YGENO-08607; No. of pages: 6; 4C: Genomics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
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Dongliang Yu a,b, Borui Pi b, Meihong Yu c, Yanfei Wang b, Zhi Ruan b, Ye Feng b,⁎, Yunsong Yu b,⁎
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Diversity and evolution of oligopeptide permease systems in staphylococcal species
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Article history: Received 24 October 2013 Accepted 24 April 2014 Available online xxxx
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Keywords: Oligopeptide permease Staphylococci Duplication Gene cluster
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Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, China State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China
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Several oligopeptide permease (Opp) systems have been found in staphylococcal species, including Opp1-4, Opp3′ and the arginine catabolic mobile element (ACME)-encoded Opp system (ACME-Opp). They confer upon bacteria the increasing fitness, but their evolutionary histories remain unclear. In this work, we performed a genome-wide identification of Opp systems in staphylococcal species. Novel Opp systems were identified, including the duplicate of Opp4 in Staphylococcus pseudintermedius and the ACME-Opp-like systems in coagulase-negative staphylococci (CoNS). Phylogenetic analysis revealed that all of the identified Opp systems were derived from Opp3 system by operon duplication during species divergence, while lateral gene transfer might also confer to the dissemination of Opp in staphylococci. In addition, we proposed an improved theory on evolution of ACME: the Opp and arginine-deiminase systems were firstly transferred from Staphylococcus haemolyticus to Staphylococcus epidermidis independently; in S. epidermidis they were assembled together and then transferred to Staphylococcus aureus. © 2014 Published by Elsevier Inc.
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1. Introduction
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Oligopeptide permease (Opp) systems are widely distributed in both gram-positive and negative bacteria. Genes encoding the Opp components are commonly organized as a cluster, including one encoding the substrate binding protein (OppA), two transmembrane proteins (OppB and OppC) which form the translocate channel, and two membrane-bound cytoplasmic ATP-binding proteins (OppD and OppF) which provide energy for peptide importing [1]. Opp systems are mainly involved in nutritional uptaking and environmental sensing [2,3], but they may play other roles such as drug resistance and virulence [4–6]. In Staphylococcus aureus, several Opp systems have been identified, namely Opp1-3 and Opp4/Opp3′. Of them, both opp4 and opp3′ operons are tandemly arranged at the downstream of opp3 operon in chromosome. However, they have varied a lot at the level of sequence, indicating that they arose through a remote duplication event [7,8]. Biological roles of Opp systems in S. aureus have been investigated: Opp2 and Opp3 participate in nickel uptaking and nitrogen utilization, respectively [7,9], while in some models Opp2 is also involved in virulence [10,11]. These Opp systems are conserved across different S. aureus isolates in
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⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Feng), [email protected] (Y. Yu).
terms of both sequence and gene organization. However, Opp systems in other staphylococcal species have not been well characterized. In addition to core chromosome-encoded Opp systems, a mobile element-encoded Opp system was identified in a communityassociated methicillin resistant S. aureus (CA-MRSA) clone USA300 (ST8-MRSA-Iva). It was originally reported as Opp3 and hereafter referred to as ACME-Opp since it is located within an arginine catabolic mobile element (ACME) [12]. Different ACMEs that have been detected in S. aureus and Staphylococcus epidermidis can be divided into three types according to the presence of arginine-deiminase system (Arc) and Opp system, i.e. type I containing both Arc and Opp systems, while types II and III containing only one of them [12–17]. ACMEs were firstly assumed to contribute to the successful spread of S. aureus, but later were proved to be unnecessary virulence factors for S. aureus [18,19]. Meanwhile, ACMEs in S. epidermidis have been found to be associated with low antibiotic resistance and low pathogenicity [20]. What is worth noting is that the function of Arc and Opp systems is independent from each other. While the ACME-Arc system allows S. aureus to thrive in acidic environments [21], the function of ACMEOpp system remains unclear. In addition, due to the high similarity of ACMEs from S. aureus and S. epidermidis, S. epidermidis is considered as the most probable donor of the ACME from S. aureus. However, it is not well described how gene transfer took place or in which manner Arc and Opp systems are assembled together [13,22]. In this work we performed genome-wide identification of Opp systems in a total of 51 isolates of seven staphylococcal species, which revealed the diversity
