# Analysis of Complete Genomes ofPropionibacterium acnes Reveals a Novel Plasmid and Increased Pseudogenes in an Acne Associated Strain

Authors: Gabriela Kasimatis; Sorel Fitz-Gibbon; Shuta Tomida; Marthew Wong; Huiying Li
Journal: BioMed Research International
 (2013)

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## Abstract

The human skin harbors a diverse community of bacteria, including the Gram-positive, anaerobic bacteriumPropionibacterium acnes. P. acnes has historically been linked to the pathogenesis of acne vulgaris, a common skin disease affecting over 80% of all adolescents in the US. To gain insight into potential P. acnes pathogenic mechanisms, we previously sequenced the complete genome of a P. acnes strain HL096PA1 that is highly associated with acne. In this study, we compared its genome to the first published complete genome KPA171202. HL096PA1 harbors a linear plasmid, pIMPLE-HL096PA1. This is the first described P. acnes plasmid. We also observed a five-fold increase of pseudogenes in HL096PA1, several of which encode proteins in carbohydrate transport and metabolism. In addition, our analysis revealed a few island-like genomic regions that are unique to HL096PA1 and a large genomic inversion spanning the ribosomal operons. Together, these findings offer a basis for understanding P. acnes virulent properties, host adaptation mechanisms, and its potential role in acne pathogenesis at the strain level. Furthermore, the plasmid identified in HL096PA1 may potentially provide a new opportunity for P. acnes genetic manipulation and targeted therapy against specific disease-associated strains.


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## Body

## 1. Introduction

Acne vulgaris, commonly called acne, is a disease of the pilosebaceous unit of the skin. It affects over 80% of all adolescents in the US [1] and persists into adulthood in 50% of the cases [2, 3]. While the etiology of the disease is undefined, four pathogenic mechanisms have been proposed: increased sebum production, changes in the follicle, hormone, and the activity of the follicular microflora [4–8]. Antibiotic treatment is one of the main acne therapies targeting the microbes living in the follicle.Propionibacterium acnes, a Gram-positive anaerobic bacterium, has been associated with acne pathogenesis, largely due to the fact that it is commonly isolated from acne lesions [9, 10] and that it can cause inflammation in the host skin. Contrarily, P. acnes is accepted as a commensal bacterium and in some cases has been shown to play a protective role against invading pathogenic colonization [11]. Our study of the human skin microbiome associated with acne demonstrated that P. acnes dominated the pilosebaceous unit in both healthy individuals and acne patients [12]. However, at the strain level, P. acnes distributions were significantly different in the two cohorts, suggesting that different P. acnes strains may contribute differently to skin health and disease. Therefore, understanding the genetic differences between acne-associated strains and other strains is essential to understanding the phenotypic differences of the strains and their different roles in acne.Complete genome sequences provide detailed insights into genetic variations among strains, which may explain their phenotypic differences. We previously sequenced a completeP. acnes genome, HL096PA1 [12]. This strain belongs to recA type IA and ribotype (RT) 5, which is highly associated with acne. It is resistant to multiple antibiotics, including tetracycline, clindamycin, and erythromycin with resistance-conferring mutations in the 16S ribosomal RNA (rRNA) gene (G1058C) and the 23S rRNA gene (A2058G). To date, HL096PA1 is the only available complete genome of acne-associated strains [12]. The first sequenced P. acnes strain with a complete genome is KPA171202 [13]. This strain belongs to recA type IB and RT1, which was not specifically associated with acne [12]. The KPA171202 genome is 2.56 M bp long with 60% GC content. 2,333 open reading frames (ORFs) are encoded. To investigate whether genomic variations among P. acnes strains can explain their differences in virulent properties, in this study we performed a detailed genome comparison of the genome of HL096PA1 to KPA171202.


## 2. Materials and Methods

### 2.1.P. acnes HL096PA1 Genome Sequencing, Assembly and Annotation

HL096PA1 was sequenced using Roche/454 FLX as previously described [12]. The genome was finished by multiple long-range PCRs combined with Sanger sequencing. Genome assembly and annotation were previously described [12]. Extensive manual inspection and editing of the genome annotation were performed. The GenBank accession numbers for HL096PA1 chromosome and plasmid pIMPLE-HL096PA1 are CP003293 and CP003294, respectively.


### 2.2. Genome Comparison

P. acnes genome visualization and sequence comparison were performed using the ARTEMIS comparison tool (http://www.sanger.ac.uk/software/ACT/) [14]. Best-BLASTp matches with a cutoff E-value of 1E-10 were used to identify HL096PA1 and KPA orthologous proteins.


### 2.3. Identification and Verification of Pseudogenes

Predicted partial or truncated HL096PA1 protein-coding ORFs were aligned to homologs or truncated proteins in the nonredundant protein database to identify pseudogenes. For pseudogene verification, primers flanking the gene regions with frameshifts were designed for suspected HL096PA1 pseudogenes. DNA fragments of 500–1,000 bp were generated using PCR. Sanger sequencing was used to sequence the full length of the amplicons to verify the frameshifts.


### 2.4. Verification of Genomic Inversion

PCR targeting the chromosomal inversion region of HL096PA1 was performed using the primer sets described in Figure3(b). Each 20 μL-reaction contained 13.3 μL molecular grade H2O, 2 μL 10X PCR buffer, 0.6 μL 50 mM MgSO4, 0.4 μL 10 mM dNTPs, 0.8 μL primers (final concentration of 0.4 μM), 0.1 μL Platinum Taq DNA Polymerase High Fidelity (Invitrogen), and 2 μL 30 ng/uL genomic DNA template. Genomic DNA extracted from HL096PA1 or KPA171202 was amplified. Thermocycling conditions were as the following: initial denaturation step of 10 minutes at 94°C, 35 cycles of 45 seconds at 94°C, 45 seconds at 54°C, and 7 minutes at 72°C and a final elongation step of 20 minutes at 72°C. The primer sequences used are the following: P1 (5′-CGCCACAGCATCACTTAAT-3′), P2 (5′-ACTTGTTACCACAAACCTATTCTT-3′), P3 (5′-CAGACCACTTCTCTAACACACA-3′), P4 (5′-GGCTTACCTCAACAATGTTAAA-3′), P5 (5′-CACGCATTGGAATTACAGAG-3′), and P6 (5′-CGAACCTACTCAGATGGAATTAC-3′).


## 2.1.P. acnes HL096PA1 Genome Sequencing, Assembly and Annotation

HL096PA1 was sequenced using Roche/454 FLX as previously described [12]. The genome was finished by multiple long-range PCRs combined with Sanger sequencing. Genome assembly and annotation were previously described [12]. Extensive manual inspection and editing of the genome annotation were performed. The GenBank accession numbers for HL096PA1 chromosome and plasmid pIMPLE-HL096PA1 are CP003293 and CP003294, respectively.


## 2.2. Genome Comparison

P. acnes genome visualization and sequence comparison were performed using the ARTEMIS comparison tool (http://www.sanger.ac.uk/software/ACT/) [14]. Best-BLASTp matches with a cutoff E-value of 1E-10 were used to identify HL096PA1 and KPA orthologous proteins.


## 2.3. Identification and Verification of Pseudogenes

Predicted partial or truncated HL096PA1 protein-coding ORFs were aligned to homologs or truncated proteins in the nonredundant protein database to identify pseudogenes. For pseudogene verification, primers flanking the gene regions with frameshifts were designed for suspected HL096PA1 pseudogenes. DNA fragments of 500–1,000 bp were generated using PCR. Sanger sequencing was used to sequence the full length of the amplicons to verify the frameshifts.


