Plasmids with a BHR are putatively important for many taxa in soil, as they can shuttle useful traits such as antibiotic-, heavy metal- or UV-resistance genes, efflux pumps, restriction modification systems, and toxin–antitoxin systems between different members of the bacterial communities in soil. Several studies have demonstrated that the transfer range of plasmids is often much broader than their replication range (Musovic et al., 2006; Baharoglu et al., 2010). Further retrospective evidence for this notion comes from sequenced genomes or plasmids of gram-positive and gram-negative bacteria that often carry identical antibiotic resistance genes or remnants of MGE (Tauch et al., 2002a).
To predict the evolutionary range of hosts in which plasmids evolved, Suzuki et al. (2010) recently explored the use of nucleotide composition for a range of plasmids. Based on the assumption that plasmids acquire the genomic signature of their hosts, the trinucleotide composition of a range of plasmids was compared with all sequenced bacterial genomes. Indeed, the signatures of several typical BHR plasmids such as IncP-1, PromA and IncU displayed signatures that were dissimilar to any chromosomal signature, suggesting that these plasmids did not fix in a particular host (Suzuki et al., 2010). Surprisingly, similar findings were also reported for IncP-9 plasmids, which are thought to have a much narrower host range.
An important feature of many conjugative BHR plasmids is that they efficiently mobilize or retro-mobilize smaller mobilizable plasmids. In fact, many conjugative BHR plasmids have been isolated based on their ability to mobilize IncQ plasmids (van Elsas et al., 1998; Gstalder et al., 2003). BHR plasmids most often reported from soil bacteria belonged either to the IncP-1 group or the recently suggested group of PromA plasmids (Van der Auwera et al., 2009). Sequence-based insights into the diversity of backbone genes of these main groups of BHR plasmids in soil bacteria revealed a strong modular organization of the backbones, comprising genes for replication, maintenance and transfer and hot spots for acquisition of accessory genes. Whereas plasmids belonging to the IncP-1 group carry a large amount of accessory genes, plasmids of the recently proposed PromA group seem to promote bacterial adaptation by efficiently sampling the soil mobilome by retrotransfer (van Elsas et al., 1998).
Plasmids belonging to the IncP-1 group
Plasmids belonging to the IncP-1 group have attracted the attention of plasmid biologists for more than 40 years, as these plasmids transfer and stably replicate in a wide range of bacterial hosts and are considered BHR plasmids (Adamczyk & Jagura-Burdzy, 2003). The first IncP-1 plasmids described were isolated from hospital specimens and thus IncP-1 plasmids were originally categorized as clinically important plasmids. Nowadays this plasmid group is considered ubiquitous and several studies using cultivation-independent methods have detected and isolated IncP-1 plasmids from soils and related ecosystems such as sewage and manure (Smalla et al., 2000a, 2006; Heuer et al., 2002; Bahl et al., 2009).
Back in the 1990s, the complete sequence of the two archetype plasmids of the IncP-1α, RP4 (Pansegrau et al., 1994) and the IncP-1β, R751 (Thorsted et al., 1998) provided the first insights from comparative genomics. The sequence comparison confirmed the conservation of plasmid backbone sequences for replication, transfer and stable inheritance between the two subgroups. The plasmid backbone of both plasmids was found to be interrupted at two sites between oriV and trfA, and between the tra and trb operons (Thorsted et al., 1998). In both plasmids, replication and transfer are globally regulated by the KorA and KorB protein encoded by genes of the central control operon. Nowadays, 28 published IncP-1 sequences are available. The sequenced plasmids originated from different geographic regions and environments, e.g. sewage, soils and river sediments. Based on 19 IncP-1 plasmids sequenced at the time, Schlüter et al. (2007) presented a comprehensive comparative genomic analysis of IncP-1 plasmids isolated from sewage. One of the most remarkable observations was the high nucleotide sequence conservation of the plasmid backbone. Based on DNA signature analysis, a more recent analysis of 25 complete backbone genomes of IncP-1 plasmids by means of newly developed bioinformatics tools demonstrated the existence of seven clades and the importance of homologous recombination as a key factor in IncP-1 plasmid backbone evolution. Genomic signature analysis data from Norberg et al. (2011) indicated that the different IncP-1 plasmids analyzed to date have adapted to hosts belonging to different species and that their backbones seem to originate from various parental plasmids.
Accessory elements were inserted at various sites of three backbone regions, the hot spots of insertion (Heuer et al., 2004). In most of the IncP-1β plasmids, accessory genes are inserted in two specific regions between the oriV and the trfA, and between the tra and the trb operons. However, the IncP-1β plasmids pB2, pB3, pTP6 and pJP4 only carry insertions in one of the hot spots (Heuer et al., 2004; Trefault et al., 2004; Smalla et al., 2006).
