pIMPLE-HL096PA1 P humerusii HL044PA1

C leptum DSM 753

Shared between P acnes and P humerusii Tad locus

50

0

100

150

200

250

Contigs with homologous genes

Contigs without homologous genes





Sequ

ence

cove

rage

HL037PA2 HL037PA3 HL044PA1

(a)

(b)

(c)

15400 17600 19800 22000 24200 26400 28600 30800 33000 35200 37400 39600 41800 44000 46200

Figure 2 The homologous regions among P acnes pIMPLE-HL096PA1 P humerusii HL044PA1 and C leptum DSM 753 (a) The 74 ORFsencoded on the plasmid pIMPLE-HL096PA1 are shown ORFs PAGK 2344ndashPAGK 2378 (blue) including the Tad locus (pink) are sharedamong all three genomes In addition several ORFs are shared between P acnes and P humerusii (orange) ORFs unique to P acnes areshown in grey Absence of P acnes homologous ORFs in P humerusii or C leptum is shown in white (b) Organizations of the ORFs sharedamong all three genomes (c)The sequence coverage of the contigs in three P humerusii strains (HL037PA2 HL037PA3 and HL044PA1) thatshare homologous genes with pIMPLE-HL096PA1 is 3ndash5-folds higher than the contigs that do not share homologous genes with pIMPLE-HL096PA1

P acnes P humerusii and C leptum or transferred from anancestor organism Further investigation into the relation-ships among these microbes that primarily reside at differentbody sites will provide insight to how genetic materials weretransferred and how this mechanism affected the virulentproperties of human commensals

Within this cluster of C leptum homologs on pIMPLE-HL096PA1 we identified a Tad (tight adhesion) locus [12]Tad locus was reported to mediate colonization pathogen-esis and tight adhesion of bacteria to host cell surfacesthrough Flp pilus assembly in Gram-positive pathogens suchas Corynebacterium dipheriae Mycobacterium tuberculosisMycobacterium bovis and Streptomyces coelicor [29ndash31] Theorganizations and annotations of the ORFs in this locusare shown in Figure 2 and Table 2 ORFs PAGK 2360ndashPAGK 2370 encode a set of Tad proteins including Flp

(fimbrial low molecular-weight proteins) pilus proteinsTadZ TadA TadB TadC and TadEThe transcription of Tadlocus in HL096PA1 appears to be controlled by a sigma 70family transcription factor (PAGK 2357) and an antisigmafactor (PAGK 2358) located immediately upstreamof the Tadlocus Sigma 70 family transcription factors are commonlyfound upstream of virulence-associated gene loci and havebeen shown to regulate virulence expression in response toparticular stress-related stimuli in several pathogenic bacteriasuch as Vibrio cholera [32] This suggests that Tad locusexpression in HL096PA1 may be induced under specificconditions leading to enhanced colonization and adhesion tohost cells While KPA171202 does not possess such a plasmidand the Tad locus majority of the RT4 and RT5 strains ofP acnes which are highly associated with acne contain ahomologous plasmid and the Tad locus [12] The unique


KPA171202

HL096PA1

ATCC11828

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(a)

P1P2

P3P4

P5P6

P1P6P5 P3

P4 P2

Primer set IV II V

Primer set I II III

KPA171202

HL096PA1 175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(b)

HL KPA HL KPA HL KPA HL KPA HL KPA

Primer set I II III IV VPrimers P1 P2 P3 P4 P5 P6 P1 P5 P2 P6

Target genome HL096PA1 Both HL096PA1 KPA171202 KPA171202Predicted size of

amplicon (bp)7073 6255 6949 7298 6830

7126

3054

1018

(c)

Figure 3 Genomic inversion in HL096PA1 (a) Genomic inversions observed in P acnes strains HL096PA1 and ATCC11828 compared to Pacnes strain KPA171202 (b) Primer sets IndashV designed to verify the genomic inversion in HL096PA1 (c) PCR amplified DNA fragments ofthe inverted regions shown on an agarose gel Primers target genomes and the predicted sizes of the amplicons are shown in the table HLstrain HL096PA1 KPA strain KPA171202


Table 2 Annotations of the Tad locus in pIMPLE-HL096PA1

ORF Tad homolog Function Pfam ID Pfam 119864-valuePAGK 2360 RcpC Flp pilus assembly protein rough colony protein-C SAF 960119864 minus 05

PAGK 2361 TadZA ATPase NA NAPAGK 2362 TadA ATPase GSPII E 150119864 minus 10

PAGK 2363 TadBC Flp pilus assembly protein GSPII F 210119864 minus 05

PAGK 2364 TadBC Flp pilus assembly protein GSPII F 0014PAGK 2365 NA Unknown NA NAPAGK 2366 TadE Minor Flp pilus assembly protein TadE 00015PAGK 2367 TadE Minor Flp pilus assembly protein TadE 0018PAGK 2368 TadE Minor Flp pilus assembly protein TadE 300119864 minus 07

PAGK 2369 TadB Flp pilus assembly protein LysM BTAD 310119864 minus 10

PAGK 2370 RcpB Flp assembly protein rough colony protein-B Pilus CpaD 053

presence of such a plasmid in acne associated strains maypotentially explain their role in acne pathogenesis and canpossibly be used as a pathogenicity marker