## 2.4. Verification of Genomic Inversion

PCR targeting the chromosomal inversion region of HL096PA1 was performed using the primer sets described in Figure3(b). Each 20 μL-reaction contained 13.3 μL molecular grade H2O, 2 μL 10X PCR buffer, 0.6 μL 50 mM MgSO4, 0.4 μL 10 mM dNTPs, 0.8 μL primers (final concentration of 0.4 μM), 0.1 μL Platinum Taq DNA Polymerase High Fidelity (Invitrogen), and 2 μL 30 ng/uL genomic DNA template. Genomic DNA extracted from HL096PA1 or KPA171202 was amplified. Thermocycling conditions were as the following: initial denaturation step of 10 minutes at 94°C, 35 cycles of 45 seconds at 94°C, 45 seconds at 54°C, and 7 minutes at 72°C and a final elongation step of 20 minutes at 72°C. The primer sequences used are the following: P1 (5′-CGCCACAGCATCACTTAAT-3′), P2 (5′-ACTTGTTACCACAAACCTATTCTT-3′), P3 (5′-CAGACCACTTCTCTAACACACA-3′), P4 (5′-GGCTTACCTCAACAATGTTAAA-3′), P5 (5′-CACGCATTGGAATTACAGAG-3′), and P6 (5′-CGAACCTACTCAGATGGAATTAC-3′).


## 3. Results and Discussion

### 3.1. General Genome Features ofP. acnes Strain HL096PA1

Similar to other sequencedP. acnes strains, HL096PA1 has a circular chromosome of 249, 4191 bp (Figure 1). It encodes three sets of 16S, 23S, and 5S rRNA operons, 45 tRNA genes, and 2,254 protein coding genes. A comparison of the general genome features of HL096PA1 and KAP171202 is shown in Table 1. Although HL096PA1 belongs to a different lineage, it shares 94% of the sequence with the genome of KPA171202. Among the proteins encoded on the HL096PA1 chromosome, 91% are orthologous to KPA171202 proteins (>90% amino acid identity in >60% sequence length). This suggests that small genomic variations or extrachromosomal elements may be responsible for different phenotypes of the two strains and their different associations with acne pathogenesis.Table 1
Comparison of the general features of HL096PA1 and KPA171202 genomes.
Features
HL096PA1
KPA171202
Chromosome
Plasmid
Chromosome
recA phylotype
IA
IB
Ribotype (RT)
5
1
Acne association
Yes
No
Size (bp)
2,494,191
56,169
2,560,265
GC content (%)
60
62
60
Coding sequences
2,254
74
2,297
Pseudogenes
79
0
14
Coding density (%)
90
83
91
tRNAs
45
0
45
RNA operons
3
0
3
Sequencing method
454/Roche
454/Roche
Sanger
Sequencing coverage
50X
8.7XFigure 1
Circular representation ofP. acnes HL096PA1 chromosome. From the outer circle to the inner circle are circle 1, ORFs on the positive strand (blue); circle 2, ORFs on the negative strand (green); circle 3, pseudogenes (red); circle 4, tRNA (gold) and rRNA (purple); circle 5, GC content (light/dark grey); circle 6, GC skew (light/dark grey). DNA Plotter [46] was used in making the figure.


### 3.2. Plasmid in HL096PA1, pIMPLE-HL096PA1

Compared to KPA171202, the most significant specific feature of HL096PA1 is a plasmid. HL096PA1 genome sequencing and assembly revealed a second large contig in addition to the chromosome, which appears to be an extrachromosomal plasmid [12]. The plasmid, named pIMPLE-HL096PA1, is 56,169 bp long. It encodes 74 ORFs (PAGK_2319–PAGK_2392) and one origin of replication (RepA). Several plasmid related proteins, including plasmid partition protein ParA (PAGK_2332) and plasmid stabilization system toxin and antitoxin proteins (PAGK_2321 and PAGK_2322), are encoded. Based on the sequencing coverage and quantitative PCR results, the copy number of the plasmid is three per genome [12]. While our attempts to isolate the plasmid in culture have been unsuccessful possibly due to the low copy number and the large size of the plasmid, we have since identified homologous plasmids in multiple other P. acnes strains that belong to the same lineage as HL096PA1 based on genome sequencing [12].The assembled sequence contig suggests that pIMPLE-HL096PA1 is a linear plasmid, with hairpin ends used for bidirectional replication. Linear plasmids with terminal inverted repeats (TIRs) and 5′ terminally attached proteins have been found in bacteria, fungi, and higher eukaryotes [15–19]. Well-studied examples of human pathogenic bacteria with such linear plasmids include Spirochaete Borrelia and Filamentous Streptomyces [16, 20–24]. TIRs have been shown to be essential for bidirectional linear plasmid replication [20, 22–24]. Similar to the linear plasmids observed in Borrelia and Streptomyces, pIMPLE-HL096PA1 contains a 20 bp short TIR with a sequence of ACGACACCAGCACCCACAAC at each end of the plasmid. Due to the difficulty in assembling the repeat sequences at the ends, the lengths of both ends of the plasmid are not fully defined but were assembled to the same. Based on the assembled plasmid sequence, the left arm TIR is located 743 bp from the start of the plasmid sequence, spanning the first ORF (PAGK_2319, an unknown protein). The right arm TIR is located 743 bp from the 3′ end of the plasmid sequence. Analysis of the sequences at the ends of the plasmid reveals potential hairpin structures within the repeat sequences. The last ORF on the plasmid, PAGK_2392, is located near the right arm TIR and encodes resolvase A (ResA). ResA may facilitate fusion and resolution of hairpin telomeres during plasmid replication. It may cut the circular-like plasmid after replication into two linear plasmids with hairpin ends, using a mechanism similar to telomere resolvase (ResT), an enzyme commonly encoded by linear plasmids and shown to facilitate resolution of hairpin telomeres during plasmid replication [25].The genes encoded on pIMPLE-HL096PA1 have little redundancy with the HL096PA1 chromosome. The plasmid shares only two genes (PAGK_2383 and PAGK_2391) with the chromosome. However, it encodes 12 genes (PAGK_2331, PAGK_2332, and PAGK_2337–PAGK_2346) homologous to the genes in KPA171202, which are absent on the HL096PA1 chromosome and are mostly hypothetical proteins with unknown functions. It also encodes an additional esterase/lipase (PAGK_2386). Lipases have been shown to contribute to growth advantage and possible pathogenicity ofP. acnes in acne. Lipases hydrolyze triacylglycerols to free fatty acids, which may reduce the invasion of other skin pathogens but may also lead to inflammation and the development of acne when over-produced [26, 27].Significant horizontal gene transfer is evident within pIMPLE-HL096PA1. Thirty-five of the 74 predicted ORFs (PAGK_2344–PAGK_2378) are highly homologous to ORFs CLOLEP_00122–CLOLEP_00166 encoded inClostridium leptum DSM 753 (E-value = 1E-100 to 0) (Figure 2(a)), a Gram-positive commensal bacterium found in the human gut. The same region is also highly homologous to the genomes of Propionibacterium humerusii strains HL037PA2, HL037PA3, HL044PA1, and P08 [28]. P. humerusii is a bacterium found on the human skin [12] and is closely related to P. acnes. It shares several additional ORFs with pIMPLE-HL096PA1 (Figure 2(a)). Based on our sequencing data of strains HL037PA2, HL037PA3, and HL044PA1, the homologous genes in these P. humerusiigenomes had a three to five folds higher sequencing coverage than other genes in the genomes (Figure 2(c)). Although these genomes are not complete genomes, the higher sequencing coverage of these genes suggests that they may also be located on an extrachromosomal mobile genetic element in P. humerusii. It is unclear whether this set of ORFs was horizontally transferred among P. acnes, P. humerusii, and C. leptum or transferred from an ancestor organism. Further investigation into the relationships among these microbes that primarily reside at different body sites will provide insight to how genetic materials were transferred and how this mechanism affected the virulent properties of human commensals.Figure 2
The homologous regions amongP. acnes pIMPLE-HL096PA1, P. humerusii HL044PA1, and C. leptum DSM 753. (a) The 74 ORFs encoded on the plasmid pIMPLE-HL096PA1 are shown. ORFs PAGK_2344–PAGK_2378 (blue), including the Tad locus (pink), are shared among all three genomes. In addition, several ORFs are shared between P. acnes and P. humerusii (orange). ORFs unique to P. acnesare shown in grey. Absence of P. acneshomologous ORFs in P. humerusii or C. leptum is shown in white. (b) Organizations of the ORFs shared among all three genomes. (c) The sequence coverage of the contigs in three P. humerusii strains (HL037PA2, HL037PA3, and HL044PA1) that share homologous genes with pIMPLE-HL096PA1 is 3–5-folds higher than the contigs that do not share homologous genes with pIMPLE-HL096PA1.Genomic inversion in HL096PA1. (a) Genomic inversions observed inP. acnes strains HL096PA1 and ATCC11828 compared to P. acnes strain KPA171202. (b) Primer sets I–V designed to verify the genomic inversion in HL096PA1. (c) PCR amplified DNA fragments of the inverted regions shown on an agarose gel. Primers, target genomes, and the predicted sizes of the amplicons are shown in the table. HL, strain HL096PA1; KPA, strain KPA171202.
(a)
(b)
(c)Within this cluster ofC. leptum homologs on pIMPLE-HL096PA1, we identified a Tad (tight adhesion) locus [12]. Tad locus was reported to mediate colonization, pathogenesis, and tight adhesion of bacteria to host cell surfaces through Flp pilus assembly in Gram-positive pathogens, such as Corynebacterium dipheriae, Mycobacterium tuberculosis, Mycobacterium bovis,andStreptomyces coelicor[29–31]. The organizations and annotations of the ORFs in this locus are shown in Figure 2 and Table 2. ORFs PAGK_2360–PAGK_2370 encode a set of Tad proteins, including Flp (fimbrial low molecular-weight proteins) pilus proteins, TadZ, TadA, TadB, TadC, and TadE. The transcription of Tad locus in HL096PA1 appears to be controlled by a sigma 70 family transcription factor (PAGK_2357) and an antisigma factor (PAGK_2358) located immediately upstream of the Tad locus. Sigma 70 family transcription factors are commonly found upstream of virulence-associated gene loci and have been shown to regulate virulence expression in response to particular stress-related stimuli in several pathogenic bacteria such as Vibrio cholera [32]. This suggests that Tad locus expression in HL096PA1 may be induced under specific conditions, leading to enhanced colonization and adhesion to host cells. While KPA171202 does not possess such a plasmid and the Tad locus, majority of the RT4 and RT5 strains of P. acnes, which are highly associated with acne, contain a homologous plasmid and the Tad locus [12]. The unique presence of such a plasmid in acne associated strains may potentially explain their role in acne pathogenesis and can possibly be used as a pathogenicity marker.Table 2
Annotations of the Tad locus in pIMPLE-HL096PA1.
ORF
Tad homolog
Function
Pfam ID
PfamE-value
PAGK_2360
RcpC
Flp pilus assembly protein, rough colony protein-C
SAF
9.60
E
-
05
PAGK_2361
TadZ/A
ATPase
N/A
N/A
PAGK_2362
TadA
ATPase
GSPII_E
1.50
E
-
10
PAGK_2363
TadB/C
Flp pilus assembly protein
GSPII_F
2.10
E
-
05
PAGK_2364
TadB/C
Flp pilus assembly protein
GSPII_F
0.014
PAGK_2365
N/A
Unknown
N/A
N/A
PAGK_2366
TadE
Minor Flp pilus assembly protein
TadE
0.0015
PAGK_2367
TadE
Minor Flp pilus assembly protein
TadE
0.018
PAGK_2368
TadE
Minor Flp pilus assembly protein
TadE
3.00
E
-
07
PAGK_2369
TadB
Flp pilus assembly protein
LysM, BTAD
3.10
E
-
10
PAGK_2370
RcpB
Flp assembly protein, rough colony protein-B
Pilus, CpaD
0.53


