SEARCH

SEARCH BY CITATION

Keywords:

  • biodegradation;
  • replication initiation (Rep) proteins;
  • incompatibility groups;
  • conjugation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

Large plasmids (‘megaplasmids’) are commonly found in members of the Alphaproteobacterial family Sphingomonadaceae (‘sphingomonads’). These plasmids contribute to the extraordinary catabolic flexibility of this group of organisms, which degrade a broad range of recalcitrant xenobiotic compounds. The genomes of several sphingomonads have been sequenced during the last years. In the course of these studies, also the sequences of several plasmids have been determined. The analysis of the published information and the sequences deposited in the public databases allowed a first classification of these plasmids into a restricted number of groups according to the proteins involved in the initiation of replication, plasmid partition and conjugation. The sequence comparisons demonstrated that the plasmids from sphingomonads encode for four main groups of replication initiation (Rep) proteins. These Rep proteins belong to the protein superfamilies RepA_C (Pfam 04796), Rep_3 (Pfam 01051), RPA (Pfam 10134) and HTH-36 (Pfam 13730). The ‘degradative megaplasmids’ pNL2, pCAR3, pSWIT02, pCHQ1, pISP0, and pISP1, which code for genes involved in the degradation of aromatic hydrocarbons, carbazole, dibenzo-p-dioxin and γ-hexachlorocyclohexane, carry Rep proteins which either belong to the RepA_C- (plasmids pNL2, pCAR3, pSWIT02), Rep-3- (plasmids pCHQ1, pISP0) or RPA-superfamily (pISP1). The classification of these ‘degradative megaplasmids’ into three groups is also supported by sequence comparisons of the proteins involved in plasmid partition (ParAB) and the organization of the three genes on the respective plasmids. All analysed ‘degradative megaplasmids’ carry genes, which might allow a conjugative transfer of the plasmids. Sequence comparisons of these genes suggest the presence of at least two types of transfer functions, which either are closer related to the tra- or vir-genes previously described for plasmids from other sources.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

The sphingomonads represent a group of Alphaproteobacteria, which encompass in our days the genera Novosphingobium, Sphingobium, Sphingomonas, Sphingopyxis, Sphingosinicella, Sphingomicrobium, Sphingorhabdus and Parasphingopyxis. These genera share a number of phenotypic traits, such as the presence of sphingolipids in their outer membranes, the formation of usually yellow-pigmented colonies and a specific pattern of polyamines (Kämpfer et al., 2012; Uchida et al., 2012; Jogler et al., 2013). Sphingomonads have been intensively studied during the last years because of their pronounced ability to degrade recalcitrant natural and xenobiotic compounds, such as various polycyclic aromatic hydrocarbons (PAHs), nonylphenols, sulphonated naphthalenes, chlorinated dibenzofurans and dibenzodioxins, carbazole, polyethylene glycols and different herbicides and pesticides (Stolz, 2009). It was shown in the last years that many sphingomonads possess (often several) plasmids and especially that rather large plasmids are common in this bacterial group. These large plasmids are commonly designated as ‘megaplasmids’ if their sizes exceed about 100 kbp (Basta et al., 2004, 2005; Aylward et al., 2013). These ‘megaplasmids’ often carry genes coding for degradative pathways, which are often found either on different replicons (as e.g. observed for the degradative pathways of γ-hexachlorocyclohexane or dibenzo-p-dioxin) or are at least organized in several transcriptional units (Stolz, 2009). Furthermore, these plasmids often undergo after transfer between different sphingomonads pronounced rearrangements (Feng et al., 1997ab; Ogram et al., 2000; Basta et al., 2004, 2005). Therefore, it seems that the maintenance, transfer and recombination of these plasmids are of major importance for the exceptional degradative capabilities of this group of bacteria.

The genomes of several sphingomonads have recently been sequenced, and therefore, also an increasing number of plasmid sequences from sphingomonads became available. It was therefore attempted to analyse the available plasmid sequence data in order to collect the currently accessible information about (degradative) plasmids in sphingomonads.

Sequenced (degradative) plasmids from sphingomonads

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

The first example of a sequenced and carefully analysed degradative plasmid from a sphingomonad was plasmid pNL1 from Sphingomonas (now Novosphingobium) aromaticivorans F199, which carries all genes required for the degradation of biphenyl, naphthalene, m-xylene and p-cresol (Romine et al., 1999). Subsequently, the sequence analysis of plasmids pCAR3 (carrying all the genes for the mineralization of carbazole), pCHQ1 (coding for the linRED genes participating in the degradation of γ-hexachlorocyclohexane) and pPDL2 (coding for a parathion hydrolase involved in organophosphate degradation) has been published (Shintani et al., 2007; Nagata et al., 2011; Pandeeti et al., 2012; Table 1).