33 Pseudogenes in HL096PA1 Pseudogenes tend to beabundant in bacterial species that have recently adaptedto eukaryotic hosts or a pathogenic lifestyle [33] Thusidentification of pseudogenes in disease associated strainsmay provide insight to their host adaptation and pathogenicmechanisms We identified 79 predicted pseudogenes inHL096PA1 genome (Table 3) In addition to confirmationof the pseudogenes based on the high-quality 454 sequencereads we selected 17 out of the 79 identified pseudogenes andconfirmed the frameshifts in all 17 genes by PCR and Sangersequencing Most pseudogenes represent recent gene disrup-tions and are usually unique to a particular genome [34]HL096PA1 and KPA171202 share only four predicted pseudo-genes Compared to the 14 frameshifted ORFs encoded in theKPA171202 genome [13] HL096PA1 harbors a significantlyincreased number of pseudogenes This may suggest a fasterreduction rate of the chromosome of this strain Sixty-oneof the 79 predicted pseudogenes in HL096PA1 contain singleinactivating mutations indicating that the majority of thesemutations were recently acquired [34]

Although the functions of the predicted pseudogenes inHL096PA1 are diverse 14 of the 79 pseudogenes belong to thefunctional group of carbohydrate transport and metabolismincluding five phosphotransferase system (PTS) componentsresponsible for carbohydrate regulation and sensing Twoof the three predicted fructose PTSs contain frameshiftssuggesting that only one fructose uptake system remainsfunctional in the genome [35] In addition HL096PA1 con-tains two pseudogenes in the only predicted galactitol PTS[35] and one disrupted component in the single glucitol PTSThis suggests that both of these systems are nonfunctional inthis strain These gene disruptions observed in the PTS com-ponents and other proteins of the carbohydrate transport andmetabolism pathwaysmay reflect an adaptation ofHL096PA1to the availability of nutrients in the pilosebaceous unit wherethe major carbon source is lipids but not carbohydrates

In addition to PTS genes multiple virulence-associatedadhesion proteins in HL096PA1 harbor frameshifts

Adhesion proteins are of special interest because of theirpathogenic potential and involvement in biofilm formationBruggemann et al reported nine putative adhesion proteinsin KPA171202 genomewhich contain thrombospondin type 3repeats PKD and SEST domains [13] We found frameshiftsin five of the nine orthologous adhesion proteins inHL096PA1 including PAGK 1592 PAGK 1593 PAGK 1821PAGK 1897 and PAGK 2110 These orthologs were alsorecently reported to contain frameshifts in P acnes strain266 [36] which is a strain more closely related to HL096PA1than to KPA171202 The differences in functional adhesionprotein genes among the strains suggest that various P acnesstrains may use antigenic variations a mechanism used bymany pathogenic bacteria to avoid the host immune system[37] as a host immune invasion mechanism

34 Genomic Islands in HL096PA1 HL096PA1 harbors sev-eral island-like genomic regions In KPA171202 11 genomicislands were reported previously [13] These regions encodeproteins associated with pathogenicity or survival mobilityand proteins of unknown function The chromosome ofHL096PA1 shares eight of the island-like regions foundin KPA171202 Three of the KPA171202 island-like regionsare absent in HL096PA1 including PPA839-878 PPA1278-1321 and PPA1576-1618 In addition to the genomic islandsdescribed by Bruggemann et al [13] an island-like region(PPA2055-2092) in KPA171202 reported later [36] is alsoabsent in HL096PA1 On the other hand HL096PA1 containstwo unique genomic islands locus 1 and locus 2 which havebeen shown to be associated with the disease state of acne[12] These two loci are absent in KPA171202 but present inall strains that belong to the same lineage as HL096 PA1[12] including almost all RT4 and RT5 strains and anothercompletely sequenced strain SK137 [36]

HL096PA1 locus 1 spans over 6Kb and encodes sevenORFs (PAGK 0121ndashPAGK 0127) This region shows evidenceof a cryptic phage-like integration through the flankingtRNAs It encodes phage-related genes such as site-specificrecombinase An antibiotic ABC transporter is also encodedin this region however it is a predicted pseudogene suggest-ing that antibiotic resistance was once conferred through thisphage integration


Table 3 Annotations of the 79 predicted pseudogenes in HL096PA1

Number ORF Start End Function Note1 PAGK 0006 7621 6061 ABC transporter ATP-binding protein Shared with KPA171202

2 PAGK 0019 23673 23196 PTS system galactitol-specific IIAcomponent

Confirmed by PCR and Sangersequencing

3 PAGK 0020 24985 23730 PTS system galactitol-specific IICcomponent


4 PAGK 0024 25937 27844 Hypothetical protein Shared with KPA1712025 PAGK 0025 28692 28418 Hypothetical protein6 PAGK 0026 29964 29294 Hypothetical protein7 PAGK 0089 100317 102505 Glycosyl hydrolase family protein8 PAGK 0123 149702 148105 Site-specific recombinase9 PAGK 0193 226656 225834 Regulator protein10 PAGK 0224 266991 265055 Putative RHS-related protein11 PAGK 0243 289824 290724 Putative ABC transporter