http://dx.doi.org/10.1016/j.ygeno.2014.04.003 0888-7543/© 2014 Published by Elsevier Inc.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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41 selected S. aureus isolates were classified into two classes according to the Opp systems they carried, i.e. class I (36 isolates) carrying Opp1-4 and class II (5 isolates) carrying Opp1-3 and Opp3′ (Fig. 1, Table S1). Multi-locus sequence typing analysis showed that the S. aureus isolates from class I had diverse genetic backgrounds, while class II contained ST398, ST93 and ST151 isolates only. Interestingly, the two S. aureus classes cannot separate their clones from each other, so that duplication of Opp3 system may have occurred several times in S. aureus independently and the duplicates, i.e. Opp3′ or Opp4, are not the major cause leading to the wide spread of ST5 and ST8 clones (Fig. 2). Seven staphylococcal species other than S. aureus were also investigated in this work, which showed a pronounced diversity of Opp systems (Table 1). For those characterized Opp systems, only Opp3 was conserved in all of the staphylococcal species. Notably, Opp3 and Opp4 were tandemly arranged in S. aureus (class I), but they were not adjacent to each other in Staphylococcus carnosus TM300 and Staphylococcus pseudintermedius ED99. Moreover, tandem duplication of Opp4 (designated Opp4′) was identified in S. pseudintermedius ED99, and operon opp4′ was directly located at the downstream of opp4 operon in chromosome. The protein identity between Opp4 components to their counterparts in Opp4′ system was 76–87%. Remarkably, both Opp4 and Opp4′ were absent from S. pseudintermedius strain HKU10-03, indicating that the acquisition of Opp4 and its subsequent duplication in ED99 both occurred recently. The Opp2 system was identified in the non-S. aureus species except Staphylococcus haemolyticus and Staphylococcus lugdunensis. In S. carnosus, S. pseudintermedius and Staphylococcus saprophyticus,
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2.3. Pseudogenes in opp gene clusters
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In this work, we identified over one hundred Opp systems in staphylococcal species. Most of them are intact and their coding genes are clustered. However, opp pseudogenes can also be identified in six S. aureus isolates and in three other non-S. aureus species. Frame shift and interruption by insert sequence (IS) were two main reasons of gene inactivation, which were responsible for eight and four pseudogenes, respectively (Table 2). Interestingly, the four IS interrupted genes were exclusively opp4 genes, indicating that opp4 was a hotspot for insertion events.
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In S. aureus, the entire structure of ACME-Opp system has been exclusively characterized in USA300 strains, i.e. FPR3757 and TCH1516, which are nearly identical [12,23]. In this work, genome-wide identification of their homologues in other staphylococcal species was performed, which found several ACME-OPPL systems in S. haemolyticus, S. lugdunensis and Staphylococcus warneri (Table 1). Notably, components of the two ACME-OPPL systems in S. haemolyticus showed varied protein identity to their S. aureus counterparts, i.e. 84–91% for ACME-OppLH1 (SH0147-151) and 49–59% for ACME-OppLH2 (SH0288-292). In contrast, ACME-OPPL systems in S. lugdunensis showed lower sequence identity to S. aureus homologues, e.g. 54–72% for ACME-OppLL1 (SLGD_00199-203) and 47–59% for ACME-OppLL2 (SLGD_00621-626). Moreover, in contrary to S. aureus and S. epidermidis, in which the ACMEs are adjacent to staphylococcal cassette chromosome (SCC) element regions, the ACME-OPPL systems in S. lugdunensis and S. haemolyticus are not contained in ACME elements and are distant from SCCmec. For example, ACME-OppLH1 and ACME-OppLH2 in S. haemolyticus are 25 kb and 165 kb away from the SCC composite island, respectively [24].
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2.1. Identification of Opp1-4 systems in staphylococci
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2.2. ACME-Opp-like (ACME-OPPL) systems in staphylococci
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Opp2 system had an extra component, Opp2A, a substrate binding 112 protein that showed 60 to 65% protein identity to Opp5A in S. aureus. 113
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of Opp systems in staphylococci systematically. Moreover, based on comparative and phylogenetic analysis, we made a more solid hypothesis about the evolution of ACME-Opp system.
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Fig. 1. Opp operons in staphylococci. a) Gene organization of opp operons identified in staphylococcal species. Coding genes for Opp2II are organized in a cluster, but for Opp2I, the oppA gene is separated from others. b) Arrangement of opp3 and opp4 operons (or their duplicates) in staphylococcal chromosomes. Operons opp3 and opp4 are tandemly arranged in Staphylococcus aureus, but not in S. carnosus and S. pseudintermedius.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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Table 2 Pseudogenes in staphylococcal opp operons. Strains
Genes
Description
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S. aureus MASA252
opp4A opp4B opp4F opp4A opp4B opp3A opp3F opp3F opp4B opp2A opp3A oppFa
ISSau1 interruption ISSau2 interruption Frame shift ISSau1 interruption IS256 interruption Frame shift Frame shift Frame shift Frame shift Frame shift Frame shift Frame shift
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S. aureus TCH60 S. S. S. S.
aureus T0131 aureus 11819-97 aureus 71193 aureus HO 5096 0412
This oppF gene is contained in ACME-Opp-like system II of S. lugdunensis HKU09-01.