Table 1. Sequenced plasmids from different sphingomonads and organization of the rep and par genes
PlasmidSize (kbp)OrganismsDegradative pathway(s) forSpecific genesGenBank numberRep-superfamilyReferencesLocalization (bp) of repA or repB (size of protein encoded)Localization (bp) of parA (size of protein encoded)Localization (bp) of parB (size of protein encoded)
  1. na, not annotated.

pNL1184Novosphingobium aromaticivorans F199Biphenyl, naphthalene, m-xylene, p-cresolAll genes necessary NC_009426.1 RepA_CRomine et al. (1999)

86 362–87 666

433 aa

84 603–85 808

401 aa

83 388–84 371

326 aa

pNL2487Novosphingobium aromaticivorans F199  NC_009427.1 RPARomine et al. (1999)

209 439–210 644

401 aa

212 040–213 242

400 aa

210 877–211 962

361 aa

pCAR3255Sphingomonas sp. KA1CarbazoleAll genes necessary NC_008308.1 RepA_CShintani et al. (2007)

200 594–201 594

433 aa

202 396–203 658

420 aa

203 777–204 757

326 aa

pCHQ1191Sphingobium japonicum UT26γ-Hexachlorocyclohexane linRED NC_014007.1 Rep_3Nagata et al. (2010)

1–1360

387 aa

1360–2562

400 aa

2607–3623

338 aa

pUT132Sphingobium japonicum UT26  NC_014005 RPANagata et al. (2010)

1–1104

367 aa

1417–2052

211 aa

na
pUT25.4Sphingobium japonicum UT26  NC_014009.1 RPANagata et al. (2010)

1–654

217 aa

nana
pPDL237 Sphingobium fuliginis Organophosphates opd NC_019376.1 RPAPandeeti et al. (2012)

19 777–20 880

367 aa

21 193–21 828

211 aa

na
pLA1188Novosphingobium pentaaromativo-rans US6-1Benz[a]pyrenePAH catabolic region AGFM01000122.1 Rep_3Luo et al. (2012)

84 694–85 854

386 aa

86 021–87 223

400 aa

87 506–88 327

273 aa

pLA260Novosphingobium pentaaromativo-rans US6-1  NZ_AGFM01000123 ?Luo et al. (2012)

2557–3339

260 aa

43 879–44 637

252 aa

42 857–43 882

341 aa

Mpl1160Novosphingobium sp. strain PP1YAromatic hydrocarbons  FR856860 RepA_CD'Argenio et al. (2011)

51 3635–51 4939

434 aa

51 5490–51 6695

401 aa

51 6867–51 7844

325 aa

Lpl192Novosphingobium sp. strain PP1Y  FR856860 RPAD'Argenio et al. (2011)

88 922–90 199

425 aa

86 441–87 634

397 aa

87 744–88 817

357 aa

Spl49Novosphingobium sp. strain PP1Y  FR856859 HTH-36D'Argenio et al. (2011)

45 176–45 961

261 aa

nana
pISP0276Sphingomonas sp. MM-1γ-Hexachlorocyclohexane linF CP004037 Rep_3Tabata et al. (2011, 2013)

4081–5244

376 aa

5439–6641

400 aa

6686–7702

338 aa

pISP1172Sphingomonas sp. MM-1γ-HexachlorocyclohexanelinA, linC CP004038 RPATabata et al. (2011, 2013)

114 750–115 880

376 aa

117 028–118 224

398 aa

115 961–117 031

356 aa

pISP254Sphingomonas sp. MM-1  CP004039 RPATabata et al. (2011, 2013)

1–1104

367 aa

1417–2052

211 aa

na
pISP344Sphingomonas sp. MM-1γ-Hexachlorocyclohexane linRES CP004040 RPATabata et al. (2011, 2013)

37 217–38 329

370  aa

38 704–39 357

217 aa

na
pISP433Sphingomonas sp. MM-1γ-HexachlorocyclohexanelinB, linC CP004041 Rep_3Tabata et al. (2011, 2013)

101–1012

303 aa

1376–2011

211 aa

na
pSWIT01310Sphingomonas wittichii RW1  CP000700 Rep_3Miller et al. (2010)

17 4080–17 5195

371 aa

4708–5361

217 aa

15 1005–15 3194

729 aa

pSWIT02223Sphingomonas wittichii RW1(Chlorinated) dibenzo-p-dioxin(s) dxnA1A2 CP000701.1 RepA_CMiller et al. (2010)

63 589–64 893

434 aa

61 838–62 989

383 aa

60 612–61 586

324 aa

pSLGP149Sphingobium sp. SYK-6  AP012223 Rep_3Masai et al. (2012)

1–1164

387 aa

1360–2562

400 aa

2610–3623

337 aa

pSPHCH01124 Sphingobium chlorophenolicum L-1   NC_015595 Rep_3Copley et al. (2012)

47 083–48 171

362 aa

45 685–46 887

400 aa

44 624–45 637

337 aa

pSY219Sphingobium chungbukense DJ77  NC_016000.1 RPAYeon & Kim (2011)

10 225–11 319

364 aa

6087–6737

216 aa

na
pYAN-15.2 Sphingobium yanoikuyae   NC_008246.1 HTH-36Ochou et al. (2008)

3757–4413

218 aa

nana
pYAN-24.9 Sphingobium yanoikuyae   NC_008247.1 Rep_3Ochou et al. (2008)

648–1541

297 aa

nana
pSM103 mini4.3Sphingopyxis macrogoltabida 103  NC_014466 RepA_CTani et al. (2011)

2644–3567

307 aa

nana
pSx-Qyy5.7Sphingobium xenophagum QYY  NC_006826 HTH-36 

1855–2562

235 aa

nana

For some other degradative plasmids from sphingomonads, currently, only the sequence data deposited in public databases are available, for example, for plasmid pSWIT02 from the dibenzo-p-dioxin degrading strain Sphingomonas wittichii RW1 (coding for the dibenzo-p-dioxin dioxygenase) or plasmids pISP0, pISP1, pISP3 and pISP4 from the γ-hexachlorocyclohexane-degrading isolate Sphingomonas sp. MM-1 (Table 1). These sequenced plasmids belong to a much larger number of degradative plasmids, and plasmids are also involved in the degradation of several PAHs, naphthalenesulphonates or polymeric polyethylenglycols and polyvinyl alcohols by sphingomonads (Fredrickson et al., 1999; Shuttleworth et al., 2000; Cho & Kim, 2001; Basta et al., 2004; Tani et al., 2007; Hu et al., 2008).