### 3.3. Pseudogenes in HL096PA1

Pseudogenes tend to be abundant in bacterial species that have recently adapted to eukaryotic hosts or a pathogenic lifestyle [33]. Thus identification of pseudogenes in disease associated strains may provide insight to their host adaptation and pathogenic mechanisms. We identified 79 predicted pseudogenes in HL096PA1 genome (Table 3). In addition to confirmation of the pseudogenes based on the high-quality 454 sequence reads, we selected 17 out of the 79 identified pseudogenes and confirmed the frameshifts in all 17 genes by PCR and Sanger sequencing. Most pseudogenes represent recent gene disruptions and are usually unique to a particular genome [34]. HL096PA1 and KPA171202 share only four predicted pseudogenes. Compared to the 14 frameshifted ORFs encoded in the KPA171202 genome [13], HL096PA1 harbors a significantly increased number of pseudogenes. This may suggest a faster reduction rate of the chromosome of this strain. Sixty-one of the 79 predicted pseudogenes in HL096PA1 contain single inactivating mutations, indicating that the majority of these mutations were recently acquired [34].Table 3
Annotations of the 79 predicted pseudogenes in HL096PA1.
Number
ORF
Start
End
Function
Note
1
PAGK_0006
7,621
6,061
ABC transporter, ATP-binding protein
Shared with KPA171202
2
PAGK_0019
23,673
23,196
PTS system, galactitol-specific IIA component
Confirmed by PCR and Sanger sequencing
3
PAGK_0020
24,985
23,730
PTS system, galactitol-specific IIC component
Confirmed by PCR and Sanger sequencing
4
PAGK_0024
25,937
27,844
Hypothetical protein
Shared with KPA171202
5
PAGK_0025
28,692
28,418
Hypothetical protein
6
PAGK_0026
29,964
29,294
Hypothetical protein
7
PAGK_0089
100,317
102,505
Glycosyl hydrolase family protein
8
PAGK_0123
149,702
148,105
Site-specific recombinase
9
PAGK_0193
226,656
225,834
Regulator protein
10
PAGK_0224
266,991
265,055
Putative RHS-related protein
11
PAGK_0243
289,824
290,724
Putative ABC transporter
12
PAGK_0352
402,744
401,670
Hypothetical protein
Confirmed by PCR and Sanger sequencing
13
PAGK_0393
441,551
441,884
2-Hydroxy acid dehydrogenase
14
PAGK_0395
444,633
446,378
Hypothetical protein
15
PAGK_0429
474,851
476,017
Putative sensor histidine kinase, two component system
16
PAGK_0440
487,718
487,064
Hypothetical protein
17
PAGK_0463
510,587
509,659
Phosphotransferase system protein mannitol/fructose-specific IIA subunit
Confirmed by PCR and Sanger sequencing
18
PAGK_0482
529,279
530,146
Myo-inositol catabolism IolH protein
19
PAGK_0573
630,324
628,497
Hypothetical protein
20
PAGK_0589
653,210
651,537
Alpha-glucosidase/amylase family protein
21
PAGK_0592
657,824
655,229
Aminopeptidase N
22
PAGK_0601
674,112
671,871
UvrD/Rep helicase
23
PAGK_0603
685,081
680,455
Hypothetical protein
24
PAGK_0604
687,943
685,068
Putative helicase
25
PAGK_0613
702,394
698,431
Hypothetical protein
26
PAGK_0720
820,262
819,153
Putative peptide ABC transporter, permease component
27
PAGK_0757
855,259
854,123
Putative lysophospholipase
Confirmed by PCR and Sanger sequencing
28
PAGK_0811
908,541
907,748
Hypothetical protein
29
PAGK_0875
986,883
986,286
Hypothetical protein
30
PAGK_0944
1,060,628
1,059,358
Prephenate dehydrogenase
31
PAGK_1004
1,127,197
1,126,813
Hypothetical protein
32
PAGK_1011
1,133,493
1,133,051
Hypothetical protein
33
PAGK_1158
1,298,563
1,297,552
Putative oxidoreductase protein
Confirmed by PCR and Sanger sequencing
34
PAGK_1160
1,299,462
1,299,708
Beta-glucosidase fragment
Confirmed by PCR and Sanger sequencing
35
PAGK_1181
1,318,788
1,319,584
Putative hydrolase
Confirmed by PCR and Sanger sequencing
36
PAGK_1233
1,374,383
1,376,290
PTS system, mannitol-specific IIABC component
Confirmed by PCR and Sanger sequencing
37
PAGK_1238
1,380,347
1,381,136
Putative metallo-beta-lactamase
38
PAGK_1242
1,386,373
1,384,720
Acetyl-coenzyme A synthetase
39
PAGK_1275
1,426,760
1,425,953
GntR family regulatory protein
40
PAGK_1276
1,426,762
1,427,445
Hypothetical protein
41
PAGK_1284
1,434,222
1,434,422
Hypothetical protein
42
PAGK_1285
1,434,707
1,435,190
Transposase, Mutator family
43
PAGK_1332
1,481,350
1,482,114
Hypothetical protein
Confirmed by PCR and Sanger sequencing
44
PAGK_1344
1,496,744
1,495,088
Hypothetical protein
45
PAGK_1347
1,501,212
1,499,580
ABC transporter ATP-binding protein
Confirmed by PCR and Sanger sequencing
46
PAGK_1354
1,508,597
1,511,439
Oxidoreductase, putative D-lactate
47
PAGK_1387
1,549,654
1,548,834
Putative GntR-family transcriptional regulator
48
PAGK_1407
1,567,445
1,569,345
Hypothetical protein
49
PAGK_1536
1,703,687
1,704,612
Putative lysophospholipase
50
PAGK_1586
1,764,348
1,765,387
Binding-protein-dependent transport system inner membrane component
Confirmed by PCR and Sanger sequencing and shared withP. acnes J139
51
PAGK_1587
1,765,384
1,766,265
Binding-protein-dependent transport system inner membrane component
Shared withP. acnes J139
52
PAGK_1592
1,770,327
1,769,760
Protein associated to putative adhesion protein
53
PAGK_1593
1,771,040
1,770,487
Protein associated to putative adhesion protein
54
PAGK_1594
1,773,223
1,771,037
Hypothetical protein
55
PAGK_1608
1,786,154
1,785,310
Hypothetical protein
56
PAGK_1643
1,820,268
1,818,959
Transporter (sodium : sulfate symporter)
Shared with KPA171202
57
PAGK_1717
1,907,494
1,905,155
Putative ABC transporter-associated permease
Confirmed by PCR and Sanger sequencing
58
PAGK_1730
1,924,158
1,920,911
Endo-beta-N-acetylglucosaminidase family protein
59
PAGK_1742
1,935,480
1,935,974
Hypothetical protein
60
PAGK_1744
1,936,979
1,938,333
Putative sialidase
61
PAGK_1760
1,952,431
1,951,577
Glycerophosphoryl diester phosphodiesterase
62
PAGK_1793
1,981,847
1,978,587
Hypothetical protein
Confirmed by PCR and Sanger sequencing
63
PAGK_1821
2,014,629
2,016,701
Hypothetical protein
Confirmed by PCR and Sanger sequencing
64
PAGK_1858
2,054,370
2,056,156
ABC transporter ATP-binding protein
65
PAGK_1895
2,103,920
2,105,327
Pyridine nucleotide-disulphide oxidoreductase
66
PAGK_1897
2,107,483
2,109,669
Hypothetical protein
Shared withP. acnes J139
67
PAGK_1966
2,185,427
2,184,028
Amidase
68
PAGK_1990
2,207,618
2,209,728
5′-Nucleotidase/2′,3′-cyclic phosphodiesterase
69
PAGK_1995
2,211,776
2,211,311
Hypothetical protein
70
PAGK_1997
2,212,029
2,212,392
Hypothetical protein
71
PAGK_2003
2,218,626
2,218,090
Hypothetical protein
72
PAGK_2008
2,222,549
2,223,462
Putative asparaginase
73
PAGK_2023
2,236,080
2,235,748
Ornithine carbamoyltransferase, catabolic
74
PAGK_2041
2,255,990
2,257,066
Putative lysophospholipase
75
PAGK_2110
2,327,052
2,328,480
Putative adhesion or S-layer protein
Confirmed by PCR and Sanger sequencing
76
PAGK_2118
2,338,399
2,337,074
Hypothetical protein
77
PAGK_2159
2,375,435
2,374,944
MarR family transcriptional regulator
Confirmed by PCR and Sanger sequencing
78
PAGK_2189
2,405,633
2,407,955
D-ornithine aminomutase E component
79
PAGK_2225
2,450,551
2,449,750
SorC family transcriptional regulator
Shared with KPA171202Although the functions of the predicted pseudogenes in HL096PA1 are diverse, 14 of the 79 pseudogenes belong to the functional group of carbohydrate transport and metabolism, including five phosphotransferase system (PTS) components responsible for carbohydrate regulation and sensing. Two of the three predicted fructose PTSs contain frameshifts, suggesting that only one fructose uptake system remains functional in the genome [35]. In addition, HL096PA1 contains two pseudogenes in the only predicted galactitol PTS [35] and one disrupted component in the single glucitol PTS. This suggests that both of these systems are nonfunctional in this strain. These gene disruptions observed in the PTS components and other proteins of the carbohydrate transport and metabolism pathways may reflect an adaptation of HL096PA1 to the availability of nutrients in the pilosebaceous unit, where the major carbon source is lipids but not carbohydrates.In addition to PTS genes, multiple virulence-associated adhesion proteins in HL096PA1 harbor frameshifts. Adhesion proteins are of special interest because of their pathogenic potential and involvement in biofilm formation. Brüggemann et al. reported nine putative adhesion proteins in KPA171202 genome which contain thrombospondin type 3 repeats, PKD and SEST domains [13]. We found frameshifts in five of the nine orthologous adhesion proteins in HL096PA1, including PAGK_1592, PAGK_1593, PAGK_1821, PAGK_1897, and PAGK_2110. These orthologs were also recently reported to contain frameshifts in P. acnes strain 266 [36], which is a strain more closely related to HL096PA1 than to KPA171202. The differences in functional adhesion protein genes among the strains suggest that various P. acnes strains may use antigenic variations, a mechanism used by many pathogenic bacteria to avoid the host immune system [37], as a host immune invasion mechanism.