12 PAGK 0352 402744 401670 Hypothetical protein Confirmed by PCR and Sangersequencing

13 PAGK 0393 441551 441884 2-Hydroxy acid dehydrogenase14 PAGK 0395 444633 446378 Hypothetical protein

15 PAGK 0429 474851 476017 Putative sensor histidine kinase twocomponent system

16 PAGK 0440 487718 487064 Hypothetical protein

17 PAGK 0463 510587 509659 Phosphotransferase system proteinmannitolfructose-specific IIA subunit


18 PAGK 0482 529279 530146 Myo-inositol catabolism IolH protein19 PAGK 0573 630324 628497 Hypothetical protein

20 PAGK 0589 653210 651537 Alpha-glucosidaseamylase familyprotein

21 PAGK 0592 657824 655229 Aminopeptidase N22 PAGK 0601 674112 671871 UvrDRep helicase23 PAGK 0603 685081 680455 Hypothetical protein24 PAGK 0604 687943 685068 Putative helicase25 PAGK 0613 702394 698431 Hypothetical protein

26 PAGK 0720 820262 819153 Putative peptide ABC transporterpermease component

27 PAGK 0757 855259 854123 Putative lysophospholipase Confirmed by PCR and Sangersequencing

28 PAGK 0811 908541 907748 Hypothetical protein29 PAGK 0875 986883 986286 Hypothetical protein30 PAGK 0944 1060628 1059358 Prephenate dehydrogenase31 PAGK 1004 1127197 1126813 Hypothetical protein32 PAGK 1011 1133493 1133051 Hypothetical protein

33 PAGK 1158 1298563 1297552 Putative oxidoreductase protein Confirmed by PCR and Sangersequencing

34 PAGK 1160 1299462 1299708 Beta-glucosidase fragment Confirmed by PCR and Sangersequencing

35 PAGK 1181 1318788 1319584 Putative hydrolase Confirmed by PCR and Sangersequencing

36 PAGK 1233 1374383 1376290 PTS system mannitol-specific IIABCcomponent


37 PAGK 1238 1380347 1381136 Putative metallo-beta-lactamase38 PAGK 1242 1386373 1384720 Acetyl-coenzyme A synthetase39 PAGK 1275 1426760 1425953 GntR family regulatory protein


Table 3 Continued

Number ORF Start End Function Note40 PAGK 1276 1426762 1427445 Hypothetical protein41 PAGK 1284 1434222 1434422 Hypothetical protein42 PAGK 1285 1434707 1435190 Transposase Mutator family



45 PAGK 1347 1501212 1499580 ABC transporter ATP-binding protein Confirmed by PCR and Sangersequencing

46 PAGK 1354 1508597 1511439 Oxidoreductase putative D-lactate

47 PAGK 1387 1549654 1548834 Putative GntR-family transcriptionalregulator

48 PAGK 1407 1567445 1569345 Hypothetical protein49 PAGK 1536 1703687 1704612 Putative lysophospholipase

50 PAGK 1586 1764348 1765387 Binding-protein-dependent transportsystem inner membrane component

Confirmed by PCR and Sangersequencing and shared with P acnes J139

51 PAGK 1587 1765384 1766265 Binding-protein-dependent transportsystem inner membrane component Shared with P acnes J139

52 PAGK 1592 1770327 1769760 Protein associated to putative adhesionprotein


54 PAGK 1594 1773223 1771037 Hypothetical protein55 PAGK 1608 1786154 1785310 Hypothetical protein56 PAGK 1643 1820268 1818959 Transporter (sodium sulfate symporter) Shared with KPA171202

57 PAGK 1717 1907494 1905155 Putative ABC transporter-associatedpermease


58 PAGK 1730 1924158 1920911 Endo-beta-N-acetylglucosaminidasefamily protein

59 PAGK 1742 1935480 1935974 Hypothetical protein60 PAGK 1744 1936979 1938333 Putative sialidase

61 PAGK 1760 1952431 1951577 Glycerophosphoryl diesterphosphodiesterase



64 PAGK 1858 2054370 2056156 ABC transporter ATP-binding protein

65 PAGK 1895 2103920 2105327 Pyridine nucleotide-disulphideoxidoreductase

66 PAGK 1897 2107483 2109669 Hypothetical protein Shared with P acnes J13967 PAGK 1966 2185427 2184028 Amidase

68 PAGK 1990 2207618 2209728 51015840-Nucleotidase2101584031015840-cyclicphosphodiesterase

69 PAGK 1995 2211776 2211311 Hypothetical protein70 PAGK 1997 2212029 2212392 Hypothetical protein71 PAGK 2003 2218626 2218090 Hypothetical protein72 PAGK 2008 2222549 2223462 Putative asparaginase

73 PAGK 2023 2236080 2235748 Ornithine carbamoyltransferasecatabolic

74 PAGK 2041 2255990 2257066 Putative lysophospholipase

75 PAGK 2110 2327052 2328480 Putative adhesion or S-layer protein Confirmed by PCR and Sangersequencing



Table 3 Continued

Number ORF Start End Function Note

77 PAGK 2159 2375435 2374944 MarR family transcriptional regulator Confirmed by PCR and Sangersequencing

78 PAGK 2189 2405633 2407955 D-ornithine aminomutase E component

79 PAGK 2225 2450551 2449750 SorC family transcriptional regulator Shared with KPA171202

HL096PA1 locus 2 spans over 20Kb and encodes23 ORFs (PAGK 0160ndashPAGK 0182) Within this genomicisland a complete streptolysin S (SLS) biosynthesis gene clus-ter (PAGK 0168ndashPAGK 0174) is encoded including CAAXamino protease family protein (PAGK 0170) streptolysinprototoxin protein SagA (PAGK 0171) cyclodehydratasegenes SagD SagC and SagB (PAGK 0172ndashPAGK 0174) andtwo hypothetical proteins (PAGK 0168 and PAGK 0169)Streptolysin biosynthetic cluster is commonly found inGram-positive mammalian pathogens such as Clostrid-ium botulinum Listeria monocytogenes and Staphylococcusaureus [38]This gene cluster is potentially capable of produc-ing a nonribosomally synthesized secreted toxin streptolysinStreptolysin is responsible for the classical beta-hemolyticphenotype of bacterial colonies grown on blood agar mediaThis gene cluster inHL096PA1may contribute to its virulenceagainst the host and to its dominant colonization againstother pilosebaceous unit dwelling microbes