It has been demonstrated for many sphingomonads with the ability to degrade xenobiotic compounds that they contain multiple plasmids. Thus, in S. aromaticivorans F199, S. wittichii RW1 and Novosphingobium pentaaromativorans US6-1, two plasmids each were found. In the γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26 and the PAHs-degrading isolate Novosphingobium sp. strain PP1Y three plasmids, in the naphthalenesulphonates-degrading strain Sphingobium xenophagum BN6 and the organophosphates-degrading Sphingobium fuligines ATCC27551 four plasmids and in the γ-hexachlorocyclohexane-degrading strain Sphingomonas sp. MM-1 even five plasmids have been detected (Table 1; Romine et al., 1999; Basta et al., 2004; D'Argenio et al., 2011; Luo et al., 2012; Pandeeti et al., 2012; Tabata et al., 2013). Furthermore, for some sphingomonads, the presence of a ‘second chromosome’ has been described. These ‘second chromosomes’ are often only slightly larger than some of the ‘megaplasmids’ and resemble in various traits (e.g. the mechanism of replication) the ‘megaplasmids’. Therefore, it appears that these ‘second chromosomes’ might have been evolved by the uptake of some essential genes by certain ‘megaplasmids’ (Copley et al., 2012; Nagata et al., 2011).

The ability of sphingomonads to host several different plasmids in a single cell is essential for the degradation of many organic compounds. Thus, it has been shown for S. japonicum UT26 and also for Sphingomonas sp. MM-1 that the genes encoding for the mineralization of γ-hexachlorocyclohexane are scattered on at least three replicons in these strains (Nagata et al., 2010, 2011; Tabata et al., 2013). Similarly, in S. wittichii RW1, only the genes coding for the initial ‘dibenzo-p-dioxin dioxygenase’ have been located on plasmid pSWIT02 (Colquhoun et al., 2012).

Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

The ability or inability of different plasmids to coexist in bacterial cells is generally correlated with the existence of different incompatibility groups. The presence of five different plasmids in Sphingomonas sp. MM-1 clearly demonstrated that there must exists at least five different incompatibility groups in sphingomonads, and it can be assumed that the pronounced rearrangements, which occur after the conjugative transfer of degradative plasmids among sphingomonads, might be (at least in certain cases) related to incompatibility phenomena (Feng et al., 1997ab; Ogram et al., 2000; Basta et al., 2004, 2005). The phenotypically defined incompatibility groups can be correlated with the sequences of the replication initiator (Rep) proteins and the proteins involved in plasmid partition (Par) (Petersen, 2011). In this context, the Rep proteins are especially important as these are responsible for the initial site specific DNA-binding and nicking activities, which represent the first steps in plasmid replication. The plasmid sequences deposited at the NCBI database originating from the genera Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis clearly demonstrated that the genes annotated as rep genes (repA or repB) almost exclusively belong to three protein superfamilies. Thus, proteins belonging to the RepA_C superfamily (Pfam 04796), Rep_3 (Pfam 01051) and RPA superfamily (Pfam 10134) were found in large numbers among the deposited sequences (Table 1). In addition, the Rep proteins from four smaller plasmids (pUT2, pYAN-1, pSx-Qyy, Spl) – which do not carry any catabolic genes – were classified to belong to the HTH-36 superfamily (Pfam 13730).

An alignment of the Rep-sequences allowed the construction of a dendrogram to visualize the relationship among the different Rep-sequences (Fig. 1). This demonstrated that the Rep proteins from the large degradative plasmids pNL1, pCAR3, pSWIT02 and Mpl can be clearly differentiated from the Rep proteins encoded by other plasmids. Thus, these Rep proteins belong to the RepA_C family and are composed of about 430 amino acids (aa). In contrast, all other annotated Rep proteins are almost consistently smaller than 400 aa and did not belong to the RepA_C superfamily (Table 1).

image

Figure 1. Phylogenetic tree of putative RepA proteins from various plasmids from different sphingomonads. The alignment of the sequences (for the relevant NCBI registration numbers, see Table 1) and the phylogenetic tree (ClustalW, UPGMA) were carried out at the Kyoto University Bioinformatics Centre (www.genome.jp).

Download figure to PowerPoint

A second group of ‘megaplasmids’ consists of plasmid pISP1 (172 kbp) from Sphingomonas sp.MM-1, pNL2 (487 kbp) from N. aromaticivorans F199 and Lpl (192 kbp) from Novosphingobium sp. strain PP1Y. These plasmids encode for Rep proteins belonging to the RPA superfamily. Obviously, the plasmids of this group (=‘Mega-RPA’) are compatible with those of the group defined above (=‘Mega-RepAC’) as plasmids pNL1 and pNL2 are found together in N. aromaticivorans F199, and plasmids Mpl and Lpl in Novosphingobium sp. strain PP1Y (Romine et al., 1999; D'Argenio et al., 2011).