### 3.4. Genomic Islands in HL096PA1

HL096PA1 harbors several island-like genomic regions. In KPA171202, 11 genomic islands were reported previously [13]. These regions encode proteins associated with pathogenicity or survival, mobility, and proteins of unknown function. The chromosome of HL096PA1 shares eight of the island-like regions found in KPA171202. Three of the KPA171202 island-like regions are absent in HL096PA1, including PPA839-878, PPA1278-1321, and PPA1576-1618. In addition to the genomic islands described by Brüggemann et al. [13], an island-like region (PPA2055-2092) in KPA171202 reported later [36] is also absent in HL096PA1. On the other hand, HL096PA1 contains two unique genomic islands, locus 1 and locus 2, which have been shown to be associated with the disease state of acne [12]. These two loci are absent in KPA171202 but present in all strains that belong to the same lineage as HL096 PA1 [12], including almost all RT4 and RT5 strains and another completely sequenced strain SK137 [36].HL096PA1 locus 1 spans over 6 Kb and encodes seven ORFs (PAGK_0121–PAGK_0127). This region shows evidence of a cryptic phage-like integration through the flanking tRNAs. It encodes phage-related genes such as site-specific recombinase. An antibiotic ABC transporter is also encoded in this region; however, it is a predicted pseudogene, suggesting that antibiotic resistance was once conferred through this phage integration.HL096PA1 locus 2 spans over 20 Kb and encodes 23 ORFs (PAGK_0160–PAGK_0182). Within this genomic island, a complete streptolysin S (SLS) biosynthesis gene cluster (PAGK_0168–PAGK_0174) is encoded, including CAAX amino protease family protein (PAGK_0170), streptolysin prototoxin protein SagA (PAGK_0171), cyclodehydratase genes SagD, SagC, and SagB (PAGK_0172–PAGK_0174), and two hypothetical proteins (PAGK_0168 and PAGK_0169). Streptolysin biosynthetic cluster is commonly found in Gram-positive mammalian pathogens such asClostridium botulinum, Listeria monocytogenes, and Staphylococcus aureus [38]. This gene cluster is potentially capable of producing a nonribosomally synthesized secreted toxin, streptolysin. Streptolysin is responsible for the classical beta-hemolytic phenotype of bacterial colonies grown on blood agar media. This gene cluster in HL096PA1 may contribute to its virulence against the host and to its dominant colonization against other pilosebaceous unit dwelling microbes.The second major group of genes encoded in locus 2 is phage defense genes. By using Pfam [39], we identified an abortive infection Abi_2 domain (pfam07751) within ORF PAGK_0180, which is involved in bacteriophage resistance via premature cell death upon phage entry. This abortive infection defense mechanism against bacterial phage is mediated by a single copy of Abi_2 domain containing protein [40]. A second copy of this Abi_2 domain containing gene, PAGK_2391, is encoded on the plasmid pIMPLE-HL096PA1. This suggests a possible common origin of locus 2 and the plasmid.Additional genomic evidence supports a possible common origin of locus 2 and the plasmid pIMPLE-HL096PA1. Located at the immediate downstream of the abortive infection defense system is ORF PAGK_0182. It is a hypothetical protein homologous to CLOLEP_00167 inC. leptum. As described above, the plasmid pIMPLE-HL096PA1 contains a cluster of genes with high similarities to C. leptum DSM753 genes CLOLEP_00122–CLOLEP_00166, while the homolog of CLOLEP_00167 lies in locus 2 on the chromosome of HL096PA1. This further suggests a possible common origin of locus 2 and the plasmid. Notably, this gene in locus 2, PAGK_0182, contains a 31 bp sequence. As reported by Fitz-Gibbon et al. [12], this 31 bp sequence was found identical to the clustered regularly interspaced short palindromic repeat (CRISPR) spacer sequence encoded in some of the type II P. acnes strains. This suggests that the genes in locus 2 may have been acquired through a phage or plasmid mechanism in RT4 and RT5 strains, while type II strains do not harbor this genomic island possibly due to the protection mechanism of CRISPR/cas system against foreign DNA.


### 3.5. Genomic Inversion in HL096PA1

While genomic inversion is commonly seen in bacterial genomes, pathogenic bacteria often exhibit a high degree of genomic rearrangement [41]. We observed a large genomic inversion in HL096PA1 when compared to KPA171202. The inversion spans the ribosomal region of 1.2 Mb ranging from the 1st set to the 3rd set of ribosomal operons (Figure 3(a)). To verify the inversion, we designed primer sets that span the beginning, middle, and the end of the inverted region (Figure 3(b)). PCR amplifications of these regions were performed. As predicted based on the inverted genomic region, HL096PA1 had strong amplifications of the regions covered by primer sets I, II, and III but had no specific amplifications using primer sets IV and V (Figure 3(c)). The inversion was further confirmed by sequencing the ends of the amplicons using the Sanger method. KPA171202 was used as a control. As predicted, KPA171202 had no specific amplifications using primer sets I and III, but strong amplifications using primer sets II, IV, and V. Among the nine currently available complete P. acnes genomes, ATCC11828 strain [42] also contains a genomic inversion. Similar to the inversion in HL096PA1, it spans a ribosomal region of 0.9 Mb including the 1st set and the 2nd set of ribosomal operons (Figure 3(a)).


## 3.1. General Genome Features ofP. acnes Strain HL096PA1

Similar to other sequencedP. acnes strains, HL096PA1 has a circular chromosome of 249, 4191 bp (Figure 1). It encodes three sets of 16S, 23S, and 5S rRNA operons, 45 tRNA genes, and 2,254 protein coding genes. A comparison of the general genome features of HL096PA1 and KAP171202 is shown in Table 1. Although HL096PA1 belongs to a different lineage, it shares 94% of the sequence with the genome of KPA171202. Among the proteins encoded on the HL096PA1 chromosome, 91% are orthologous to KPA171202 proteins (>90% amino acid identity in >60% sequence length). This suggests that small genomic variations or extrachromosomal elements may be responsible for different phenotypes of the two strains and their different associations with acne pathogenesis.Table 1
Comparison of the general features of HL096PA1 and KPA171202 genomes.
Features
HL096PA1
KPA171202
Chromosome
Plasmid
Chromosome
recA phylotype
IA
IB
Ribotype (RT)
5
1
Acne association
Yes
No
Size (bp)
2,494,191
56,169
2,560,265
GC content (%)
60
62
60
Coding sequences
2,254
74
2,297
Pseudogenes
79
0
14
Coding density (%)
90
83
91
tRNAs
45
0
45
RNA operons
3
0
3
Sequencing method
454/Roche
454/Roche
Sanger
Sequencing coverage
50X
8.7XFigure 1
Circular representation ofP. acnes HL096PA1 chromosome. From the outer circle to the inner circle are circle 1, ORFs on the positive strand (blue); circle 2, ORFs on the negative strand (green); circle 3, pseudogenes (red); circle 4, tRNA (gold) and rRNA (purple); circle 5, GC content (light/dark grey); circle 6, GC skew (light/dark grey). DNA Plotter [46] was used in making the figure.