The second major group of genes encoded in locus 2 isphage defense genes By using Pfam [39] we identified anabortive infection Abi 2 domain (pfam07751) within ORFPAGK 0180 which is involved in bacteriophage resistancevia premature cell death upon phage entry This abortiveinfection defensemechanism against bacterial phage ismedi-ated by a single copy of Abi 2 domain containing protein[40] A second copy of this Abi 2 domain containing genePAGK 2391 is encoded on the plasmid pIMPLE-HL096PA1This suggests a possible common origin of locus 2 and theplasmid

Additional genomic evidence supports a possible com-mon origin of locus 2 and the plasmid pIMPLE-HL096PA1Located at the immediate downstream of the abortive infec-tion defense system is ORF PAGK 0182 It is a hypotheticalprotein homologous to CLOLEP 00167 in C leptum Asdescribed above the plasmid pIMPLE-HL096PA1 contains acluster of genes with high similarities to C leptum DSM753genes CLOLEP 00122ndashCLOLEP 00166 while the homologof CLOLEP 00167 lies in locus 2 on the chromosome ofHL096PA1 This further suggests a possible common originof locus 2 and the plasmid Notably this gene in locus 2PAGK 0182 contains a 31 bp sequence As reported by Fitz-Gibbon et al [12] this 31 bp sequence was found identical tothe clustered regularly interspaced short palindromic repeat(CRISPR) spacer sequence encoded in some of the type IIP acnes strains This suggests that the genes in locus 2 mayhave been acquired through a phage or plasmid mechanismin RT4 and RT5 strains while type II strains do not harborthis genomic island possibly due to the protectionmechanismof CRISPRcas system against foreign DNA

35 Genomic Inversion in HL096PA1 While genomic inver-sion is commonly seen in bacterial genomes pathogenic bac-teria often exhibit a high degree of genomic rearrangement[41] We observed a large genomic inversion in HL096PA1when compared to KPA171202 The inversion spans theribosomal region of 12Mb ranging from the 1st set to the3rd set of ribosomal operons (Figure 3(a)) To verify theinversion we designed primer sets that span the beginningmiddle and the end of the inverted region (Figure 3(b)) PCRamplifications of these regions were performed As predictedbased on the inverted genomic region HL096PA1 had strongamplifications of the regions covered by primer sets I IIand III but had no specific amplifications using primer setsIV and V (Figure 3(c)) The inversion was further confirmedby sequencing the ends of the amplicons using the Sangermethod KPA171202 was used as a control As predictedKPA171202 had no specific amplifications using primer setsI and III but strong amplifications using primer sets II IVand V Among the nine currently available complete P acnesgenomes ATCC11828 strain [42] also contains a genomicinversion Similar to the inversion in HL096PA1 it spans aribosomal region of 09Mb including the 1st set and the 2ndset of ribosomal operons (Figure 3(a))

4 Conclusions

Our recent study of the skin microbiome has shown thatP acnes resides in the pilosebaceous unit of both healthyskin and acne affected skin [12] challenging the traditionalconcept applied in clinical practice that allP acnes contributesto acne pathogenesis Moreover several studies of the Pacnes strain populations on the skin suggested that acne-associated P acnes subpopulations exist and may correlatewith the genomic variations of the strains [12 22 36 4344] Our genome sequencing and comparison of a largenumber of strains revealed significant disease- or health-associated genetic elements [12] however detailed analysesof extrachromosomal elements pseudogenes and genomestructure variations were limited due to unfinished genomesequences The comparison between two complete genomesHL096PA1 and KPA171202 described in this study allowed usto better understand the strain-specific genomic variationsin plasmid pseudogenes genomic islands and genomicinversion that may be associated with disease pathogenesis

In this comparison we identified several strain-specificgenetic elements that are potentially associated with in-creased virulence ofHL096PA1 in acne Present inHL096PA1and absent in KPA171202 is the first described P acnesplasmid carrying a tight adhesion locus which has been


shown to enhance colonization and biofilm formation inseveral human pathogens [29ndash31] Further investigation ofthis P acnes plasmid will provide insight on its pathogenicpotential Compared to KPA171202 we observed a morethan five-fold increase in the number of pseudogenes inHL096PA1 which suggests a potential mechanism of thisstrain to host adaptation HL096PA1 contains two genomicislands that are unique to acne-associated strains and maybe associated with increased virulence of this lineage in acne[12] In addition a large genomic inversion was observedin HL096PA1 Together these genetic differences revealed bycomplete genome comparison provide insights into the com-plexity of the strain variations of this organism and supportthe existence of specificP acnes subpopulationswhichmay beassociated with acne pathogenesis [36 45] Furthermore thefirst P acnes plasmid identified in HL096PA1 may potentiallyprovide a new opportunity for genetic manipulation of thisorganism The knowledge gained from this comparativestudy may potentially lead to novel diagnostic and targetedtherapeutic approaches for acne against a specific diseaseassociated lineage of P acnes

