Zentrum für Genomforschung, Universität Bielefeld, D-33615 Bielefeld, Germany1
Lehrstuhl für Genetik, Universität Bielefeld, D-33615 Bielefeld, Germany2
Plant Research International, 6700AA Wageningen, The Netherlands3
Biologische Bundesanstalt für Land-und Forstwirtschaft, D-38104 Braunschweig, Germany4
School for Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK5
NERC Institute of Virology and Environmental Microbiology, Oxford OX1 3SR, UK6
Department of Biology, University of Idaho, Moscow, ID 83844, USA7
Cardiff School of Biosciences, University of Wales, Cardiff CF10 3TL, UK8
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland9
Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland10
Robert Koch-Institut, Bereich Wernigerode, D-38855 Wernigerode, Germany11
Max-Planck-Institut für Molekulare Genetik, Dahlem, D-14195 Berlin, Germany12
Author for correspondence: Christopher M. Thomas. Tel: +44 121 414 5903. Fax: +44 121 414 5925. e-mail: c.m.thomas{at}bham.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: bacterial genome, exogenous plasmid isolation, plasmid stability, conjugative transfer, horizontal gene spread
The GenBank accession number for the pIPO2T sequence reported in this paper is AJ297913.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In plant-related environments, such as the rhizosphere, conjugation has been shown to be a key mechanism that can facilitate the gene flow between Gram-negative bacteria (van Elsas et al., 1988 ; Smit et al., 1991
, 1993
; Pukall et al., 1996
; Lilley & Bailey, 1997
). Thus, studies with broad-host-range plasmids of incompatibility groups IncP-1, IncN and IncW, as well as studies with the phytosphere-isolated plasmid pQM, showed that rates of transfer between pseudomonads were greater in the rhizospheres of wheat and sugar beet, respectively, than in bulk soil (van Elsas et al., 1988
; Lilley et al., 1994
). These results indicate that naturally occurring conjugative plasmid transfer may be a frequent process with significance for the adaptive capacity of rhizospheric bacterial communities. It is therefore likely that naturally occurring self-transmissible plasmids carrying the genes necessary for conjugative DNA transfer play a major role in gene-exchange processes in plant-related microbial communities (van Elsas et al., 2000a
).
In previous work describing the capacity of plant-associated bacterial populations to effect gene transfer, a set of cryptic plasmids with gene-mobilizing capacity was isolated from bacterial populations of the wheat rhizosphere (van Elsas et al., 1998 ). These plasmids were isolated by a triparental mating system (triparental exogenous isolation) allowing the isolation of mobile genetic elements by virtue of their capacity to mobilize non-selftransferable plasmids (Hill et al., 1992
, 1996
; Smalla et al., 2000
). The system consisted of an E. coli donor strain carrying the mobilizable but non-selftransferable IncQ plasmid pMOL187, and a plasmidless rifampicin-resistant Ralstonia eutropha recipient strain (Top et al., 1994
). Plasmid pMOL187 contains the czc gene cassette, encoding resistances against ions of cadmium, zinc and cobalt, which can only be expressed in the R. eutropha recipient strain. Following a multi-partner mating between indigenous wheat rhizosphere bacteria and the two partners of the mating system, plasmids with broad-host-range mobilizing capacity can be obtained in the recipient via co-transfer with pMOL187 (Top et al., 1994
). This procedure, thus, selects naturally occurring plasmids primarily on the basis of their capacity to mobilize and self-transfer, i.e. to efficiently produce conjugation bridges between the mating partners employed.
One of the plasmids obtained from bacterial populations of the wheat rhizosphere, termed pIPO2, had an estimated size of about 38 kb and was not classifiable into any known group by replicon typing via PCR and hybridization with replicon-specific probes (van Elsas et al., 1998 ). The plasmid could mobilize IncQ plasmids to many different Gram-negative bacterial species and had a broad self-transfer range among the
, ß and
subclasses of the Proteobacteria (van Elsas et al., 1998
). Plasmid pIPO2 was able to mobilize the IncQ plasmid pIE723 to these hosts at considerable frequencies under rhizosphere conditions in the field (van Elsas et al., 1998
). The presence of pIPO2 enabled both Pseudomonas fluorescens (van Elsas et al., 1998
) and R. eutropha (Szpirer et al., 1999
) to capture selectable IncQ plasmids from other hosts via retrotransfer.