## 3.2. Plasmid in HL096PA1, pIMPLE-HL096PA1

Compared to KPA171202, the most significant specific feature of HL096PA1 is a plasmid. HL096PA1 genome sequencing and assembly revealed a second large contig in addition to the chromosome, which appears to be an extrachromosomal plasmid [12]. The plasmid, named pIMPLE-HL096PA1, is 56,169 bp long. It encodes 74 ORFs (PAGK_2319–PAGK_2392) and one origin of replication (RepA). Several plasmid related proteins, including plasmid partition protein ParA (PAGK_2332) and plasmid stabilization system toxin and antitoxin proteins (PAGK_2321 and PAGK_2322), are encoded. Based on the sequencing coverage and quantitative PCR results, the copy number of the plasmid is three per genome [12]. While our attempts to isolate the plasmid in culture have been unsuccessful possibly due to the low copy number and the large size of the plasmid, we have since identified homologous plasmids in multiple other P. acnes strains that belong to the same lineage as HL096PA1 based on genome sequencing [12].The assembled sequence contig suggests that pIMPLE-HL096PA1 is a linear plasmid, with hairpin ends used for bidirectional replication. Linear plasmids with terminal inverted repeats (TIRs) and 5′ terminally attached proteins have been found in bacteria, fungi, and higher eukaryotes [15–19]. Well-studied examples of human pathogenic bacteria with such linear plasmids include Spirochaete Borrelia and Filamentous Streptomyces [16, 20–24]. TIRs have been shown to be essential for bidirectional linear plasmid replication [20, 22–24]. Similar to the linear plasmids observed in Borrelia and Streptomyces, pIMPLE-HL096PA1 contains a 20 bp short TIR with a sequence of ACGACACCAGCACCCACAAC at each end of the plasmid. Due to the difficulty in assembling the repeat sequences at the ends, the lengths of both ends of the plasmid are not fully defined but were assembled to the same. Based on the assembled plasmid sequence, the left arm TIR is located 743 bp from the start of the plasmid sequence, spanning the first ORF (PAGK_2319, an unknown protein). The right arm TIR is located 743 bp from the 3′ end of the plasmid sequence. Analysis of the sequences at the ends of the plasmid reveals potential hairpin structures within the repeat sequences. The last ORF on the plasmid, PAGK_2392, is located near the right arm TIR and encodes resolvase A (ResA). ResA may facilitate fusion and resolution of hairpin telomeres during plasmid replication. It may cut the circular-like plasmid after replication into two linear plasmids with hairpin ends, using a mechanism similar to telomere resolvase (ResT), an enzyme commonly encoded by linear plasmids and shown to facilitate resolution of hairpin telomeres during plasmid replication [25].The genes encoded on pIMPLE-HL096PA1 have little redundancy with the HL096PA1 chromosome. The plasmid shares only two genes (PAGK_2383 and PAGK_2391) with the chromosome. However, it encodes 12 genes (PAGK_2331, PAGK_2332, and PAGK_2337–PAGK_2346) homologous to the genes in KPA171202, which are absent on the HL096PA1 chromosome and are mostly hypothetical proteins with unknown functions. It also encodes an additional esterase/lipase (PAGK_2386). Lipases have been shown to contribute to growth advantage and possible pathogenicity ofP. acnes in acne. Lipases hydrolyze triacylglycerols to free fatty acids, which may reduce the invasion of other skin pathogens but may also lead to inflammation and the development of acne when over-produced [26, 27].Significant horizontal gene transfer is evident within pIMPLE-HL096PA1. Thirty-five of the 74 predicted ORFs (PAGK_2344–PAGK_2378) are highly homologous to ORFs CLOLEP_00122–CLOLEP_00166 encoded inClostridium leptum DSM 753 (E-value = 1E-100 to 0) (Figure 2(a)), a Gram-positive commensal bacterium found in the human gut. The same region is also highly homologous to the genomes of Propionibacterium humerusii strains HL037PA2, HL037PA3, HL044PA1, and P08 [28]. P. humerusii is a bacterium found on the human skin [12] and is closely related to P. acnes. It shares several additional ORFs with pIMPLE-HL096PA1 (Figure 2(a)). Based on our sequencing data of strains HL037PA2, HL037PA3, and HL044PA1, the homologous genes in these P. humerusiigenomes had a three to five folds higher sequencing coverage than other genes in the genomes (Figure 2(c)). Although these genomes are not complete genomes, the higher sequencing coverage of these genes suggests that they may also be located on an extrachromosomal mobile genetic element in P. humerusii. It is unclear whether this set of ORFs was horizontally transferred among P. acnes, P. humerusii, and C. leptum or transferred from an ancestor organism. Further investigation into the relationships among these microbes that primarily reside at different body sites will provide insight to how genetic materials were transferred and how this mechanism affected the virulent properties of human commensals.Figure 2
The homologous regions amongP. acnes pIMPLE-HL096PA1, P. humerusii HL044PA1, and C. leptum DSM 753. (a) The 74 ORFs encoded on the plasmid pIMPLE-HL096PA1 are shown. ORFs PAGK_2344–PAGK_2378 (blue), including the Tad locus (pink), are shared among all three genomes. In addition, several ORFs are shared between P. acnes and P. humerusii (orange). ORFs unique to P. acnesare shown in grey. Absence of P. acneshomologous ORFs in P. humerusii or C. leptum is shown in white. (b) Organizations of the ORFs shared among all three genomes. (c) The sequence coverage of the contigs in three P. humerusii strains (HL037PA2, HL037PA3, and HL044PA1) that share homologous genes with pIMPLE-HL096PA1 is 3–5-folds higher than the contigs that do not share homologous genes with pIMPLE-HL096PA1.Genomic inversion in HL096PA1. (a) Genomic inversions observed inP. acnes strains HL096PA1 and ATCC11828 compared to P. acnes strain KPA171202. (b) Primer sets I–V designed to verify the genomic inversion in HL096PA1. (c) PCR amplified DNA fragments of the inverted regions shown on an agarose gel. Primers, target genomes, and the predicted sizes of the amplicons are shown in the table. HL, strain HL096PA1; KPA, strain KPA171202.
(a)
(b)
(c)Within this cluster ofC. leptum homologs on pIMPLE-HL096PA1, we identified a Tad (tight adhesion) locus [12]. Tad locus was reported to mediate colonization, pathogenesis, and tight adhesion of bacteria to host cell surfaces through Flp pilus assembly in Gram-positive pathogens, such as Corynebacterium dipheriae, Mycobacterium tuberculosis, Mycobacterium bovis,andStreptomyces coelicor[29–31]. The organizations and annotations of the ORFs in this locus are shown in Figure 2 and Table 2. ORFs PAGK_2360–PAGK_2370 encode a set of Tad proteins, including Flp (fimbrial low molecular-weight proteins) pilus proteins, TadZ, TadA, TadB, TadC, and TadE. The transcription of Tad locus in HL096PA1 appears to be controlled by a sigma 70 family transcription factor (PAGK_2357) and an antisigma factor (PAGK_2358) located immediately upstream of the Tad locus. Sigma 70 family transcription factors are commonly found upstream of virulence-associated gene loci and have been shown to regulate virulence expression in response to particular stress-related stimuli in several pathogenic bacteria such as Vibrio cholera [32]. This suggests that Tad locus expression in HL096PA1 may be induced under specific conditions, leading to enhanced colonization and adhesion to host cells. While KPA171202 does not possess such a plasmid and the Tad locus, majority of the RT4 and RT5 strains of P. acnes, which are highly associated with acne, contain a homologous plasmid and the Tad locus [12]. The unique presence of such a plasmid in acne associated strains may potentially explain their role in acne pathogenesis and can possibly be used as a pathogenicity marker.Table 2
Annotations of the Tad locus in pIMPLE-HL096PA1.
ORF
Tad homolog
Function
Pfam ID
PfamE-value
PAGK_2360
RcpC
Flp pilus assembly protein, rough colony protein-C
SAF
9.60
E
-
05
PAGK_2361
TadZ/A
ATPase
N/A
N/A
PAGK_2362
TadA
ATPase
GSPII_E
1.50
E
-
10
PAGK_2363
TadB/C
Flp pilus assembly protein
GSPII_F
2.10
E
-
05
PAGK_2364
TadB/C
Flp pilus assembly protein
GSPII_F
0.014
PAGK_2365
N/A
Unknown
N/A
N/A
PAGK_2366
TadE
Minor Flp pilus assembly protein
TadE
0.0015
PAGK_2367
TadE
Minor Flp pilus assembly protein
TadE
0.018
PAGK_2368
TadE
Minor Flp pilus assembly protein
TadE
3.00
E
-
07
PAGK_2369
TadB
Flp pilus assembly protein
LysM, BTAD
3.10
E
-
10
PAGK_2370
RcpB
Flp assembly protein, rough colony protein-B
Pilus, CpaD
0.53