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S. carnosus TM300 S. warneri SG1 S. lugdunensis HKU09-01
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3. Discussion
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phylogenetic relationship of Opp systems in staphylococcal species (Fig. 3). Based on the tree we had the following findings. Firstly, Opp5A in S. aureus and S. epidermidis belonged to the same clad as Opp2A in S. saprophyticus and S. carnosus (CoNS), as well as S. pseudintermedius (CoPS), implying that Opp5A was derived from Opp2A. This hypothesis was also supported by the evidence that Opp5A and Opp2-BCDF form a nickel transport system in S. aureus (designated NikA and NikBCDE, respectively) [9]. Secondly, OppA proteins in ACME-Opp and ACME-OppL1 systems were clustered together, which were close to ACME-OPPL2 systems but distinct from other clads. So ACMEOpp system in S. aureus was probably evolved from the ACME-OPPL1 through operon duplication in the early period of staphylococci divergence. Meanwhile, the deviation of ACME-OppL1 and -OppL2 also suggested a secondary duplication occurring in the remote past but it might be not a common feature. Moreover, although extensive co-linearity between staphylococcal species has not been found, small-scaled co-linearity around the opp operons was observed in this work. The flanked genes of opp3 operon were conserved in all the investigated staphylococcal species, even in Macrococcus caseolyticus, another member of the family Staphylococcaceae [25]. For operons encoding systems Opp1 and Opp2, their environmental structures were also conserved across staphylococci, with the exception that in S. pseudintermedius the flanked regions of Opp2 operon were different from other staphylococci. Notably, although opp4 operons were identified in S. carnosus and S. pseudintermedius, they were not tandemly arranged at the downstream of opp3 operon, indicating that they were generated from remote duplication followed by recombination events, or perhaps they were acquired by lateral gene transfer from other staphylococci.
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Fig. 2. Multi-locus sequence typing analysis of selected Staphylococcus aureus strains. The clones carrying Opp1234 systems are colored in light blue, and the clones carrying Opp1233′ systems are colored in dark green. Solid lines indicate one mis-matched allele between connected clones, whereas dash lines indicate two or more mis-matched alleles between connected clones. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Phylogenetic analysis of Opp systems
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All of the characterized Opp systems contain two permeases and two ATP-binding proteins, while the substrate binding protein OppA is unique. In this work, OppA protein was used for reconstructing the
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Table 1 Opp systems in staphylococcal species other than S. aureus.
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A variety of Opp systems were identified in staphylococcal 178 species in this work. Besides the previously characterized Opp1-4, 179
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Strains
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ACME-Opp-like
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carnosus TM300 epidermidis RP62A epidermidis ATCC 12228 haemolyticus JCSC1435 lugdunensis HKU09-01 lugdunensis N920143 pseudintermedius HKU10-03 pseudintermedius ED99 saprophyticus ATCC 15305 warneri SG1
– ABCDF ABCDF – ABCDF ABCDF ABCDF ABCDF – ABCDF
ABCDF BCDF BCDF – – – ABCDF ABCDF ABCDF BCDF
BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA
ADFBC – – – – – – ADFBC – –
– – – – – – – ADFBC – –
– – – ABCDF (2)a ABCDF (2) ABCDF (2) – – – ABCDF
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Investigated strains of S. haemolyticus and S. lugdunensis encoded two ACME-Opp-like systems.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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Opp3′ and ACME-Opp in S. aureus, duplication of Opp4 was found in S. pseudintermedius ED99, and ACME-OPPL systems were identified in several CoNS. These findings suggest that novel Opp systems are generated along with the species divergence of staphylococci. Regardless of its original roles in adaptation, e.g. increasing the efficiency of peptide transport [7], the duplicates of Opp systems in S. aureus are involved in different pathways. For example, the substrate binding proteins in Opp systems are developed to sense different substrates [26]. Duplication is a common mechanism in producing new genes in microbial evolution for environmental adaptation [27]. In S. aureus, many virulence genes were created in this manner, exemplified by the paralogous genes located within the genomic islands vSaα and vSaβ, which encode for exotoxins, lipoproteins, serine proteases, enterotoxins, and lantibiotic bacteriocins [28]. In this work, both recent duplication, e.g. Opp33′ in S. aureus and Opp44′ in S. pseudintermedius, and remote duplication, e.g. Opp34 in S. aureus, were observed. However, the duplicated Opp systems are not always functional, as inferred from the opp pseudogenes identified in the staphylococcal species. Lateral gene transfer is the other important mechanism for staphylococci acquiring novel Opp systems. The Opp4 system was present in S. pseudintermedius ED99 but absent from S. pseudintermedius HKU1003. Likewise, its components showed much higher similarity to Opp4 in other staphylococcal species than Opp3 in S. pseudintermedius.