A third group of large degradative plasmids was identified among the plasmids that possess a Rep protein belonging to the Rep_3 superfamily. This group (=‘Mega-Rep3’) encompasses in addition to plasmids pCHQ1 and pISP0 (carrying genes for the degradation of γ-hexachlorocyclohexane) and pLA1 (carrying genes for the degradation of PAHs) also some other rather large (124–310 kbp) plasmids (e.g. pSLGP, pSPHCH01, pSWIT01), which presumably are not involved in the degradation of organic compounds, although the relevant annotations suggest that plasmids pSLPG and pSPHCH01 carry several genes, which are related to the resistance against toxic metals such as Cu or Hg. Unfortunately, there is some confusion in the annotation of the genes encoding the rep genes in this group. Thus, these genes have been annotated as repA in the case of plasmids pCHQ1, pLA1 and pSLGP, but as repB for plasmids pISP0, pSWIT01 and pSPHCH01. This differentiation is not reflected by the phylogenetic trees obtained in the course of the sequence comparisons and thus should be avoided (see e.g. Fig. 1). The coexistence of plasmids pSWIT01 and pSWIT02 in S. wittichii RW1 suggests that also the plasmids belonging to the ‘Mega-RPA-group’ (pSWIT02) and ‘Mega-Rep3-group’ (pSWIT01) represent different incompatibility groups within the sphingomonads.

The sequence comparisons also suggested that the smaller plasmids in general code for Rep proteins which either belong to the HTH-36 superfamily or the RPA superfamily (Table 1). [But it should be kept in mind that Pfam 10134 (=RPA superfamily) and Pfam 01051(=Rep_3 superfamily) define closely related sequences.] The dendrogram also suggested that pISP2 and pUT1, pISP3 and pSY2, and pISP4 and pYAN-2, respectively, carry closely related Rep proteins. As plasmids pISP2, pISP3 and pISP4 are able to coexist in Sphingomonas sp. MM-1, this might indicate that these groups represent three additional ‘incompatibility groups’ within the sphingomonads which might mainly enclose smaller plasmids.

The identification of Rep proteins belonging to the RepA_C-, Rep_3- and RPA-superfamilies clearly demonstrated that the plasmids from sphingomonads are closely related to plasmids from other bacterial groups. Thus, RepA proteins belonging to the RepA_C family have previously been described for plasmids from the incompatibility group IncW. The members of this incompatibility group (e.g. plasmids R388 or pSa) are known as broad-host-range plasmids and have already been isolated from Alphaproteobacteria (Fernández-Lopez et al., 2006). Similarly, Rep proteins belonging to the Rep_3 family have been identified in broad-host-range plasmids belonging to the IncN family (such as e.g. plasmid R46). Furthermore, a recent ‘metagenomic’ survey of rep genes obtained from activated sludge communities demonstrated that these three types of rep genes are rather prevalent among the rep genes observed in these complex communities (Sentchilo et al., 2013).

Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

The analysis of the large ‘megaplasmids’ pNL1 and pCAR3 had demonstrated that on these plasmids, parA and parB genes are located in close proximity to the repA genes (Romine et al., 1999; Shintani et al., 2007). Par (=partition) proteins are encoded by various plasmids and are essential for the proper partition of (especially larger) plasmids to the bacterial daughter cells. In these systems, ParB binds in a sequence-specific way to the plasmid DNA, and ParA is acting as an ATPase involved in plasmid partition (Funnell & Slavcev, 2004).

Sequence comparisons demonstrated that parA and parB genes are present in close proximity to the respective repA genes not only in pNL1 and pCAR3 but also on the two other groups of large plasmids identified above (Table 1). To further confirm the suggested classification of the ‘megaplasmids’ from sphingomonads, phylogenetic trees were constructed derived from the RepA, ParA and ParB sequences. These comparisons demonstrated for the three independently constructed dendrograms, a rather similar organization (Fig. 2). Thus, in all three dendrograms, pCAR3, pNL1, pSWIT02 and Mpl (=‘Mega-RepAC’) clustered together. Furthermore, also pCHQ1, pSLCP, pSPHCH01, pISP0 and pLA1 (=‘Mega-Rep3’) formed a clearly defined cluster. There was only some variability regarding the ‘Mega-RPA’-group, as the ParA and ParB sequences from plasmid pISP1 did not cluster together with the sequences from plasmids pNL2 and Lpl in the dendrograms. Nevertheless, the relevant sequences from these three plasmids were always clearly separated from the two other groups (Fig. 2).

image

Figure 2. Phylogenetic trees of putative RepA, ParA and ParB proteins from the megaplasmids from different sphingomonads. The alignment of the sequences (for the relevant NCBI registration numbers, see Table 1) and the phylogenetic tree (ClustalW, UPGMA) were carried out at the Kyoto University Bioinformatics Centre (www.genome.jp).

Download figure to PowerPoint

For the smaller plasmids pUT1, pISP2, pISP3, pISP4 and pDL2, only parA genes had been annotated in close proximity to the respective repA genes. The parA genes from these plasmids are significantly smaller compared with those found in the three groups of ‘megaplasmids’ and encode only for proteins of about 210 aa (Table 1). The sequence comparisons showed for plasmids pUT1, pISP2 and pPDL2 that in each case between the genes annotated as repA or parA, an additional small open reading frame (ORF) was present. These ORFs coded for proteins of 94–95 amino acid residues. An alignment of these sequences from pUT1, pISP2 and pPDL2 (YP_003543404, YP_006965787, YP_006965787) demonstrated that the encoded proteins are almost completely identical (92 identical amino acid residues). The conservation of the sequence and the position of these ORFs suggest that the encoded small proteins function as ParB. Similar combinations of ParA proteins with sizes of about 210–220 aa and ParB proteins with sizes of 70–95 aa have previously been described for plasmids related to plasmid pTAR from Agrobacterium tumefaciens (Kalnin et al., 2000; Funnell & Slavcev, 2004).