Conflict of Interests

The authors declare no direct financial interests

Authorsrsquo Contribution

Gabriela Kasimatis performed genome sequence gap-fillingmanual annotation and genome comparison and identifiedand confirmed the pseudogenes in HL096PA1 Sorel Fitz-Gibbon assembled the HL096PA1 genome and performedautomated annotation Shuta Tomida performed genomeannotation and genome comparison and deposited the gen-ome sequence MarthewWong performed experiments veri-fying genomic inversion Gabriela Kasimatis Shuta Tomidaand Huiying Li wrote the paper Huiying Li designed anddirected the project

Acknowledgments

The authors thank Lin Lin for her technical support in fin-ishing theHL096PA1 genomeThey also thankAnya Loncaricand Dr Robert Modlin at UCLA for providing HL096PA1strain This research was partly funded by one of the demon-stration projects by the NIH Human Microbiome Projectunder Grant no UH2AR057503 from NIAMS

References

[1] O L Aldana D B Holland and W J Cunliffe ldquoVariation inpilosebaceous duct keratinocyte proliferation in acne patientsrdquoDermatology vol 196 no 1 pp 98ndash99 1998

[2] W J Cunliffe ldquoManagement of adult acne and acne variantsrdquoJournal of Cutaneous Medicine and Surgery vol 2 supplement3 pp S7ndashS13 1998

[3] CN Collier J CHarperWC CantrellWWang KW Fosterand B E Elewski ldquoThe prevalence of acne in adults 20 years and

olderrdquo Journal of the American Academy of Dermatology vol 58no 1 pp 56ndash59 2008

[4] W J Cunliffe ldquoLooking back to the futuremdashacnerdquoDermatologyvol 204 no 3 pp 167ndash172 2002

[5] G F Webster ldquoInflammation in acne vulgarisrdquo Journal of theAmerican Academy of Dermatology vol 33 no 2 I pp 247ndash2531995

[6] H GollnickW Cunliffe D Berson et al ldquoManagement of acnea report from a global alliance to improve outcomes in acnerdquoJournal of the American Academy of Dermatology vol 49 no 1pp S1ndashS37 2003

[7] U Jappe ldquoPathological mechanisms of acne with special em-phasis on Propionibacterium acnes and related therapyrdquo ActaDermato-Venereologica vol 83 no 4 pp 241ndash248 2003

[8] D Thiboutot H Gollnick V Bettoli et al ldquoNew insights intothe management of acne an update from the Global Allianceto Improve Outcomes in Acne Grouprdquo Journal of the AmericanAcademy of Dermatology vol 60 no 5 pp S1ndashS50 2009

[9] E A Grice H H Kong S Conlan et al ldquoTopographical andtemporal diversity of the human skinmicrobiomerdquo Science vol324 no 5931 pp 1190ndash1192 2009

[10] A E Till V Goulden W J Cunliffe and K T Holland ldquoThecutaneous microflora of adolescent persistent and late-onsetacne patients does not differrdquo British Journal of Dermatologyvol 142 no 5 pp 885ndash892 2000

[11] E A Grice and J A Segre ldquoThe skin microbiomerdquo NatureReviews Microbiology vol 9 no 4 pp 244ndash253 2011

[12] S Fitz-Gibbon S Tomida B H Chiu et al ldquoPropionibacteriumacnes strain populations in the human skin microbiome associ-ated with acnerdquo Journal of Investigative Dermatology 2013

[13] H Bruggemann A Henne F Hoster et al ldquoThe completegenome sequence of Propionibacterium acnes a commensal ofhuman skinrdquo Science vol 305 no 5684 pp 671ndash673 2004

[14] T J Carver KM RutherfordM BerrimanM A RajandreamB G Barrell and J Parkhill ldquoACT the Artemis comparisontoolrdquo Bioinformatics vol 21 no 16 pp 3422ndash3423 2005

[15] A J F Griffiths ldquoNatural plasmids of filamentous fungirdquoMicro-biological Reviews vol 59 no 4 pp 673ndash685 1995

[16] J Hinnebusch and K Tilly ldquoLinear plasmids and chromosomesin bacteriardquoMolecular Microbiology vol 10 no 5 pp 917ndash9221993

[17] F Meinhardt F Kempken J Kamper and K Esser ldquoLinearplasmids among eukaryotes fundamentals and applicationrdquoCurrent Genetics vol 17 no 2 pp 89ndash95 1990

[18] M Rohe J Schrunder P Tudzynski and F Meinhardt ldquoPhy-logenetic relationships of linear protein-primed replicatinggenomesrdquo Current Genetics vol 21 no 2 pp 173ndash176 1992

[19] T J Hosted TWang and A C Horan ldquoCharacterization of theStreptomyces lavendulae IMRU 3455 linear plasmid pSLV45rdquoMicrobiology vol 150 no 6 pp 1819ndash1827 2004

[20] J Hinnebusch and A G Barbour ldquoLinear plasmids of Borreliaburgdorferi have a telomeric structure and sequence similar tothose of a eukaryotic virusrdquo Journal of Bacteriology vol 173 no22 pp 7233ndash7239 1991