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Fig. 3. Phylogenetic analysis of Opp systems in staphylococcal species. This neighbor-joining tree was constructed based on the aligned amino acid sequences of OppA. The OppA in Macrococcus caseolyticus was used as outgroup. The scale bar denotes the rate of nucleotide substitution.
All lines of evidence strongly suggest that it is lateral gene transfer that has given rise to Opp4 system in strain ED99. ACMEs have been exclusively characterized in S. aureus and S. epidermidis, although its spread in other species like S. haemolyticus was described [29]. In this work, several ACME-OPPL systems were identified in non-S. aureus staphylococci. In S. haemolyticus, ACME-OppLH1 was inferred to be the recent ancestor of ACMEOpp system in S. aureus and S. epidermidis because of their remarkably high sequence similarity. The characterized ACMEs in S. aureus and S. epidermidis were linked to the SCC elements and performed as composite islands. However, ACME-OppL1 in all sequenced S. haemolyticus isolates was not adjacent to the SCC region, and it was not bound together with Arc system. These findings, together with the significant genetic diversity of ACMEs observed in S. epidermidis, suggest that the major components of ACMEs were firstly transferred from S. haemolyticus to S. epidermidis, where the Arc and Opp systems were assembled together, and were then transferred to S. aureus. The ancestors of ACME-Opp systems are native content in S. haemolyticus and have not demonstrated a role in bacteria fitness. However, after being transferred to S. epidermidis and S. aureus as components of ACMEs, ACME-Opp systems have already made certain contribution to clone success, indicating that its recent divergence from S. haemolyticus to S. epidermidis/S. aureus has assigned novel functions. Finally, the typing analysis can't separate class I and II S. aureus from
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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5. Materials and methods
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5.1. Selected bacterial genomes
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Fifty-one completely sequenced staphylococci were investigated in this work, including S. aureus (41 isolates), S. carnosus (1 isolate), S. epidermidis (2 isolates), S. haemolyticus (1 isolate), S. lugdunensis (2 isolates), S. pseudintermedius (2 isolates), S. saprophyticus (1 isolate) and S. warneri (1 isolate) (Table S2). Their genome sequences and the annotation were retrieved from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/).
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5.2. Identification of opp genes
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According to the description by Hiron et al. [7], opp genes from S. aureus NCTC 8325 were collected, i.e. components of opp1-4 and the additional opp5A. ACME-opp genes were collected from S. aureus USA300_FPR3757 (YP_492791-YP_492795) [12]. These 25 genes were then used as the seeds for sequence based homologue search by using BLASTP. The candidate opp genes were collected with the parameters E-value b 1e−3, identity N 25% and the query coverage N 80%. In order to identify the extensively diverged homologues, Hidden Markov Model (HMM) was also used in greedy search of potential opp genes. HMM profiles for three protein families, i.e. SBP_bac_5 (PF00496), BPD_transp_1 (PF00528) and ABC_tran (PF00005), were retrieved from PFAM database (http://pfam.janelia.org/), and the HMM search was performed using HMMER3.0 with gathering thresholds [30]. Manual checking was subsequently carried out to determine the Opp systems.
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5.3. Identification of pseudogenes
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Candidate pseudogenes were identified by tBLASTn and the frame shift was determined using GeneWise (www.ebi.ac.uk). Insertion sequences in opp operons were classified using IS finder database (www-is.biotoul.fr).
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5.4. Phylogenetic analysis
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Multiple sequence alignment was performed using ClustalW [31]. MEGA 4 was used to construct the neighbor-joining tree of OppA, which was bootstrapped with 1000 replicates [32]. The minimum split tree of selected S. aureus isolates was generated by Bionumeric software (Applied Maths, Belgium), with the allelic profiles of seven housekeeping genes being retrieved from multi-locus sequence typing database (www.mlst.net).
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This work was supported by the Ministry of Health of the People's Republic of China (no. 201002021) and the National Science Foundation of China (no. 81201248 & no. 81201327). The authors would like to thank Dr Sheng Donglai for his critical reading and language improvement of the manuscript prior to submission.
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Acknowledgments
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Firstly, our work performed a systematic analysis of Opp systems in staphylococcal species and classified the known and newly found Opp systems into six families, which was helpful to resolve the currently confused nomenclature of Opp systems. Secondly, phylogenetic analysis and comparative genomics suggested again that Opp3 was the ancestor of other Opp systems and also revealed that operon duplication and lateral gene transfer played a crucial role in the generation of new Opp systems in staphylococcal species. Thirdly, this work revealed the origin of S. aureus encoded ACME-Opp system and promoted the understanding of ACME evolution. When these findings indicate the crucial role of Opp systems in staphylococci adaptation, nevertheless, whether their function has been diverged in different species or what's the exact role of ACME-Opp/-like systems remains to be illustrated in future study.
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each other, indicating that, in contrary to ACME-Opp, Opp3′ and Opp4 were not involved in clonal spread.