Organization of the rep and par genes on the different types of plasmids

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

It has been suggested recently that the structural coupling of a repA (or repB) gene together with a parAB operon, an origin of replication (oriV) and a palindromic centromere seems to be typical for replicons from Alphaproteobacteria. In this context, it also has been suggested that the replicons from this group of bacteria could be classified into only four different systems. Thus, in three basic types of replicons, a parAB operon is found together with repA, repB or dnaA type replicase genes. The fourth type of replicon are the repABC-type operons, which are specifically found in Alphaproteobacteria and which can be differentiated from the three other groups as in this system the oriV is situated within the replicase gene (Petersen, 2011).

The comparison of the organization of the three relevant genes on the plasmids from sphingomonads demonstrated that the three groups of ‘megaplasmids’ identified in the course of the sequence comparisons of the individual rep and par genes also corresponded with the gene organization. Thus, in the group of plasmids consisting of pNL1, pCAR3, pSWIT02 and Mpl (‘Mega-RepAC’), the repA genes are always transcribed in the opposite direction to the parAB genes (Fig. 3). For pNL1 and pCAR3, it has been previously shown that in the sequence space between the repA and parA genes, several 16–17 bp long repeats are present. This indicates that these repeats function as iterons and thus are the DNA sequences to which the RepA proteins bind (Romine et al., 1999; Shintani et al., 2007). A search for similar iterons at the corresponding position of plasmid pSWIT02 (using the program repfind; http://zlab.bu.edu/repfind) identified three copies of a 14 bp DNA sequence, which was part of the 16 bp iteron found at the respective site in pCAR3. Thus, it can be concluded that the plasmids belonging to the ‘Mega-RepAC-family’ belong to the RepA-group of Alphaproteobacterial replicons as previously defined by Petersen (2011).

image

Figure 3. Organization of the replication and partitioning modules from the three types of ‘megaplasmids’ from sphingomonads identified in this study.

Download figure to PowerPoint

The ‘Mega-RPA’ group (consisting of plasmids pNL2, pISP1 and Lpl) demonstrated the gene order parA, parB, repA (Fig. 3). This is the same gene order as found in the repABC operons. Unfortunately, the nomenclature of the genes participating in the repABC operons is different from the nomenclature used for the three other types of replicons. Thus, in the case of the repABC operons, RepA and RepB have sequence similarities to proteins involved in active segregation of plasmids – and thus are equivalent to ParA and ParB in the other systems – and RepC is the replication initiator protein – and thus is equivalent to RepA in the other systems (Cevallos et al., 2008). The repABC plasmids show in addition to the highly conserved gene order also further characteristics. Thus, it had been shown that in the large intergenic sequence between repB and repC, a gene is present which codes for a small antisense RNA which is involved in the control of plasmid replication (Cevallos et al., 2008). Therefore, the sequence space between the genes coding for the parB and repA genes of plasmids pNL2, pISP1 and Lpl were analysed and compared with the respective sequences encoding for the antisense RNAs from various plasmids belonging to the repABC family (Venkova-Canoca et al., 2004), but no significant sequence homologies were detected. This suggested that the plasmids of ‘Mega-RPA-group’ do not belong to the repABC plasmids.

The plasmids belonging to the ‘Mega-Rep3’-group (pCHQ1, pISP0, pLA1, pSLGP, pSPHCH01) demonstrated a conserved gene order repA, parA, parB, and all three genes were transcribed in the same orientation (Fig. 3). An analysis of the sequence space between the repA and parA genes of plasmids pISP0, pLA1, pSLGP and pSPHCH01 (165–195 bp; see Table 1) did not identify any significant repeated sequences.

Thus, it can be concluded that the organization of the rep and par genes on the ‘megaplasmids’ from sphingomonads belonging to the ‘Mega-RPA-’ and ‘Mega-Rep3-’ groups differs significantly from those previously described for plasmids from other Alphaproteobacteria by Petersen (2011).

Transfer functions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

There are only very few studies available, which analysed the transferability of the ‘degradative megaplasmids’ from sphingomonads. In these studies, it was shown for plasmids pNL1 and pCHQ1 (inter alia using plasmid derivatives carrying antibiotic resistance markers) that the transfer (or the ability to establish in a different genetic background) of these plasmids seems to be basically restricted to bacteria belonging to the Sphingomonadaceae (Romine et al., 1999; Basta et al., 2004, 2005; Nagata et al., 2006).