The most straightforward explanation of the evolutionary history of the completely sequenced IncP-1β plasmids is the existence of a common ancestor which was free of acquired genetic elements (Heuer et al., 2004). This ancestor probably still exists in microbial communities and occasionally acquires accessory genes by transposition events. Indeed, recently, two cryptic IncP-1β plasmids were reported that do not carry any accessory elements, except two small open reading frames (ORFs) of unknown function: plasmids pA1 from Sphingomonas (Harada et al., 2006) and pBP134 from Bordetella pertussis (Kamachi et al., 2006).
Sota et al. (2007) determined experimentally that transposon insertions in the oriV-trfA and in the tra-trb regions of pB136 displayed a higher stability than in other regions. The authors suggested that the high structural similarity of IncP-1 plasmids might be due to a region-specific insertion combined with selection for stable and transferable plasmids.
Although IncP-1 plasmids were shown experimentally to have a wide host range, stable replication in the absence of selective pressure is strain-specific (DeGelder et al., 2007; Heuer et al., 2007; Sota et al., 2010). Similarly, the permissiveness of genetically virtually identical Dickeya sp. isolates from a single field site for the uptake of IncP-1 plasmids differed by several orders of magnitude (Heuer et al., 2010).
Sequencing also revealed that there are more subgroups of IncP-1 plasmids out there than previously assumed. Several exogenously isolated plasmids from soils have been assigned to novel subgroups such as IncP-1γ, IncP-1δ, IncP-1ε and the IncP-1ξ that were previously only represented by one completely sequenced plasmid, pQKH54 (Haines et al., 2006), pEST1044 (Vedler et al., 2004) and pKJK5 (Bahl et al., 2007), respectively.
Although Schlüter et al. (2007) discussed IncP-1ε as a tentative group, we have recently shown that plasmids belonging to this group could be frequently captured from manure (Binh et al., 2008) and manure-treated soils (Heuer et al., 2012). The data obtained by cultivation-independent methods point to an important role of the IncP-1ε subgroup in the agro-ecosystem, as all IncP-1ε plasmids captured from soil carry multiple antibiotic resistance and thus could easily enter the food chain via plant-associated bacteria.
Sequence analysis of three additional plasmids exogenously isolated from soil that were grouped based on their trfA sequence to the IncP-1ε group revealed that, except for their accessory genes, these plasmids were nearly identical to the previously sequenced plasmid pKJK5 that was also captured exogenously from agricultural soil in Denmark (Heuer et al., 2012). Sequence comparison also showed that the primers developed by Götz et al. (1996) based on the sequences of RP4 and R751, were not optimal for amplification of the more recently discovered IncP-1 groups (Götz et al., 1996).
New trfA-targeting primers were recently proposed by Bahl et al. (2009). The complete sequences of nine IncP-1 plasmids isolated from soil bacteria of various geographic origins revealed that IncP-1 plasmids are also important shuttles of beneficial traits in soil bacteria (Trefault et al., 2004; Vedler et al., 2004; Smalla et al., 2006; Bahl et al., 2007; Ma et al., 2007). Table 3 lists these completely sequenced IncP-1 plasmids from soil, and 12 additional genomes from plasmids isolated from Norwegian agricultural soils were recently published (Sen et al., 2011).
Table 3. IncP-1 plasmids from soil with a complete published sequence
|Plasmid (size)||Core||Source accessory genes||GenBank accession|
|pJP4 (87.7 kb)||βR751|| |
Cupriavidus necator JMP134, soil (Australia); 2,4-d and 3-chlorobenzoate
| AY365053 |
|pADP-1 (108.8 kb)||βR751|| |
Pseudomonas sp. ADP from herbicide-contaminated agricultural soil
(USA); atrazine degradation, mercury resistance
| U66917 |
|pAMMD1 (43.6 kb)||βR751|| |
Plant growth promoting biocontrol strain Burkholderia ambifaria AMMD
| CP000443 |
|pA81 (98.2 kb)||βpB4|| |
Achromobacter xylosoxidans A8 from PCB-contaminated soil (Czech Republic);
chlorobenzoic acids degradation, heavy metal resistance
| AJ515144 |
|pA1 (46.6 kb)||βpB4|| |
Sphingomonas sp. A1 from ditch soil (Japan); no accessory genes except two
| AB231906 |
|pKJK5 (54.4 kb)||ε|| |
Exogenously isolated from manured soil (Denmark); confers resistance to
tetracycline and trimethoprim
| AM261282 |
|pEST4011 (77.0 kb)||δ|| |
Achromobacter xylosoxidans EST4002 from agricultural soil (Estonia);
| AY540995 |
|pIJB1 (99.4 kb)||δ|| |
Burkholderia cepacia 2a isolated from garden soil (UK); 2,4-d and malonate
catabolism, mercury resistance
| DQ065837 |
|pAKD4 (56.8 kb)||δ||Exogenously isolated from mercury polluted soils (Norway); mercury resistancea|| GQ983559 |
The complete sequence of plasmids pSB102 and pIPO2, both captured directly from rhizosphere bacteria by exogenous plasmid isolation, revealed striking similarities of the plasmids (Schneiker et al., 2001; Tauch et al., 2002b). Furthermore, the overall organization of the plasmids pIPO2 and pSB102 was very similar to plasmid pXF51 from the plant pathogen Xylella fastidiosa. Given the similarities between these plasmids, a novel family of BHR plasmids was proposed because both pIPO2 and pSB102 transferred to a wide range of gram-negative bacteria but did not belong to any of the known BHR plasmid groups revealed originally by PCR and probing, and confirmed by sequencing. Whereas plasmid pSB102 confers resistance to mercury compounds and was captured from rhizobacteria based on the acquired mercury resistance of Sinorhizobium meliloti, plasmid pIPO2 is a cryptic plasmid that was isolated due to its gene-mobilizing activity from wheat rhizobacteria. Comparative genomics based on their complete sequences enabled Tauch et al. (2002b) to discover that plasmids pSB102, pIPO2 and pXF51 had a strikingly similar overall genetic organization and led to the proposal of a new family of environmental BHR plasmids. The compact transfer region of all three plasmids is related to components of the transfer regions of the Ti and IncP-1 plasmids. However, the proposed mating pair formation system of all three plasmids strikingly displayed the highest homology to the type IV secretion system of the mammalian pathogen Brucella spp. (Schneiker et al., 2001; Tauch et al., 2002b). The replication and maintenance function possessed varying homologies to deduced protein sequences of different BHR plasmids.