## 3.3. Pseudogenes in HL096PA1

Pseudogenes tend to be abundant in bacterial species that have recently adapted to eukaryotic hosts or a pathogenic lifestyle [33]. Thus identification of pseudogenes in disease associated strains may provide insight to their host adaptation and pathogenic mechanisms. We identified 79 predicted pseudogenes in HL096PA1 genome (Table 3). In addition to confirmation of the pseudogenes based on the high-quality 454 sequence reads, we selected 17 out of the 79 identified pseudogenes and confirmed the frameshifts in all 17 genes by PCR and Sanger sequencing. Most pseudogenes represent recent gene disruptions and are usually unique to a particular genome [34]. HL096PA1 and KPA171202 share only four predicted pseudogenes. Compared to the 14 frameshifted ORFs encoded in the KPA171202 genome [13], HL096PA1 harbors a significantly increased number of pseudogenes. This may suggest a faster reduction rate of the chromosome of this strain. Sixty-one of the 79 predicted pseudogenes in HL096PA1 contain single inactivating mutations, indicating that the majority of these mutations were recently acquired [34].Table 3
Annotations of the 79 predicted pseudogenes in HL096PA1.
Number
ORF
Start
End
Function
Note
1
PAGK_0006
7,621
6,061
ABC transporter, ATP-binding protein
Shared with KPA171202
2
PAGK_0019
23,673
23,196
PTS system, galactitol-specific IIA component
Confirmed by PCR and Sanger sequencing
3
PAGK_0020
24,985
23,730
PTS system, galactitol-specific IIC component
Confirmed by PCR and Sanger sequencing
4
PAGK_0024
25,937
27,844
Hypothetical protein
Shared with KPA171202
5
PAGK_0025
28,692
28,418
Hypothetical protein
6
PAGK_0026
29,964
29,294
Hypothetical protein
7
PAGK_0089
100,317
102,505
Glycosyl hydrolase family protein
8
PAGK_0123
149,702
148,105
Site-specific recombinase
9
PAGK_0193
226,656
225,834
Regulator protein
10
PAGK_0224
266,991
265,055
Putative RHS-related protein
11
PAGK_0243
289,824
290,724
Putative ABC transporter
12
PAGK_0352
402,744
401,670
Hypothetical protein
Confirmed by PCR and Sanger sequencing
13
PAGK_0393
441,551
441,884
2-Hydroxy acid dehydrogenase
14
PAGK_0395
444,633
446,378
Hypothetical protein
15
PAGK_0429
474,851
476,017
Putative sensor histidine kinase, two component system
16
PAGK_0440
487,718
487,064
Hypothetical protein
17
PAGK_0463
510,587
509,659
Phosphotransferase system protein mannitol/fructose-specific IIA subunit
Confirmed by PCR and Sanger sequencing
18
PAGK_0482
529,279
530,146
Myo-inositol catabolism IolH protein
19
PAGK_0573
630,324
628,497
Hypothetical protein
20
PAGK_0589
653,210
651,537
Alpha-glucosidase/amylase family protein
21
PAGK_0592
657,824
655,229
Aminopeptidase N
22
PAGK_0601
674,112
671,871
UvrD/Rep helicase
23
PAGK_0603
685,081
680,455
Hypothetical protein
24
PAGK_0604
687,943
685,068
Putative helicase
25
PAGK_0613
702,394
698,431
Hypothetical protein
26
PAGK_0720
820,262
819,153
Putative peptide ABC transporter, permease component
27
PAGK_0757
855,259
854,123
Putative lysophospholipase
Confirmed by PCR and Sanger sequencing
28
PAGK_0811
908,541
907,748
Hypothetical protein
29
PAGK_0875
986,883
986,286
Hypothetical protein
30
PAGK_0944
1,060,628
1,059,358
Prephenate dehydrogenase
31
PAGK_1004
1,127,197
1,126,813
Hypothetical protein
32
PAGK_1011
1,133,493
1,133,051
Hypothetical protein
33
PAGK_1158
1,298,563
1,297,552
Putative oxidoreductase protein
Confirmed by PCR and Sanger sequencing
34
PAGK_1160
1,299,462
1,299,708
Beta-glucosidase fragment
Confirmed by PCR and Sanger sequencing
35
PAGK_1181
1,318,788
1,319,584
Putative hydrolase
Confirmed by PCR and Sanger sequencing
36
PAGK_1233
1,374,383
1,376,290
PTS system, mannitol-specific IIABC component
Confirmed by PCR and Sanger sequencing
37
PAGK_1238
1,380,347
1,381,136
Putative metallo-beta-lactamase
38
PAGK_1242
1,386,373
1,384,720
Acetyl-coenzyme A synthetase
39
PAGK_1275
1,426,760
1,425,953
GntR family regulatory protein
40
PAGK_1276
1,426,762
1,427,445
Hypothetical protein
41
PAGK_1284
1,434,222
1,434,422
Hypothetical protein
42
PAGK_1285
1,434,707
1,435,190
Transposase, Mutator family
43
PAGK_1332
1,481,350
1,482,114
Hypothetical protein
Confirmed by PCR and Sanger sequencing
44
PAGK_1344
1,496,744
1,495,088
Hypothetical protein
45
PAGK_1347
1,501,212
1,499,580
ABC transporter ATP-binding protein
Confirmed by PCR and Sanger sequencing
46
PAGK_1354
1,508,597
1,511,439
Oxidoreductase, putative D-lactate
47
PAGK_1387
1,549,654
1,548,834
Putative GntR-family transcriptional regulator
48
PAGK_1407
1,567,445
1,569,345
Hypothetical protein
49
PAGK_1536
1,703,687
1,704,612
Putative lysophospholipase
50
PAGK_1586
1,764,348
1,765,387
Binding-protein-dependent transport system inner membrane component
Confirmed by PCR and Sanger sequencing and shared withP. acnes J139
51
PAGK_1587
1,765,384
1,766,265
Binding-protein-dependent transport system inner membrane component
Shared withP. acnes J139
52
PAGK_1592
1,770,327
1,769,760
Protein associated to putative adhesion protein
53
PAGK_1593
1,771,040
1,770,487
Protein associated to putative adhesion protein
54
PAGK_1594
1,773,223
1,771,037
Hypothetical protein
55
PAGK_1608
1,786,154
1,785,310
Hypothetical protein
56
PAGK_1643
1,820,268
1,818,959
Transporter (sodium : sulfate symporter)
Shared with KPA171202
57
PAGK_1717
1,907,494
1,905,155
Putative ABC transporter-associated permease
Confirmed by PCR and Sanger sequencing
58
PAGK_1730
1,924,158
1,920,911
Endo-beta-N-acetylglucosaminidase family protein
59
PAGK_1742
1,935,480
1,935,974
Hypothetical protein
60
PAGK_1744
1,936,979
1,938,333
Putative sialidase
61
PAGK_1760
1,952,431
1,951,577
Glycerophosphoryl diester phosphodiesterase
62
PAGK_1793
1,981,847
1,978,587
Hypothetical protein
Confirmed by PCR and Sanger sequencing
63
PAGK_1821
2,014,629
2,016,701
Hypothetical protein
Confirmed by PCR and Sanger sequencing
64
PAGK_1858
2,054,370
2,056,156
ABC transporter ATP-binding protein
65
PAGK_1895
2,103,920
2,105,327
Pyridine nucleotide-disulphide oxidoreductase
66
PAGK_1897
2,107,483
2,109,669
Hypothetical protein
Shared withP. acnes J139
67
PAGK_1966
2,185,427
2,184,028
Amidase
68
PAGK_1990
2,207,618
2,209,728
5′-Nucleotidase/2′,3′-cyclic phosphodiesterase
69
PAGK_1995
2,211,776
2,211,311
Hypothetical protein
70
PAGK_1997
2,212,029
2,212,392
Hypothetical protein
71
PAGK_2003
2,218,626
2,218,090
Hypothetical protein
72
PAGK_2008
2,222,549
2,223,462
Putative asparaginase
73
PAGK_2023
2,236,080
2,235,748
Ornithine carbamoyltransferase, catabolic
74
PAGK_2041
2,255,990
2,257,066
Putative lysophospholipase
75
PAGK_2110
2,327,052
2,328,480
Putative adhesion or S-layer protein
Confirmed by PCR and Sanger sequencing
76
PAGK_2118
2,338,399
2,337,074
Hypothetical protein
77
PAGK_2159
2,375,435
2,374,944
MarR family transcriptional regulator
Confirmed by PCR and Sanger sequencing
78
PAGK_2189
2,405,633
2,407,955
D-ornithine aminomutase E component
79
PAGK_2225
2,450,551
2,449,750
SorC family transcriptional regulator
Shared with KPA171202Although the functions of the predicted pseudogenes in HL096PA1 are diverse, 14 of the 79 pseudogenes belong to the functional group of carbohydrate transport and metabolism, including five phosphotransferase system (PTS) components responsible for carbohydrate regulation and sensing. Two of the three predicted fructose PTSs contain frameshifts, suggesting that only one fructose uptake system remains functional in the genome [35]. In addition, HL096PA1 contains two pseudogenes in the only predicted galactitol PTS [35] and one disrupted component in the single glucitol PTS. This suggests that both of these systems are nonfunctional in this strain. These gene disruptions observed in the PTS components and other proteins of the carbohydrate transport and metabolism pathways may reflect an adaptation of HL096PA1 to the availability of nutrients in the pilosebaceous unit, where the major carbon source is lipids but not carbohydrates.In addition to PTS genes, multiple virulence-associated adhesion proteins in HL096PA1 harbor frameshifts. Adhesion proteins are of special interest because of their pathogenic potential and involvement in biofilm formation. Brüggemann et al. reported nine putative adhesion proteins in KPA171202 genome which contain thrombospondin type 3 repeats, PKD and SEST domains [13]. We found frameshifts in five of the nine orthologous adhesion proteins in HL096PA1, including PAGK_1592, PAGK_1593, PAGK_1821, PAGK_1897, and PAGK_2110. These orthologs were also recently reported to contain frameshifts in P. acnes strain 266 [36], which is a strain more closely related to HL096PA1 than to KPA171202. The differences in functional adhesion protein genes among the strains suggest that various P. acnes strains may use antigenic variations, a mechanism used by many pathogenic bacteria to avoid the host immune system [37], as a host immune invasion mechanism.