The conjugative systems of Gram-negative bacteria show in general three essential components: a type IV secretion system which spans the cell envelope and is responsible for the synthesis of the conjugative pili; the relaxosome which is a complex of proteins that process the DNA at the origin of transfer (oriT) and the coupling protein, which connects the two entities together (Lawley et al., 2004). The type IV secretion systems are rather complex and usually require more than 10 different proteins, which are involved in functions such as the synthesis of the pili and the formation of pores through the inner and outer membranes and the cell walls of the donor and recipient cells. Historically, the proteins/genes involved have been designated differently for different plasmids (especially when these plasmids belong to different incompatibility groups). Thus, the relevant proteins have been designated as Tra(X) for plasmids belonging to the incompatibility groups IncF1 and IncN, as Trb(X) for plasmids from the IncPα group, Trw(X) for IncW plasmids or VirB(1–10) for Ti plasmids (Lawley et al., 2004). Therefore, the sequences of the sphingomonad plasmids were analysed for the presence of annotated tra, trh, trb, trw or vir genes. This demonstrated that only in plasmids with sizes of c. 50–310 kbp gene clusters with 10 or more genes annotated as tra, trb, trw or vir are present.

Plasmids pNL1 and pCAR3 (from the ‘Mega-RepAC group’) carried the genes required for conjugative transfer on parts of the plasmids with a length of about 20 kb. These genes have been annotated for pNL1 and pCAR3 mostly as tra genes (traL, traE, traK, traB, traC, traW, traU, traN, traF, traH, traG, traI, traH). The genes are organized on both plasmids in the same organization which clearly resembles the gene order of the tra genes previously found on the F-plasmid. The functionalibility of these genes has been experimentally demonstrated for pNL1, as this plasmid has been conjugatively transferred among different sphingomonads. In contrast, no experimental evidence for a conjugative transfer of plasmid pCAR3 could be demonstrated (Romine et al., 1999; Shintani et al., 2007). Also plasmid pSWIT02 (which also belongs to the ‘Mega-RepAC-group’) carries genes coding for conjugative functions, but these genes had been annotated as vir genes. The annotated sequence of plasmid pSWIT02 suggested that this vir-operon consisted of the genes virB1virB11 (NCBI registry numbers ABQ71617ABQ71626). The organization of these genes is identical to the organization of the homologous genes on the Ti plasmid from A. tumefaciens and the Tra-systems of broad-host-range plasmids belonging to the IncN and IncW incompatibility groups. In addition, also the gene encoding the ‘coupling protein’ VirD4 could be identified in direct neighbourhood to the vir genes. Thus, on plasmid pSWIT02, all genes are present which allow the conjugative transfer of broad-host-range plasmids.

The absence/presence of certain genes and also the organization of conserved genes suggested that the conjugative system of plasmid pSWIT02 is different to that of plasmids pNL1 and pCAR3. Thus, the conjugative systems from plasmids pNL1 and pCAR3 are closer related to the system present on the F-plasmid, and the transfer functions encoded by plasmid pSWIT02 are closer related to the Ti plasmid or the IncN/IncW plasmids.

The plasmids belonging to the ‘Mega-Rep3-group’ (pCHQ1, pSLCP, pSPHCH01, pISP0, pLA1) all seem to carry a full set of conjugative genes. The respective gene clusters had been annotated for plasmids pISP0, pSLPG, pSPHCH01 as vir genes, and all required genes (virB1virB11, virD2 and virD4 with some exceptions regarding virB7) have been annotated. In contrast, on plasmids pCHQ1 and pLA1, the isofunctional genes had been annotated as trb or tra genes. Furthermore, the respective gene clusters from pCHQ1 and pLA1 also included traW, traU/trbC, traN, traF, traH, traG, which are specifically found in plasmids related to the F-plasmid and which do not have homologous genes in the vir-operon (Lawley et al., 2004).

Significant differences in the conjugative systems are also observed for the plasmids belonging to the ‘Mega-RPA-group’. Thus, plasmid pISP1 carries a large cluster of tra genes (traL, traE, traK, traB, traC, traW, traU, traN, traF, traH, traG, traI, traH; NCBI registry numbers YP_007618159YP_0076181751617). These tra genes show the same organization as those found on plasmids pNL1 and pCAR3 (and also the F-plasmid). In contrast, the two other plasmids from this group (pNL2 and Mpl) do not code for ‘conjugative genes’. It is conspicuous that these plasmids are the largest sequenced plasmids from sphingomonads (pNL2 = 487 kb; Mpl = 1160 kb). Thus, it might be speculated that these plasmids are somehow too large for a conjugative transfer (and thus might be on the ‘way to become a second chromosome’).

The analysis of the organization of the genes involved in the conjugative transfer of the plasmids from sphingomonads suggested that these genes are inherited independently from the rep/par systems. This was also confirmed by sequence comparisons between the genes encoding the pilins (traA, trbC or virB2), pore-forming proteins from the outer membrane (traL, trbD or virB3) or the coupling proteins (traD, traG or virD4). Thus, it was found that according to the pilins, the conjugative systems can be clearly separated as the pilins from plasmids pCAR3, pNL1 (‘Mega-RepAC’), pISP1 (‘Mega-RPA’), pLA1 and pSWIT01 (‘Mega-Rep3’) consist of 247–262 aa. In contrast, the pilins from plasmids pSWIT02 (‘Mega-RepAC’), pCHQ1, pSLPG, pSPHCH01, pISP0 (‘Mega-Rep3’) and pLA2 are composed of only 100–115 amino acids. This difference resulted in the respective phylogenetic trees in the formation of two clearly separated branches (Fig. 4a). Rather similar phylogenetic trees are also obtained for the comparisons of the pore-forming proteins and the coupling proteins (Fig. 4b and c).

image

Figure 4. Phylogenetic trees of putative TraA, TrbC or VirB2 proteins (a), TraL, TrbD or VirB3 proteins (b) or TraD, TraG or VirD4 proteins (c) from the megaplasmids from different sphingomonads. The alignment of the sequences (for the relevant NCBI registration numbers, see Table 1) and the phylogenetic tree (ClustalW, UPGMA) were carried out at the Kyoto University Bioinformatics Centre (www.genome.jp).