[21] A G Barbour and C F Garon ldquoLinear plasmids of the bac-terium Borrelia burgdorferi have covalently closed endsrdquo Sci-ence vol 237 no 4813 pp 409ndash411 1987

[22] A McDowell E Barnard I Nagy et al ldquoAn expanded mul-tilocus sequence typing scheme for propionibacterium acnesinvestigation of lsquopathogenicrsquo lsquocommensarsquo and antibiotic resistantstrainsrdquo PLOS ONE vol 7 Article ID e41480 2012


[23] C W Chen T W Yu Y S Lin H M Kieser and D AHopwood ldquoThe conjugative plasmid SLP2 of Streptomyces livi-dans is a 50 kb linear moleculerdquoMolecular Microbiology vol 7no 6 pp 925ndash932 1993

[24] B Gravius D Glocker J Pigac K Pandza D Hranueli and JCullum ldquoThe 387 kb linear plasmid pPZG101 of Streptomycesrimosus and its interactions with the chromosomerdquoMicrobiol-ogy vol 140 part 9 pp 2271ndash2277 1994

[25] KKobryn andGChaconas ldquoFusion of hairpin telomeres by theB burgdorferi telomere resolvaseResT implications for shapinga genome in fluxrdquo Molecular Cell vol 17 no 6 pp 783ndash7912005

[26] R R Marples D T Downing and A M Kligman ldquoControlof free fatty acids in human surface lipids by Corynebacteriumacnesrdquo Journal of Investigative Dermatology vol 56 no 2 pp127ndash131 1971

[27] T Coenye E Peeters and H J Nelis ldquoBiofilm formation byPropionibacterium acnes is associated with increased resistanceto antimicrobial agents and increased production of putativevirulence factorsrdquo Research in Microbiology vol 158 no 4 pp386ndash392 2007

[28] S M Butler-Wu D J Sengupta W Kittichotirat F A Matsenand R E Bumgarner ldquoGenome sequence of a novel speciesPropionibacterium humerusiirdquo Journal of Bacteriology vol 193no 14 p 3678 2011

[29] H C Schreiner K Sinatra J B Kaplan et al ldquoTight-adherencegenes of Actinobacillus actinomycetemcomitans are requiredfor virulence in a rat modelrdquo Proceedings of the National Acad-emy of Sciences of the United States of America vol 100 no 12pp 7295ndash7300 2003

[30] S C Kachlany P J Planet M K Bhattacharjee et al ldquoNon-specific adherence by Actinobacillus actinomycetemcomitansrequires genes widespread in Bacteria and Archaeardquo Journal ofBacteriology vol 182 no 21 pp 6169ndash6176 2000

[31] M Tomich P J Planet andD H Figurski ldquoThe tad locus post-cards from the widespread colonization islandrdquoNature ReviewsMicrobiology vol 5 no 5 pp 363ndash375 2007

[32] M J Kazmierczak M Wiedmann and K J Boor ldquoAlternativesigma factors and their roles in bacterial virulencerdquo Microbiol-ogy and Molecular Biology Reviews vol 69 no 4 pp 527ndash5432005

[33] S G E Andersson and C G Kurland ldquoReductive evolution ofresident genomesrdquo Trends inMicrobiology vol 6 no 7 pp 263ndash268 1998

[34] H Ochman ldquoGenomes on the shrinkrdquo Proceedings of the Na-tional Academy of Sciences of the United States of America vol102 no 34 pp 11959ndash11960 2005

[35] R D Barabote andM H Saier Jr ldquoComparative genomic anal-yses of the bacterial phosphotransferase systemrdquo MicrobiologyandMolecular Biology Reviews vol 69 no 4 pp 608ndash634 2005

[36] E Brzuszkiewicz J Weiner A Wollherr et al et al ldquoCom-parative genomics and transcriptomics of Propionibacteriumacnesrdquo PLOS ONE vol 6 Article ID e21581 2011

[37] M W Van Der Woude and A J Baumler ldquoPhase and antigenicvariation in bacteriardquo Clinical Microbiology Reviews vol 17 no3 pp 581ndash611 2004

[38] S W Lee D A Mitchell A L Markley et al ldquoDiscovery of awidely distributed toxin biosynthetic gene clusterrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5879ndash5884 2008

[39] R D Finn J Mistry J Tate et al ldquoThe Pfam protein familiesdatabaserdquo Nucleic Acids Research vol 38 no 1 pp D211ndashD2222009

[40] M C Chopin A Chopin and E Bidnenko ldquoPhage abortiveinfection in lactococci variations on a themerdquo Current Opinionin Microbiology vol 8 no 4 pp 473ndash479 2005

[41] A EDarling IMiklos andMARagan ldquoDynamics of genomerearrangement in bacterial populationsrdquo PLoS Genetics vol 4no 7 Article ID e1000128 2008

[42] B Horvath J Hunyadkurti A Voros et al ldquoGenome sequenceof Propionibacterium acnes type II strain ATCC 11828rdquo Journalof Bacteriology vol 194 pp 202ndash203 2012

[43] H B Lomholt and M Kilian ldquoPopulation genetic analysis ofPropionibacterium acnes identifies a subpopulation and epide-mic clones associated with acnerdquo PLOS ONE vol 5 no 8Article ID e12277 2010