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Contents lists available at ScienceDirect
Genomics journal homepage: www.elsevier.com/locate/ygeno
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Dongliang Yu a,b, Borui Pi b, Meihong Yu c, Yanfei Wang b, Zhi Ruan b, Ye Feng b,⁎, Yunsong Yu b,⁎
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Diversity and evolution of oligopeptide permease systems in staphylococcal species
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Article history: Received 24 October 2013 Accepted 24 April 2014 Available online xxxx
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Keywords: Oligopeptide permease Staphylococci Duplication Gene cluster
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Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, China State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China
a b s t r a c t
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Several oligopeptide permease (Opp) systems have been found in staphylococcal species, including Opp1-4, Opp3′ and the arginine catabolic mobile element (ACME)-encoded Opp system (ACME-Opp). They confer upon bacteria the increasing fitness, but their evolutionary histories remain unclear. In this work, we performed a genome-wide identification of Opp systems in staphylococcal species. Novel Opp systems were identified, including the duplicate of Opp4 in Staphylococcus pseudintermedius and the ACME-Opp-like systems in coagulase-negative staphylococci (CoNS). Phylogenetic analysis revealed that all of the identified Opp systems were derived from Opp3 system by operon duplication during species divergence, while lateral gene transfer might also confer to the dissemination of Opp in staphylococci. In addition, we proposed an improved theory on evolution of ACME: the Opp and arginine-deiminase systems were firstly transferred from Staphylococcus haemolyticus to Staphylococcus epidermidis independently; in S. epidermidis they were assembled together and then transferred to Staphylococcus aureus. © 2014 Published by Elsevier Inc.
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1. Introduction
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Oligopeptide permease (Opp) systems are widely distributed in both gram-positive and negative bacteria. Genes encoding the Opp components are commonly organized as a cluster, including one encoding the substrate binding protein (OppA), two transmembrane proteins (OppB and OppC) which form the translocate channel, and two membrane-bound cytoplasmic ATP-binding proteins (OppD and OppF) which provide energy for peptide importing [1]. Opp systems are mainly involved in nutritional uptaking and environmental sensing [2,3], but they may play other roles such as drug resistance and virulence [4–6]. In Staphylococcus aureus, several Opp systems have been identified, namely Opp1-3 and Opp4/Opp3′. Of them, both opp4 and opp3′ operons are tandemly arranged at the downstream of opp3 operon in chromosome. However, they have varied a lot at the level of sequence, indicating that they arose through a remote duplication event [7,8]. Biological roles of Opp systems in S. aureus have been investigated: Opp2 and Opp3 participate in nickel uptaking and nitrogen utilization, respectively [7,9], while in some models Opp2 is also involved in virulence [10,11]. These Opp systems are conserved across different S. aureus isolates in
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⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Feng), [email protected] (Y. Yu).
terms of both sequence and gene organization. However, Opp systems in other staphylococcal species have not been well characterized. In addition to core chromosome-encoded Opp systems, a mobile element-encoded Opp system was identified in a communityassociated methicillin resistant S. aureus (CA-MRSA) clone USA300 (ST8-MRSA-Iva). It was originally reported as Opp3 and hereafter referred to as ACME-Opp since it is located within an arginine catabolic mobile element (ACME) [12]. Different ACMEs that have been detected in S. aureus and Staphylococcus epidermidis can be divided into three types according to the presence of arginine-deiminase system (Arc) and Opp system, i.e. type I containing both Arc and Opp systems, while types II and III containing only one of them [12–17]. ACMEs were firstly assumed to contribute to the successful spread of S. aureus, but later were proved to be unnecessary virulence factors for S. aureus [18,19]. Meanwhile, ACMEs in S. epidermidis have been found to be associated with low antibiotic resistance and low pathogenicity [20]. What is worth noting is that the function of Arc and Opp systems is independent from each other. While the ACME-Arc system allows S. aureus to thrive in acidic environments [21], the function of ACMEOpp system remains unclear. In addition, due to the high similarity of ACMEs from S. aureus and S. epidermidis, S. epidermidis is considered as the most probable donor of the ACME from S. aureus. However, it is not well described how gene transfer took place or in which manner Arc and Opp systems are assembled together [13,22]. In this work we performed genome-wide identification of Opp systems in a total of 51 isolates of seven staphylococcal species, which revealed the diversity