Download figure to PowerPoint

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References

The ‘degradative megaplasmids’ from sphingomonads can be differentiated according to their rep and par genes into three major groups, which presumably represent different incompatibility groups. The DNA sequences suggest that most of these plasmids are conjugative and that the transfer functions evolve largely independently from the respective plasmid replication systems. The rep/par- and tra/vir-systems of these plasmids are clearly homologous to isofunctional systems found in other Gram-negative bacteria. This suggests that factors independent of the basic functions of plasmid transfer and maintenance are responsible for the specific occurrence of these ‘megaplasmids’ among the sphingomonads. A possible explanation for the restricted transfer of these plasmids to other bacterial groups might be related to the specific prevalence of sphingolipids in the outer membranes of sphingomonads, which might interfere with the conjugative transfer of plasmid DNA to nonsphingomonads.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Sequenced (degradative) plasmids from sphingomonads
  5. Comparison of the replication initiation (Rep) proteins encoded by plasmids from sphingomonads
  6. Comparison of the partition (Par) proteins encoded by plasmids from sphingomonads
  7. Organization of the rep and par genes on the different types of plasmids
  8. Transfer functions
  9. Conclusions
  10. References
  • Aylward FO, McDonald BR, Adams SM, Valenzuela A, Schmidt RA, Goodwin LA, Woyke T, Currie CR, Suene G & Poulsen M (2013) Comparison of 26 sphingomonad genomes reveals diverse environmental adptations and biodegradative capabilities. Appl Environ Microbiol 79: 37243733.
  • Basta T, Keck A, Klein J & Stolz A (2004) Detection and characterization of conjugative degradative plasmids in xenobiotics degrading Sphingomonas strains. J Bacteriol 186: 38623872.
  • Basta T, Bürger S & Stolz A (2005) Structural and replicative diversity of large plasmids from polycyclic aromatic compounds and xenobiotics degrading Sphingomonas strains. Microbiology 151: 20252037.
  • Cevallos MA, Cervantes-Rivera R & Gutiérrez-Rios RM (2008) The repABC plasmid family. Plasmid 60: 1937.
  • Cho J-C & Kim S-J (2001) Detection of mega plasmid from polycyclic aromatic hydrocarbon degrading Sphingomonas sp. strain KS14. J Mol Microbiol Biotechnol 3: 503506.
  • Colquhoun DR, Hartmann EM & Halden RU (2012) Proteomic profiling of the dioxin-degrading bacterium Spingomonas wittichii RW1. J Biomed Biotechnol, doi:10.1155/2012/408690.
  • Copley SD, Rokicki J, Turner P, Daligault H, Nolan M & Lund M (2012) The whole genome sequence of Sphingobium chlorophenolicum L-1: insights into the evolution of the pentachlorophenol degradation pathway. Genome Biol Evol 4: 184198.
  • D'Argenio V, Petrillo M, Cantiello P, Naso B, Cozzuto L, Notomista E, Paolella G, Di Donato A & Salvatore F (2011) De novo sequencing and assembly of the whole genome of Novosphingobium sp. strain PP1Y. J Bacteriol 193: 4296.
  • Feng X, Ou L-T & Ogram A (1997a) Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. CF-06. Appl Environ Microbiol 63: 13321337.
  • Feng X, Ou L-T & Ogram A (1997b) Cloning and sequence analysis of a novel insertion element from plasmids harbored by the carbofuran-degrading bacterium, Sphingomonas sp. CF-06. Plasmid 37: 169179.
  • Fernández-Lopez R, Garcillán-Barcia MP, Revilla C, LázaroM Vielva L & de la Cruz F (2006) Dynamics of the IncW genetic backbone imply general trends in conjugative plasmid evolution. FEMS Microbiol Rev 30: 942966.
  • Fredrickson JK, Balkwill DL, Romine MF & Shi T (1999) Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp. J Ind Microbiol Biotechnol 23: 273283.
  • Funnell BE & Slavcev RA (2004) Partition systems of bacterial plasmids. Plasmid Biology (Funnell BE & Phillips GJ, eds), pp. 81103. ASM Press, Washington, DC.
  • Hu X, Mamoto R, Jujioka Y, Tani A, Kimbara K & Kawai F (2008) The pva operon is located on the megaplasmid of Sphingopyxis sp. strain 113P and is constitutively expressed, although expression is enhanced by PVA. Appl Microbiol Biotechnol 78: 685693.
  • Jogler M, Chen H, Simon J, Rohde M, Busse HJ, Klenk HP, Tindall BJ & Overmann J (2013) Description of Sphingorhabdus planktonica gen. nov., sp. nov. and reclassification of three related members of the genus Sphingopyxis in the genus Sphingorhabdus gen. nov. Int J Syst Evol Microbiol 63: 13421349.
  • Kalnin K, Stegalkina S & Yarmolinsky M (2000) pTAR-encoded proteins in plasmid partitioning. J Bacteriol 182: 18891894.