In this study, we describe the complete nucleotide sequence and genetic organization of plasmid pIPO2, on the basis of the mini-Tn5::luxAB::tet-tagged derivative pIPO2T. This plasmid is proposed to belong to a new family of environmental broad-host-range plasmids including pSB102 and pXF51, each of which are plasmids isolated from plant-associated bacteria carrying a similarly organized set of genes necessary for bacterial conjugation. Plasmid pSB102 was isolated from the bacterial community of the alfalfa rhizosphere (Schneiker et al., 2001 ), and pXF51 is a plasmid isolated from the plant pathogen Xylella fastidiosa (Simpson et al., 2000
; Marques et al., 2001
). The sequence information of pIPO2 was used to design specific primers that were used for PCR to determine in what ecological niches pIPO2-like plasmids could be found.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid isolation, shotgun-library construction and DNA sequencing.
Plasmid DNA of pIPO2T was isolated from P. fluorescens R2f by means of the alkaline-SDS lysis and column purification procedure, according to the NucleoBond Plasmid Purification Protocol (Macherey-Nagel, Düren, Germany). Ultrasonication was used in a common ultrasonicator (at maximal amplitude). A time-course was done (0251020 s) with subsamples of the pIPO2 DNA preparation, and the treated subsamples were run on an agarose gel to determine the mean fragment sizes. The time resulting in mean fragment sizes of around 1000 bp was used to treat the full pIPO2 DNA preparation. The shotgun library was constructed in the vector pBluescript II SK- by using 0.61.2 kb size fractions of the pIPO2T DNA. Sequencing was performed in the different laboratories of participating authors and was completed at the University of Bielefeld with the following strategy. Templates for DNA sequencing were prepared by automated alkaline lysis of E. coli clones carrying recombinant plasmids by means of the QIAGEN BioRobot 9600. Cycle-sequencing reactions were carried out using purified template DNA, the Prism Ready Reaction Dye Deoxy Termination Kit and fluorescently labelled forward and reverse primers. The products of cycle sequencing were sequenced by running them on a Prism ABI 377 automated DNA sequencer (Applied Biosystems). Computer-assisted assembly of the random shotgun sequences was carried out with the STADEN sequence analysis package (Staden, 1996 ). Gaps in the pIPO2T sequence were closed by primer walking with oligonucleotides designed to contig ends (Staden, 1996
), using pIPO2T DNA as template. Final assembly and editing of the DNA sequence data resulted in a single, circular molecule with a total length of 45319 bp.
DNA sequence analysis and annotation.
The complete DNA sequence of pIPO2T was initially analysed with the automated genome interpretation system GenDB (Center for Genome Research, Bielefeld, Germany). Evaluation of the data collection in the GenDB database files was performed with the STADEN software package (Staden, 1996 ). In addition, features from searches against the nucleotide database at NCBI with the BLAST-N algorithm, version 2.0 (Altschul et al., 1997
), were analysed and incorporated into the sequence annotation. The complete sequence of pIPO2T was searched for
70-dependent promoters, using the Neural Network Promoter Prediction approach (Reese et al., 1996
). Repeat regions within the pIPO2T nucleotide sequence were identified with the REPuter software (Kurtz & Schleiermacher, 1999
).
Amino acid sequence similarities were calculated with the ALIGN computer program (Myers & Miller, 1988 ). Prediction of signal peptides and transmembrane helical segments was performed with the neural network systems SignalP (Nielsen et al., 1997
) and PredictProtein (Rost et al., 1995
), respectively. Protein sequence alignment of TraR was done using the CLUSTAL W software, version 1.4, with the BLOSUM series as protein weight matrix; shadings were drawn following the Gonnet PAM250 matrix. The annotated DNA sequence of pIPO2T was re-oriented in such a way that the orientation and order of genes is the same as in the pSB102 sequence (Schneiker et al., 2001
).
Distribution of bacteria with pIPO2.
To determine the distribution of pIPO2, PCR analysis of environmental total microbial community DNA was performed as described previously (van Elsas et al., 1998 ). The samples from which DNA was extracted were as follows.
Seven-day-old wheat.
Triticum aestivum cv. Sicco grown in Flevo silt loam soil in microcosms, Wageningen, The Netherlands (van Elsas et al., 1998 ).
Twenty-three-day-old rice.
Oryza sativa L. japonica Zhongzao 9037 grown in Flevo silt loam soil in microcosms, Wageningen, The Netherlands (Lin et al., 2000 ).
Thirty-day-old maize.
Zea mais grown in Varzea, as well as in Cerrado field soils in Sete Lagoas, Minas Gerais, Brazil (Rosado et al., 1998 ).
Mature wheat.
Triticum aestivum cv. Sicco grown in Flevo silt loam soil microplots, Wageningen, The Netherlands (van Overbeek et al., 1995 ).