[44] A McDowell A Gao E Barnard et al ldquoA novel multilocussequence typing scheme for the opportunistic pathogen pro-pionibacteriumacnes and characterization of type I cell surface-associated antigensrdquoMicrobiology vol 157 no 7 pp 1990ndash20032011

[45] I Nagy A Pivarcsi A Koreck M Szell E Urban and LKemeny ldquoDistinct strains of Propionibacterium acnes induceselective human 120573-defensin-2 and interleukin-8 expression inhuman keratinocytes through toll-like receptorsrdquo Journal ofInvestigative Dermatology vol 124 no 5 pp 931ndash938 2005

[46] T Carver N Thomson A Bleasby M Berriman and JParkhill ldquoDNAPlotter circular and linear interactive genomevisualizationrdquo Bioinformatics vol 25 no 1 pp 119ndash120 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


to recA type IA and ribotype (RT) 5 which is highly associ-ated with acne It is resistant tomultiple antibiotics includingtetracycline clindamycin and erythromycin with resistance-conferring mutations in the 16S ribosomal RNA (rRNA)gene (G1058C) and the 23S rRNA gene (A2058G) To dateHL096PA1 is the only available complete genome of acne-associated strains [12]The first sequenced P acnes strain witha complete genome is KPA171202 [13] This strain belongs torecA type IB and RT1 which was not specifically associatedwith acne [12] The KPA171202 genome is 256Mbp longwith 60 GC content 2333 open reading frames (ORFs) areencoded To investigate whether genomic variations amongP acnes strains can explain their differences in virulentproperties in this study we performed a detailed genomecomparison of the genome of HL096PA1 to KPA171202

2 Materials and Methods

21 P acnes HL096PA1 Genome Sequencing Assembly andAnnotation HL096PA1 was sequenced using Roche454FLX as previously described [12]The genomewas finished bymultiple long-range PCRs combinedwith Sanger sequencingGenome assembly and annotation were previously described[12] Extensive manual inspection and editing of the genomeannotation were performed The GenBank accession num-bers for HL096PA1 chromosome and plasmid pIMPLE-HL096PA1 are CP003293 and CP003294 respectively

22 Genome Comparison P acnes genome visualization andsequence comparison were performed using the ARTEMIScomparison tool (httpwwwsangeracuksoftwareACT)[14] Best-BLASTp matches with a cutoff E-value of 1119864 minus 10were used to identify HL096PA1 and KPA orthologous pro-teins

23 Identification and Verification of Pseudogenes Predictedpartial or truncated HL096PA1 protein-coding ORFs werealigned to homologs or truncated proteins in the nonredun-dant protein database to identify pseudogenes For pseu-dogene verification primers flanking the gene regions withframeshifts were designed for suspected HL096PA1 pseudo-genes DNA fragments of 500ndash1000 bp were generated usingPCR Sanger sequencing was used to sequence the full lengthof the amplicons to verify the frameshifts

24 Verification of Genomic Inversion PCR targeting thechromosomal inversion region of HL096PA1 was performedusing the primer sets described in Figure 3(b) Each 20120583L-reaction contained 133 120583L molecular grade H

2O 2 120583L 10X

PCR buffer 06 120583L 50mM MgSO4 04 120583L 10mM dNTPs

08120583L primers (final concentration of 04 120583M) 01 120583L Plat-inum Taq DNA Polymerase High Fidelity (Invitrogen)and 2120583L 30 nguL genomic DNA template Genomic DNAextracted fromHL096PA1 or KPA171202was amplifiedTher-mocycling conditions were as the following initial denatu-ration step of 10 minutes at 94∘C 35 cycles of 45 seconds at94∘C 45 seconds at 54∘C and 7 minutes at 72∘C and a finalelongation step of 20 minutes at 72∘C The primer sequences

Table 1 Comparison of the general features of HL096PA1 andKPA171202 genomes

Features HL096PA1 KPA171202Chromosome Plasmid Chromosome

recA phylotype IA IBRibotype (RT) 5 1Acne association Yes NoSize (bp) 2494191 56169 2560265GC content () 60 62 60Coding sequences 2254 74 2297Pseudogenes 79 0 14Coding density () 90 83 91tRNAs 45 0 45RNA operons 3 0 3Sequencing method 454Roche 454Roche SangerSequencing coverage 50X 87X

used are the following P1 (51015840-CGCCACAGCATCACT-TAAT-31015840) P2 (51015840-ACTTGTTACCACAAACCTATTCTT-31015840) P3 (51015840-CAGACCACTTCTCTAACACACA-31015840) P4 (51015840-GGCTTACCTCAACAATGTTAAA-31015840) P5 (51015840-CACGCA-TTGGAATTACAGAG-31015840) and P6 (51015840-CGAACCTACTCA-GATGGAATTAC-31015840)

3 Results and Discussion

31 General Genome Features of P acnes Strain HL096PA1Similar to other sequenced P acnes strains HL096PA1 hasa circular chromosome of 249 4191 bp (Figure 1) It encodesthree sets of 16S 23S and 5S rRNA operons 45 tRNA genesand 2254 protein coding genes A comparison of the generalgenome features of HL096PA1 and KAP171202 is shown inTable 1 Although HL096PA1 belongs to a different lineage itshares 94 of the sequence with the genome of KPA171202Among the proteins encoded on theHL096PA1 chromosome91 are orthologous to KPA171202 proteins (gt90 aminoacid identity in gt60 sequence length) This suggests thatsmall genomic variations or extrachromosomal elementsmaybe responsible for different phenotypes of the two strains andtheir different associations with acne pathogenesis