  • Kämpfer P, Arun AB, Young CC, Busse HJ, Kassmannhuber J, Rosselló-Móra R, Geueke B, Rekha PD & Chen WM (2012) Sphingomicrobium lutaoense gen. nov., sp. nov., isolated from a coastal hot spring. Int J Syst Evol Microbiol 62: 13261330.
  • Lawley T, Wilkins BM & Frost LS (2004) Bacterial conjugation in Gram-negative bacteria. Plasmid Biology (Funnell BE & Phillips GJ, eds), pp. 81103. ASM Press, Washington, DC.
  • Luo YR, Kang SG, Kim S-J, Li N, Lee J-H & Kwon KK (2012) Genome sequence of benzo[a]pyrene-degrading bacterium Novosphingobium pentaaromativorans US6-1. J Bacteriol 194: 907.
  • Masai E, Kamimura N, Kasai D et al. (2012) Complete genome sequence of Sphingobium sp. strain SYK-6, a degrader of lignin-derived biaryls and monoaryls. J Bacteriol 194: 534535.
  • Miller TD, Delcher AL, Salzberg SL, Saunders E, Detter JC & Halden RU (2010) Genome sequence of the dioxin-mineralizing bacterium Sphingomonas wittichii RW1. J Bacteriol 192: 61016102.
  • Nagata Y, Kamakura M, Endo R, Miyazaki R, Ohtsubo Y & Tsuda M (2006) Distribution of γ-hexachlorocyclohexane-degrading genes on three replicons in Sphingobium japonicum UT26. FEMS Microbiol Lett 256: 112118.
  • Nagata Y, Ohtsubo Y, Endo R, Ichikawa N, Ankai A, Oguchi A, Fukui S, Fujita N & Tsuda M (2010) Complete genome sequence of the representative γ-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26. J Bacteriol 192: 58525853.
  • Nagata Y, Natsui S, Endo R, Ohtsubo Y, Ichiwaka N, Ankai A, Oguchi A, Fukui S, Fujita N & Tsuda M (2011) Genomic organization and genomic structural rearrangements of Sphingobium japonicum UT26, an archetypal γ-hexachlorocyclohexane-degrading bacterium. Enzyme Microb Technol 49: 499508.
  • Ochou M, Saito M & Kurusu Y (2008) Characterization of two compatible small plasmids from Sphingobium yanoikuyae. Biosci Biotechnol Biochem 72: 11301133.
  • Ogram A, Duan Y-P, Trabue SL, Feng X, Castro H & Ou L-T (2000) Carbofuran degradation mediated by three related plasmid systems. FEMS Microbiol Ecol 32: 197203.
  • Pandeeti EV, Longkumer T, Chakka D et al. (2012) Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Spingobium fuliginis ATCC 27551. G3 (Bethesda) 2: 15411554.
  • Petersen J (2011) Phylogeny and compatibility: plasmid classification in the genomics era. Arch Microbiol 193: 313321.
  • Romine MF, Stillwell LC, Wong K-K, Thurston SJ, Sisk EC, Sensen C, Gaasterland T, Fredrickson JK & Saffer JD (1999) Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199. J Bacteriol 181: 15851602.
  • Sentchilo V, Mayer AP, Guy L, Miyazaki R, Tringe SG, Barry K, Malfatti S, Goessmann A, Robinson-Rechavi M & van der Meer JR (2013) Community-wide plasmid gene mobilization and selection. ISME J 7: 11731186.
  • Shintani M, Urata M, Inoue K, Eto K, Habe H, Omori T, Yamane H & Nojiri H (2007) The Sphingomonas plasmid pCAR3 is involved in complete mineralization of carbazole. J Bacteriol 189: 20072020.
  • Shuttleworth KL, Sung J, Kim E & Cerniglia CE (2000) Physiological and genetic comparison of two aromatic hydrocarbon-degrading Sphingomonas strains. Mol Cells 10: 199205.
  • Stolz A (2009) Molecular characteristics of xenobiotics-degrading sphingomonads. Appl Microbiol Biotechnol 81: 793810.
  • Tabata M, Endo R, Ohtsubo Y, Kumar A, Tsuda M & Nagata Y (2011) The lin genes for γ-hexachlorocyclohexane degradation in Sphingomonas sp. strain MM-1 proved to be dispersed across multiple plasmids. Biosci Biotechnol Biochem 15: 466472.
  • Tabata M, Ohtsubo Y, Ohhata S, Tsuda M & Nagata Y (2013) Complete genome sequence of the γ-hexachlorocyclohexane-degrading bacterium Sphingomonas sp. strain MM-1. Genome Announc 1: e0024713. doi:10.1128/genomeA.00247-13.
  • Tani A, Charoenpanich J, Mori T, Takeichi M, Kimbara K & Kawai F (2007) Structure and conservation of a polyethylene glycol-degradative operon in sphingomonads. Microbiology 153: 338346.
  • Tani A, Tanaka A, Minami T, Kimbara K & Kawai F (2011) Characterization of a cryptic plasmid, pSM103mini, from polyethylene-glycol degrading Sphingopyxis macrogoltabida strain 103. Biosci Biotechnol Biochem 75: 295298.
  • Uchida H, Hamana K, Miyazaki M, Yoshida T & Nogi Y (2012) Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine annelid worm. Int J Syst Evol Microbiol 62: 22242228.
  • Venkova-Canoca T, Soberón NE, Ramirez-Romero MA & Cevallos MA (2004) Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem-loop structure. Mol Microbiol 54: 14311444.
  • Yeon SM & Kim YC (2011) Complete sequence and organization of the Sphingobium chungbukense DJ77 pSY2 plasmid. J Microbiol 49: 684688.