Mature oats.
Avena sativa var. Gigant grown in Wildekamp loamy sand (KCl; pH 5·3; 3% organic matter) field soil, Wageningen, The Netherlands.
Tomato.
Lycopersicon esculentum cv. Moneymaker grown in Ede loamy sand soil in microcosms, Wageningen, The Netherlands.
Potato.
Solanum tuberosum cv. Desirée grown in Ede loamy sand soil microcosms, Wageningen, The Netherlands.
Cauliflower.
Brassica oleracea L. grown in Ens loam field soil, Ens, Noordoostpolder, The Netherlands.
Ramanas (white radish).
Raphanus sativus grown in Ens loam field soil, Ens, Noordoostpolder, The Netherlands.
Flevo silt loam, Wageningen, The Netherlands.
Described in van Elsas et al. (1998) .
Flevo silt loam treated with petroleum/dibenzothiophene.
In soil microcosms, Wageningen, The Netherlands (Duarte et al., 2001 ).
Lovinkhoeve.
Silt loam soil (KCl; pH 7·2; 2% organic matter) sampled from arable land at Lovinkhoeve experimental field, Flevopolder, The Netherlands, eight times over one year, on average once every 6 weeks. It should be noted that five time points with positive responses were found in summer and autumn. The samples were provided by Dr E. Smit, RIVM, Bilthoven, The Netherlands.
Ede loamy sand.
Obtained from a field microplot (KCl; pH 5·5; 3% organic matter), Wageningen, The Netherlands.
Hellendoorn loamy sand.
ILS field soil (KCl; pH 5·25·5; 4% organic matter) obtained from Hellendoorn, Overijssel, The Netherlands (van Elsas et al., 2000b ).
Westmaas clay.
Arable field soil collected at Westmaas, Noordholland, The Netherlands.
A soil.
Oil-polluted soil collected in Amsterdam, The Netherlands.
FiOS.
High-organic matter forest soil obtained from Viikki, Helsinki, Finland.
Wildekamp soil.
See above under mature oats.
Guaira.
Soil obtained from arable land with maize in Guaira, Parana, Brazil (Rosado et al., 1998 ).
Manure.
Pig and cow manure obtained from the BBA Braunschweig experimental farm, Germany.
Seawater.
Obtained from Fleves island and Eretria, Greece. Courtesy of Dr A. Karagouni, Athens, Greece.
Wastewater.
Obtained from Athens wastewater outflow. Courtesy of Dr A. Karagouni, Athens, Greece.
Environmental DNA was obtained using a direct DNA extraction method originally described by Smalla et al. (1993) and modified by van Elsas & Smalla (1995)
. Briefly, the protocol consisted of a homogenization step, a cell lysis step on the basis of bead beating, and a few extraction and purification steps, the final ones of which were commonly based on the Wizard DNA clean-up system (Promega). For all habitats investigated (Table 1
), this method yielded preparations of DNA amplifiable by PCR. Two PCR primer systems were employed to screen for the presence of pIPO2-specific sequences. The first system (I), based on primers pIPO2f and pIPO2r, amplified a product of 305 bp (from position 44091 to 44396 on the pIPO2 sequence, covering ORF33 and a little of ORF32) and was previously shown to be specific for pIPO2 (van Elsas et al., 1998
). The second system (II) was designed on the basis of the sequence of the putative repA gene. The primers pIPO2rep-x2 (5'-CGCAACCTTGCCACATCG-3') and pIPOr-y2 (5'-TAGGTAGCTCATGCGATAGG-3') amplified a product of 546 bp. PCR with environmental DNA was performed using 1 µl (520 ng) of each DNA extract. To assess the template quality of the isolated environmental DNA, controls were performed with each extract spiked with serially diluted pIPO2 DNA. The PCR conditions applied in system I were as described previously (van Elsas et al., 1998
). Thermal cycling in system II was performed as follows: denaturation at 94 °C for 3 min; 40 cycles at 94 °C for 1 min, 5055 °C for 90 s and 72 °C for 1 min; final extension at 72 °C for 20 min. PCR products were run on 1·2% (w/v) agarose gels (Sambrook et al., 1989
), and ethidium-bromide-stained bands were visualized under UV illumination. Gels were blotted to nylon membrane filters, and blots were hybridized at high stringency according to the protocol supplied by the manufacturer (Roche Diagnostics). Digoxigenin-labelled amplicons produced on plasmid pIPO2 with systems I (probe I) and II (probe II) were used as specific probes. Signals were detected following exposure of X-ray films, according to standard procedures. PCR results were considered positive and specific if a hybridization signal indicating a product of expected size with the specific probe was observed. All habitats were tested by fresh isolation of DNA followed by PCR a minimum of two times.