http://dx.doi.org/10.1016/j.ygeno.2014.04.003 0888-7543/© 2014 Published by Elsevier Inc.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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41 selected S. aureus isolates were classified into two classes according to the Opp systems they carried, i.e. class I (36 isolates) carrying Opp1-4 and class II (5 isolates) carrying Opp1-3 and Opp3′ (Fig. 1, Table S1). Multi-locus sequence typing analysis showed that the S. aureus isolates from class I had diverse genetic backgrounds, while class II contained ST398, ST93 and ST151 isolates only. Interestingly, the two S. aureus classes cannot separate their clones from each other, so that duplication of Opp3 system may have occurred several times in S. aureus independently and the duplicates, i.e. Opp3′ or Opp4, are not the major cause leading to the wide spread of ST5 and ST8 clones (Fig. 2). Seven staphylococcal species other than S. aureus were also investigated in this work, which showed a pronounced diversity of Opp systems (Table 1). For those characterized Opp systems, only Opp3 was conserved in all of the staphylococcal species. Notably, Opp3 and Opp4 were tandemly arranged in S. aureus (class I), but they were not adjacent to each other in Staphylococcus carnosus TM300 and Staphylococcus pseudintermedius ED99. Moreover, tandem duplication of Opp4 (designated Opp4′) was identified in S. pseudintermedius ED99, and operon opp4′ was directly located at the downstream of opp4 operon in chromosome. The protein identity between Opp4 components to their counterparts in Opp4′ system was 76–87%. Remarkably, both Opp4 and Opp4′ were absent from S. pseudintermedius strain HKU10-03, indicating that the acquisition of Opp4 and its subsequent duplication in ED99 both occurred recently. The Opp2 system was identified in the non-S. aureus species except Staphylococcus haemolyticus and Staphylococcus lugdunensis. In S. carnosus, S. pseudintermedius and Staphylococcus saprophyticus,
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2.3. Pseudogenes in opp gene clusters
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In this work, we identified over one hundred Opp systems in staphylococcal species. Most of them are intact and their coding genes are clustered. However, opp pseudogenes can also be identified in six S. aureus isolates and in three other non-S. aureus species. Frame shift and interruption by insert sequence (IS) were two main reasons of gene inactivation, which were responsible for eight and four pseudogenes, respectively (Table 2). Interestingly, the four IS interrupted genes were exclusively opp4 genes, indicating that opp4 was a hotspot for insertion events.
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In S. aureus, the entire structure of ACME-Opp system has been exclusively characterized in USA300 strains, i.e. FPR3757 and TCH1516, which are nearly identical [12,23]. In this work, genome-wide identification of their homologues in other staphylococcal species was performed, which found several ACME-OPPL systems in S. haemolyticus, S. lugdunensis and Staphylococcus warneri (Table 1). Notably, components of the two ACME-OPPL systems in S. haemolyticus showed varied protein identity to their S. aureus counterparts, i.e. 84–91% for ACME-OppLH1 (SH0147-151) and 49–59% for ACME-OppLH2 (SH0288-292). In contrast, ACME-OPPL systems in S. lugdunensis showed lower sequence identity to S. aureus homologues, e.g. 54–72% for ACME-OppLL1 (SLGD_00199-203) and 47–59% for ACME-OppLL2 (SLGD_00621-626). Moreover, in contrary to S. aureus and S. epidermidis, in which the ACMEs are adjacent to staphylococcal cassette chromosome (SCC) element regions, the ACME-OPPL systems in S. lugdunensis and S. haemolyticus are not contained in ACME elements and are distant from SCCmec. For example, ACME-OppLH1 and ACME-OppLH2 in S. haemolyticus are 25 kb and 165 kb away from the SCC composite island, respectively [24].
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Opp2 system had an extra component, Opp2A, a substrate binding 112 protein that showed 60 to 65% protein identity to Opp5A in S. aureus. 113
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of Opp systems in staphylococci systematically. Moreover, based on comparative and phylogenetic analysis, we made a more solid hypothesis about the evolution of ACME-Opp system.
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Fig. 1. Opp operons in staphylococci. a) Gene organization of opp operons identified in staphylococcal species. Coding genes for Opp2II are organized in a cluster, but for Opp2I, the oppA gene is separated from others. b) Arrangement of opp3 and opp4 operons (or their duplicates) in staphylococcal chromosomes. Operons opp3 and opp4 are tandemly arranged in Staphylococcus aureus, but not in S. carnosus and S. pseudintermedius.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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Table 2 Pseudogenes in staphylococcal opp operons. Strains
Genes
Description
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S. aureus MASA252
opp4A opp4B opp4F opp4A opp4B opp3A opp3F opp3F opp4B opp2A opp3A oppFa
ISSau1 interruption ISSau2 interruption Frame shift ISSau1 interruption IS256 interruption Frame shift Frame shift Frame shift Frame shift Frame shift Frame shift Frame shift
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S. aureus TCH60 S. S. S. S.
aureus T0131 aureus 11819-97 aureus 71193 aureus HO 5096 0412
This oppF gene is contained in ACME-Opp-like system II of S. lugdunensis HKU09-01.