## 3.4. Genomic Islands in HL096PA1

HL096PA1 harbors several island-like genomic regions. In KPA171202, 11 genomic islands were reported previously [13]. These regions encode proteins associated with pathogenicity or survival, mobility, and proteins of unknown function. The chromosome of HL096PA1 shares eight of the island-like regions found in KPA171202. Three of the KPA171202 island-like regions are absent in HL096PA1, including PPA839-878, PPA1278-1321, and PPA1576-1618. In addition to the genomic islands described by Brüggemann et al. [13], an island-like region (PPA2055-2092) in KPA171202 reported later [36] is also absent in HL096PA1. On the other hand, HL096PA1 contains two unique genomic islands, locus 1 and locus 2, which have been shown to be associated with the disease state of acne [12]. These two loci are absent in KPA171202 but present in all strains that belong to the same lineage as HL096 PA1 [12], including almost all RT4 and RT5 strains and another completely sequenced strain SK137 [36].HL096PA1 locus 1 spans over 6 Kb and encodes seven ORFs (PAGK_0121–PAGK_0127). This region shows evidence of a cryptic phage-like integration through the flanking tRNAs. It encodes phage-related genes such as site-specific recombinase. An antibiotic ABC transporter is also encoded in this region; however, it is a predicted pseudogene, suggesting that antibiotic resistance was once conferred through this phage integration.HL096PA1 locus 2 spans over 20 Kb and encodes 23 ORFs (PAGK_0160–PAGK_0182). Within this genomic island, a complete streptolysin S (SLS) biosynthesis gene cluster (PAGK_0168–PAGK_0174) is encoded, including CAAX amino protease family protein (PAGK_0170), streptolysin prototoxin protein SagA (PAGK_0171), cyclodehydratase genes SagD, SagC, and SagB (PAGK_0172–PAGK_0174), and two hypothetical proteins (PAGK_0168 and PAGK_0169). Streptolysin biosynthetic cluster is commonly found in Gram-positive mammalian pathogens such asClostridium botulinum, Listeria monocytogenes, and Staphylococcus aureus [38]. This gene cluster is potentially capable of producing a nonribosomally synthesized secreted toxin, streptolysin. Streptolysin is responsible for the classical beta-hemolytic phenotype of bacterial colonies grown on blood agar media. This gene cluster in HL096PA1 may contribute to its virulence against the host and to its dominant colonization against other pilosebaceous unit dwelling microbes.The second major group of genes encoded in locus 2 is phage defense genes. By using Pfam [39], we identified an abortive infection Abi_2 domain (pfam07751) within ORF PAGK_0180, which is involved in bacteriophage resistance via premature cell death upon phage entry. This abortive infection defense mechanism against bacterial phage is mediated by a single copy of Abi_2 domain containing protein [40]. A second copy of this Abi_2 domain containing gene, PAGK_2391, is encoded on the plasmid pIMPLE-HL096PA1. This suggests a possible common origin of locus 2 and the plasmid.Additional genomic evidence supports a possible common origin of locus 2 and the plasmid pIMPLE-HL096PA1. Located at the immediate downstream of the abortive infection defense system is ORF PAGK_0182. It is a hypothetical protein homologous to CLOLEP_00167 inC. leptum. As described above, the plasmid pIMPLE-HL096PA1 contains a cluster of genes with high similarities to C. leptum DSM753 genes CLOLEP_00122–CLOLEP_00166, while the homolog of CLOLEP_00167 lies in locus 2 on the chromosome of HL096PA1. This further suggests a possible common origin of locus 2 and the plasmid. Notably, this gene in locus 2, PAGK_0182, contains a 31 bp sequence. As reported by Fitz-Gibbon et al. [12], this 31 bp sequence was found identical to the clustered regularly interspaced short palindromic repeat (CRISPR) spacer sequence encoded in some of the type II P. acnes strains. This suggests that the genes in locus 2 may have been acquired through a phage or plasmid mechanism in RT4 and RT5 strains, while type II strains do not harbor this genomic island possibly due to the protection mechanism of CRISPR/cas system against foreign DNA.


## 3.5. Genomic Inversion in HL096PA1

While genomic inversion is commonly seen in bacterial genomes, pathogenic bacteria often exhibit a high degree of genomic rearrangement [41]. We observed a large genomic inversion in HL096PA1 when compared to KPA171202. The inversion spans the ribosomal region of 1.2 Mb ranging from the 1st set to the 3rd set of ribosomal operons (Figure 3(a)). To verify the inversion, we designed primer sets that span the beginning, middle, and the end of the inverted region (Figure 3(b)). PCR amplifications of these regions were performed. As predicted based on the inverted genomic region, HL096PA1 had strong amplifications of the regions covered by primer sets I, II, and III but had no specific amplifications using primer sets IV and V (Figure 3(c)). The inversion was further confirmed by sequencing the ends of the amplicons using the Sanger method. KPA171202 was used as a control. As predicted, KPA171202 had no specific amplifications using primer sets I and III, but strong amplifications using primer sets II, IV, and V. Among the nine currently available complete P. acnes genomes, ATCC11828 strain [42] also contains a genomic inversion. Similar to the inversion in HL096PA1, it spans a ribosomal region of 0.9 Mb including the 1st set and the 2nd set 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 healthy skin and acne affected skin [12], challenging the traditional concept applied in clinical practice that all P. acnes contributes to acne pathogenesis. Moreover, several studies of the P. acnes strain populations on the skin suggested that acne-associated P. acnes subpopulations exist and may correlate with the genomic variations of the strains [12, 22, 36, 43, 44]. Our genome sequencing and comparison of a large number of strains revealed significant disease- or health-associated genetic elements [12]; however, detailed analyses of extrachromosomal elements, pseudogenes, and genome structure variations were limited due to unfinished genome sequences. The comparison between two complete genomes HL096PA1 and KPA171202 described in this study allowed us to better understand the strain-specific genomic variations in plasmid, pseudogenes, genomic islands, and genomic inversion that may be associated with disease pathogenesis.In this comparison, we identified several strain-specific genetic elements that are potentially associated with increased virulence of HL096PA1 in acne. Present in HL096PA1 and absent in KPA171202 is the first describedP. acnes plasmid carrying a tight adhesion locus, which has been shown to enhance colonization and biofilm formation in several human pathogens [29–31]. Further investigation of this P. acnes plasmid will provide insight on its pathogenic potential. Compared to KPA171202, we observed a more than five-fold increase in the number of pseudogenes in HL096PA1, which suggests a potential mechanism of this strain to host adaptation. HL096PA1 contains two genomic islands that are unique to acne-associated strains and may be associated with increased virulence of this lineage in acne [12]. In addition, a large genomic inversion was observed in HL096PA1. Together these genetic differences revealed by complete genome comparison provide insights into the complexity of the strain variations of this organism and support the existence of specific P. acnes subpopulations which may be associated with acne pathogenesis [36, 45]. Furthermore, the first P. acnes plasmid identified in HL096PA1 may potentially provide a new opportunity for genetic manipulation of this organism. The knowledge gained from this comparative study may potentially lead to novel diagnostic and targeted therapeutic approaches for acne against a specific disease associated lineage of P. acnes.



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*Source: 918320-2013-05-13.xml*