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Within the cluster ORF30ORF34 are two genes whose deduced polypeptide products showed significant similarity to the ParA and ParB proteins of plasmid RP4 (Table 2), which are part of a complex multimer resolution/post-segregational killing system (reviewed by Pansegrau et al., 1994a
). The ORF34 protein of pIPO2, deduced from joining the 5' and 3' sequence fragments of pIPO2T (Table 2
), contains a potential helixturnhelix motif at its C-terminal end (position 190211; Dodd & Egan, 1990
). The protein is likely to function in dimer resolution, since its amino acid sequence shows homology to resolvases of the Tn3 family. However, the disruption of orf34, caused by the tagging procedure with the mini-Tn5::luxABtet transposon in pIPO2T, did not result in plasmid instability in P. fluorescens. The fact that pIPO2T was stably maintained in P. fluorescens R2f, even without tetracycline selection, suggests that orf34 is dispensible for stable plasmid inheritance in at least some bacterial hosts (van Elsas et al., 1998
). Conversely, the low rate of transfer from P. fluorescens R2f to E. coli as well as to Pseudomonas corrugata, coupled with the consistently poor growth of the putative E. coli transconjugants, indicated poor maintenance of the pIPO2T plasmid in these hosts (van Elsas et al., 1998
). An apparent redundancy of parA-like auxiliary functions would not be unprecedented: IncP-9 plasmids also possess an apparently cryptic putative resolvase (res) gene adjacent to their rep gene (Greated et al., 2000
).
In addition, pIPO2 carries a putative operon (ORF26ORF22) whose key deduced proteins show amino acid sequence similarity to proteins from the central control region of IncP-1 plasmids (Table 2; Fig. 2
). Both the incC and korB gene products have been shown to be required for the segregational stabilization (partitioning) phenotype, in instances where they have been studied in detail (reviewed by Gerdes et al., 2000
; Bignell & Thomas, 2001
). KorB belongs to the ParB family of DNA-binding proteins that bind the cis-acting centromere-like sequence, and IncC belongs to the ParA family of ATPases needed for symmetric distribution of the KorBDNA complexes. Note that ParA and ParB here should not be confused with ParA and ParB referred to in the previous paragraph. Centromere-like sequences in other systems are normally found upstream or downstream of the incC operon, appearing as one or more inverted or direct repeats. The most obvious candidate for a centromere-like region is therefore the cluster of direct repeats upstream of the ssb promoter (Fig. 2
). An unusual feature of this putative operon is the inclusion of a gene encoding a single-stranded DNA-binding protein (Ssb). ssb genes are generally associated with replication or transfer regions rather than with partitioning genes (Golub & Low, 1986
; Jovanovic et al., 1992
). A comparison between the organization of the ssb operon in plasmid pIPO2 and the recently described rhizobial plasmid pSB102 (Schneiker et al., 2001
) revealed complete collinearity. In addition, there is considerable sequence conservation at the -35 and -10 sites (Fig. 2
), and the repeat sequences upstream of the putative operon were conserved between the two plasmids.
|
Putative conjugative transfer region
More than half of the plasmid, i.e. approximately 23 kb, is occupied by the putative transfer region, delimited by ORF1/ORF2 (traA) and a sequence region proposed to function as the transfer origin (oriT) of pIPO2 in between ORF21 and ORF22 (Figs 1 and 3
). The proposed oriT resembles the T-border sequences (5'-TATCCTGC-3', see Fig. 3
) which flank the T-DNA segment of the agrobacterial Ti plasmids, a region that is transferred from bacteria to plant cells by a conjugation-like process. These so-called nick region sequences are part of the IncP-1 oriT region and contain eight conserved base pairs, which have been called the nic-site (for a review, see Pansegrau & Lanka, 1996
). The orientation of the nic-site in the lower strand (reverse complementary strand) of pIPO2 predicts that the transfer (tra) genes would be transmitted into the recipient cell last, as observed for most other conjugation systems (each of the plasmids listed in Fig. 3
). This is the same as the pIPO2-related plasmids pSB102 and pXF51 (Marques et al., 2001
; Schneiker et al., 2001
), which have significant sequence similarity with pIPO2 at the proposed nic region and carry inverted repeat structures (Fig. 3
) which might be important for target recognition during DNA processing. Each of the 17 reading frames in between traA and oriT has significant sequence similarity to a conjugative transfer gene product and/or to a product of a type IV secretion system (Christie & Vogel, 2000
; Zechner et al., 2000
), suggesting a single and unique transfer region. Potential Tra components of pIPO2 fall into two classes, the DNA processing functions (orfs 3, 8, 15, 17 and 20, highlighted in green in Fig. 1
; Table 2
) and the mating pair formation functions (orfs 2, 47, 914, 18 and 19, highlighted in light green in Fig. 1
; Table 2
), as outlined below.