32 Plasmid in HL096PA1 pIMPLE-HL096PA1 Comparedto KPA171202 the most significant specific feature ofHL096PA1 is a plasmid HL096PA1 genome sequencing andassembly revealed a second large contig in addition to thechromosome which appears to be an extrachromosomalplasmid [12] The plasmid named pIMPLE-HL096PA1is 56169 bp long It encodes 74 ORFs (PAGK 2319ndashPAGK 2392) and one origin of replication (RepA) Severalplasmid related proteins including plasmid partition proteinParA (PAGK 2332) and plasmid stabilization system toxinand antitoxin proteins (PAGK 2321 and PAGK 2322) areencoded Based on the sequencing coverage and quantitativePCR results the copy number of the plasmid is three pergenome [12] While our attempts to isolate the plasmid inculture have been unsuccessful possibly due to the low copy


HL096PA1


2400000 0

240000

480000

720000

960000

12000001440000

1680000

1920000

2160000









PAGK 2

PAGK 2376


PAGK 2378



PAGK 2353

PAGK PAG



PAGK 23

ORF (PAGK) 2319

2320

2321

2322

2323

2324

2325

2326

2327

2328

2329

2330

2331

2332

2333

2334

2335

2336

2337

2338

2339

2340

2341

2342

2343

2344

2345

2346

2347

2348

2349

2350

2351

2352

2353

2354

2355

2356

2357

2358

2359

2360

2361

2362

2363

2364

2365

2366

2367

2368

2369

2370

2371

2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

2391

2392


C leptum DSM 753


50

0

100

150

200

250







Sequ

ence

cove

rage


(a)

(b)

(c)

15400 17600 19800 22000 24200 26400 28600 30800 33000 35200 37400 39600 41800 44000 46200






KPA171202

HL096PA1

ATCC11828

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(a)

P1P2

P3P4

P5P6

P1P6P5 P3

P4 P2

Primer set IV II V

Primer set I II III

KPA171202

HL096PA1 175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(b)




amplicon (bp)7073 6255 6949 7298 6830

7126

3054

1018

(c)












































Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


HL096PA1


2400000 0

240000

480000

720000

960000

12000001440000

1680000

1920000

2160000









PAGK 2

PAGK 2376


PAGK 2378



PAGK 2353

PAGK PAG



PAGK 23

ORF (PAGK) 2319

2320

2321

2322

2323

2324

2325

2326

2327

2328

2329

2330

2331

2332

2333

2334

2335

2336

2337

2338

2339

2340

2341

2342

2343

2344

2345

2346

2347

2348

2349

2350

2351

2352

2353

2354

2355

2356

2357

2358

2359

2360

2361

2362

2363

2364

2365

2366

2367

2368

2369

2370

2371

2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

2391

2392


C leptum DSM 753


50

0

100

150

200

250







Sequ

ence

cove

rage


(a)

(b)

(c)

15400 17600 19800 22000 24200 26400 28600 30800 33000 35200 37400 39600 41800 44000 46200






KPA171202

HL096PA1

ATCC11828

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(a)

P1P2

P3P4

P5P6

P1P6P5 P3

P4 P2

Primer set IV II V

Primer set I II III

KPA171202

HL096PA1 175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(b)




amplicon (bp)7073 6255 6949 7298 6830

7126

3054

1018

(c)












































Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




PAGK 2

PAGK 2376


PAGK 2378



PAGK 2353

PAGK PAG



PAGK 23

ORF (PAGK) 2319

2320

2321

2322

2323

2324

2325

2326

2327

2328

2329

2330

2331

2332

2333

2334

2335

2336

2337

2338

2339

2340

2341

2342

2343

2344

2345

2346

2347

2348

2349

2350

2351

2352

2353

2354

2355

2356

2357

2358

2359

2360

2361

2362

2363

2364

2365

2366

2367

2368

2369

2370

2371

2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

2391

2392


C leptum DSM 753


50

0

100

150

200

250







Sequ

ence

cove

rage


(a)

(b)

(c)

15400 17600 19800 22000 24200 26400 28600 30800 33000 35200 37400 39600 41800 44000 46200






KPA171202

HL096PA1

ATCC11828

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(a)

P1P2

P3P4

P5P6

P1P6P5 P3

P4 P2

Primer set IV II V

Primer set I II III

KPA171202

HL096PA1 175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(b)




amplicon (bp)7073 6255 6949 7298 6830

7126

3054

1018

(c)












































Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


KPA171202

HL096PA1

ATCC11828

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(a)

P1P2

P3P4

P5P6

P1P6P5 P3

P4 P2

Primer set IV II V

Primer set I II III

KPA171202

HL096PA1 175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

175000 350000 525000 700000 875000 1050000 1225000 1400000 1575000 1750000 1925000 2100000 2275000 2450000

(b)




amplicon (bp)7073 6255 6949 7298 6830

7126

3054

1018

(c)












































Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued































Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued









4 Conclusions









Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Analysis of Complete Genomes of ...pagk 2348 pagk 2352 pagk 2355 pagk 2346 pagk 2351 pagk 2354 ... 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374 2375 2376