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S. carnosus TM300 S. warneri SG1 S. lugdunensis HKU09-01
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phylogenetic relationship of Opp systems in staphylococcal species (Fig. 3). Based on the tree we had the following findings. Firstly, Opp5A in S. aureus and S. epidermidis belonged to the same clad as Opp2A in S. saprophyticus and S. carnosus (CoNS), as well as S. pseudintermedius (CoPS), implying that Opp5A was derived from Opp2A. This hypothesis was also supported by the evidence that Opp5A and Opp2-BCDF form a nickel transport system in S. aureus (designated NikA and NikBCDE, respectively) [9]. Secondly, OppA proteins in ACME-Opp and ACME-OppL1 systems were clustered together, which were close to ACME-OPPL2 systems but distinct from other clads. So ACMEOpp system in S. aureus was probably evolved from the ACME-OPPL1 through operon duplication in the early period of staphylococci divergence. Meanwhile, the deviation of ACME-OppL1 and -OppL2 also suggested a secondary duplication occurring in the remote past but it might be not a common feature. Moreover, although extensive co-linearity between staphylococcal species has not been found, small-scaled co-linearity around the opp operons was observed in this work. The flanked genes of opp3 operon were conserved in all the investigated staphylococcal species, even in Macrococcus caseolyticus, another member of the family Staphylococcaceae [25]. For operons encoding systems Opp1 and Opp2, their environmental structures were also conserved across staphylococci, with the exception that in S. pseudintermedius the flanked regions of Opp2 operon were different from other staphylococci. Notably, although opp4 operons were identified in S. carnosus and S. pseudintermedius, they were not tandemly arranged at the downstream of opp3 operon, indicating that they were generated from remote duplication followed by recombination events, or perhaps they were acquired by lateral gene transfer from other staphylococci.
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Fig. 2. Multi-locus sequence typing analysis of selected Staphylococcus aureus strains. The clones carrying Opp1234 systems are colored in light blue, and the clones carrying Opp1233′ systems are colored in dark green. Solid lines indicate one mis-matched allele between connected clones, whereas dash lines indicate two or more mis-matched alleles between connected clones. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Phylogenetic analysis of Opp systems
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All of the characterized Opp systems contain two permeases and two ATP-binding proteins, while the substrate binding protein OppA is unique. In this work, OppA protein was used for reconstructing the
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Table 1 Opp systems in staphylococcal species other than S. aureus.
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A variety of Opp systems were identified in staphylococcal 178 species in this work. Besides the previously characterized Opp1-4, 179
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Strains
Opp1
Opp2
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Opp4
Opp4′
ACME-Opp-like
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S. S. S. S. S. S. S. S. S. S.
carnosus TM300 epidermidis RP62A epidermidis ATCC 12228 haemolyticus JCSC1435 lugdunensis HKU09-01 lugdunensis N920143 pseudintermedius HKU10-03 pseudintermedius ED99 saprophyticus ATCC 15305 warneri SG1
– ABCDF ABCDF – ABCDF ABCDF ABCDF ABCDF – ABCDF
ABCDF BCDF BCDF – – – ABCDF ABCDF ABCDF BCDF
BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA BCDFA
ADFBC – – – – – – ADFBC – –
– – – – – – – ADFBC – –
– – – ABCDF (2)a ABCDF (2) ABCDF (2) – – – ABCDF
t1:14
a
Investigated strains of S. haemolyticus and S. lugdunensis encoded two ACME-Opp-like systems.
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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Opp3′ and ACME-Opp in S. aureus, duplication of Opp4 was found in S. pseudintermedius ED99, and ACME-OPPL systems were identified in several CoNS. These findings suggest that novel Opp systems are generated along with the species divergence of staphylococci. Regardless of its original roles in adaptation, e.g. increasing the efficiency of peptide transport [7], the duplicates of Opp systems in S. aureus are involved in different pathways. For example, the substrate binding proteins in Opp systems are developed to sense different substrates [26]. Duplication is a common mechanism in producing new genes in microbial evolution for environmental adaptation [27]. In S. aureus, many virulence genes were created in this manner, exemplified by the paralogous genes located within the genomic islands vSaα and vSaβ, which encode for exotoxins, lipoproteins, serine proteases, enterotoxins, and lantibiotic bacteriocins [28]. In this work, both recent duplication, e.g. Opp33′ in S. aureus and Opp44′ in S. pseudintermedius, and remote duplication, e.g. Opp34 in S. aureus, were observed. However, the duplicated Opp systems are not always functional, as inferred from the opp pseudogenes identified in the staphylococcal species. Lateral gene transfer is the other important mechanism for staphylococci acquiring novel Opp systems. The Opp4 system was present in S. pseudintermedius ED99 but absent from S. pseudintermedius HKU1003. Likewise, its components showed much higher similarity to Opp4 in other staphylococcal species than Opp3 in S. pseudintermedius.