The presence of long repetitive sequences is another interesting feature shared by pSB102, pIPO2 and pXF51. In pIPO2 and pSB102 at least one pair of inverted repeats was found flanking a varying number of different ORFs of presently unknown function (Tauch et al., 2002b). Direct and inverted repeats promote rearrangements and thus contribute to the evolution of plasmids. The analysis of the complete genome sequence of Collimonas fungivorans led to the discovery of plasmid pTer331, which had a genetic organization very similar to that of plasmid pIPO2, and for many ORFs the deduced amino acid identity was above 90% (Mela et al., 2008).
Recently, analysis of the complete sequence of plasmid pMOL98, a miniTn5-tagged derivative of pES1 isolated in a triparental mating from a hydrocarbon-polluted soil (Gstalder et al., 2003), confirmed what was posited earlier, i.e. that pMOL98 is another member of the pIPO2-like plasmid family (Van der Auwera et al., 2009).
Another candidate belonging to this group of plasmids, pMRAD02, was recently discovered in a sequencing project of Methylobacterium radiotolerans JCM2831 isolated from unpolished rice in Japan. All plasmids belonging to the pIPO2 family have in common the backbone modules comprising genes for replication, maintenance and transfer. The evolutionary relatedness of pIPO2, pTER331, pSB102, pMOL98 and pMRAD02 was recently assessed based on six concatenated backbone proteins (Van der Auwera et al., 2009). Curiously, the plasmids captured in or isolated from Betaproteobacteria (plasmids pIPO2 and pMOL98 were both captured in a triparental mating in strains belonging to the genus Cupriavidus; pTER331 originated from C. fungivorans) were more similar to each other than to plasmids captured in or isolated from Alphaproteobacteria (pSB102 was captured in S. meliloti; pMRAD02 originated from M. radiotolerans).
Despite their overall highly similar organization, the sequences of the repA gene of pIPO2, pSP102 and pXF51 share only low nucleotide identities. The sequence divergence of the repA is certainly advantageous for the development of specific primer systems. Two primer sets targeting the repA gene of pIPO2 were used to determine the environmental distribution of replicons carrying this rep gene. Interestingly, PCR amplification from total community DNA in combination with Southern blot hybridization showed that the pIPO2 repA gene sequences were mainly detected in the rhizosphere of various crop plants and in previously planted agricultural soils (Tauch et al., 2002b). However, to detect pIPO2-like plasmids primers it would be more advantageous to target sequences of the transfer module, as these genes are better conserved among the pIPO2-like plasmids. A high stability of pTer331 in C. fungivorans was observed during cultivation over 35 generations in liquid King's B medium, indicating an active stable partitioning system. Despite different experimental attempts to elucidate a putative role of pTer331, such as an improved rhizosphere competence, no phenotype trait except the ability to mobilize or retro-mobilize the IncQ plasmid pSM1890 could be attributed to the presence of the plasmid. Thus pIPO2, pTer331 and most likely pEES1 are cryptic plasmids, and it is assumed that these cryptic plasmids contribute to the adaptability of their hosts by promoting sampling of the soil mobilome (Mela et al., 2008; Van der Auwera et al., 2009).
The occurrence of these highly related plasmids in soils from various geographic regions and their ability to mobilize and retro-mobilize suggests an important role in plasmid-mediated adaptability of soil bacteria. Van der Auwera et al. (2009) proposed naming BHR-plasmids belonging to the pIPO2 family PromA in accordance with the traditional BHR-plasmids PromN (IncN), PromP (IncP-1), PromU (IncU) and PromW (IncW). Although plasmid nomenclature and classification will remain problematic, the increasing number of sequenced plasmid replicons will certainly facilitate a more reliable classification and, even more importantly, provide the tools for studying their ecology and importance in adaptation of soil bacterial populations to changing environments.