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Fig. 3. Phylogenetic analysis of Opp systems in staphylococcal species. This neighbor-joining tree was constructed based on the aligned amino acid sequences of OppA. The OppA in Macrococcus caseolyticus was used as outgroup. The scale bar denotes the rate of nucleotide substitution.
All lines of evidence strongly suggest that it is lateral gene transfer that has given rise to Opp4 system in strain ED99. ACMEs have been exclusively characterized in S. aureus and S. epidermidis, although its spread in other species like S. haemolyticus was described [29]. In this work, several ACME-OPPL systems were identified in non-S. aureus staphylococci. In S. haemolyticus, ACME-OppLH1 was inferred to be the recent ancestor of ACMEOpp system in S. aureus and S. epidermidis because of their remarkably high sequence similarity. The characterized ACMEs in S. aureus and S. epidermidis were linked to the SCC elements and performed as composite islands. However, ACME-OppL1 in all sequenced S. haemolyticus isolates was not adjacent to the SCC region, and it was not bound together with Arc system. These findings, together with the significant genetic diversity of ACMEs observed in S. epidermidis, suggest that the major components of ACMEs were firstly transferred from S. haemolyticus to S. epidermidis, where the Arc and Opp systems were assembled together, and were then transferred to S. aureus. The ancestors of ACME-Opp systems are native content in S. haemolyticus and have not demonstrated a role in bacteria fitness. However, after being transferred to S. epidermidis and S. aureus as components of ACMEs, ACME-Opp systems have already made certain contribution to clone success, indicating that its recent divergence from S. haemolyticus to S. epidermidis/S. aureus has assigned novel functions. Finally, the typing analysis can't separate class I and II S. aureus from
Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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5. Materials and methods
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5.1. Selected bacterial genomes
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Fifty-one completely sequenced staphylococci were investigated in this work, including S. aureus (41 isolates), S. carnosus (1 isolate), S. epidermidis (2 isolates), S. haemolyticus (1 isolate), S. lugdunensis (2 isolates), S. pseudintermedius (2 isolates), S. saprophyticus (1 isolate) and S. warneri (1 isolate) (Table S2). Their genome sequences and the annotation were retrieved from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/).
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According to the description by Hiron et al. [7], opp genes from S. aureus NCTC 8325 were collected, i.e. components of opp1-4 and the additional opp5A. ACME-opp genes were collected from S. aureus USA300_FPR3757 (YP_492791-YP_492795) [12]. These 25 genes were then used as the seeds for sequence based homologue search by using BLASTP. The candidate opp genes were collected with the parameters E-value b 1e−3, identity N 25% and the query coverage N 80%. In order to identify the extensively diverged homologues, Hidden Markov Model (HMM) was also used in greedy search of potential opp genes. HMM profiles for three protein families, i.e. SBP_bac_5 (PF00496), BPD_transp_1 (PF00528) and ABC_tran (PF00005), were retrieved from PFAM database (http://pfam.janelia.org/), and the HMM search was performed using HMMER3.0 with gathering thresholds [30]. Manual checking was subsequently carried out to determine the Opp systems.
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Candidate pseudogenes were identified by tBLASTn and the frame shift was determined using GeneWise (www.ebi.ac.uk). Insertion sequences in opp operons were classified using IS finder database (www-is.biotoul.fr).
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Multiple sequence alignment was performed using ClustalW [31]. MEGA 4 was used to construct the neighbor-joining tree of OppA, which was bootstrapped with 1000 replicates [32]. The minimum split tree of selected S. aureus isolates was generated by Bionumeric software (Applied Maths, Belgium), with the allelic profiles of seven housekeeping genes being retrieved from multi-locus sequence typing database (www.mlst.net).
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This work was supported by the Ministry of Health of the People's Republic of China (no. 201002021) and the National Science Foundation of China (no. 81201248 & no. 81201327). The authors would like to thank Dr Sheng Donglai for his critical reading and language improvement of the manuscript prior to submission.
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Firstly, our work performed a systematic analysis of Opp systems in staphylococcal species and classified the known and newly found Opp systems into six families, which was helpful to resolve the currently confused nomenclature of Opp systems. Secondly, phylogenetic analysis and comparative genomics suggested again that Opp3 was the ancestor of other Opp systems and also revealed that operon duplication and lateral gene transfer played a crucial role in the generation of new Opp systems in staphylococcal species. Thirdly, this work revealed the origin of S. aureus encoded ACME-Opp system and promoted the understanding of ACME evolution. When these findings indicate the crucial role of Opp systems in staphylococci adaptation, nevertheless, whether their function has been diverged in different species or what's the exact role of ACME-Opp/-like systems remains to be illustrated in future study.
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each other, indicating that, in contrary to ACME-Opp, Opp3′ and Opp4 were not involved in clonal spread.
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Please cite this article as: D. Yu, et al., Genomics (2014), http://dx.doi.org/10.1016/j.ygeno.2014.04.003
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