|
|
Signal sequence prediction (Nielsen et al., 1997 ) revealed that nine proteins encoded by pIPO2, including TraC, TraE and TraK, possess a signal peptide, like the corresponding proteins of the Ti plasmid (Fig. 5
). Three proteins, including those encoded by TraG (the proposed IncN-like entry exclusion function Eex) and TraI, contain signal sequences that resemble the signal peptidase II cleavage site found in lipoprotein precursors (Pugsley, 1993
).
|
|
According to its sequence, the pIPO2 protein TraM belongs to the VirB11 family of traffic/secretion NTPases (Krause et al., 2000a ; Planet et al., 2001
). A few members of this superfamily [Ti VirB11, R388 TrwD, RP4 TrbB and the Helicobacter pylori 0525 (Cag
) protein] have been shown to hydrolyse NTPs, to form hexamers of identical subunits in solution, and seem to be associated with the cytoplasmic membrane (Krause et al., 2000a
, b
). The three-dimensional structure of the H. pylori protein Cag
shows the form of a six-clawed grapple, which is supposed to open and close upon ATP binding/hydrolysis (Yeo et al., 2000
). This property suggests that the hexamers may function as chaperones or have pore character, and that they may be involved in substrate transmission into target cells.
Plasmid pIPO2 belongs to a new family of environmentally important plasmids
The gene organization of the putative conjugative transfer region (Tra region) of pIPO2 has been found to be conserved in pSB102 (Schneiker et al., 2001 ) and in the X. fastidiosa plasmid pXF51 (Marques et al., 2001
), indicating that the three Tra systems belong to the same class (Fig. 7
). The interruption of the Mpf gene clusters by the proposed DNA processing gene traB/top next to traA/traL is unique for pIPO2, pSB102 and also for pXF51. The sequence-related traE gene has been found in IncP-1 plasmids in the Tra1 region (which encodes DNA processing genes) as a tra gene that is non-essential at least for intraspecific E. coli matings (Lessl et al., 1993
), even in the absence of chromosomally encoded topA and topB genes (G. Schröder & E. Lanka, unpublished data). Complementation by other chromosome-encoded topoisomerases, for instance, the topoisomerase IV (Deibler et al., 2001
), may be the reason for this finding.
|
The compact transfer regions of the three plasmids pIPO2, pSB102 and pXF51 contain elements related to components of the Ti plasmid T-DNA transfer system and the conjugative transfer system of IncP-1 plasmids. This modular design principle of the combination of functional units of different phylogenetic origin apparently is valid for most of the conjugative transfer systems (Pansegrau & Lanka, 1996 ; Zechner et al., 2000
). Although related to the Ti plasmid VirB proteins, the proposed mating pair formation components of the three plasmids are much more closely related to proteins of the type IV secretion system of Brucella spp. (Table 2
). The latter are thought to be responsible for the secretion or delivery to mammalian cells of as yet unknown substrates, most likely proteinaceous toxins.
An additional common feature of pIPO2 and pSB102 is the existence of a replication initiator protein, RepA, which is embedded by putative ORFs of unknown function. However, both plasmids, pIPO2 and pSB102, lack iteron-like structures supposed to function as part of the vegetative origin, i.e. as binding sites for RepA. Two regions in pIPO2 and pSB102 contain clusters of predicted ORFs with unknown functions. Strikingly, many of the ORFs of unknown function are related between pIPO2 and pSB102, suggesting a possible involvement of these ORFs in host/plasmid functioning in natural habitats.
In conclusion, plasmid pIPO2 fits well into the new class of environmental plasmids defined by plasmid pSB102 (Schneiker et al., 2001 ). Interestingly, pIPO2, as well as pSB102 and pXF51, resides in plant-associated bacteria. This further supports the hypothesis that all three plasmids might be variants of an archetypical plasmid class associated with phytosphere bacteria.
Determination of the distribution of plasmid pIPO2
Plasmid pIPO2 was initially isolated by the E. coli/R.eutropha-based triparental exogenous plasmid isolation system (Top et al., 1994 ), selecting for plasmids with IncQ plasmid mobilizing capacity. pIPO2 was hereby isolated from extracts of soil associated with wheat seedlings, at just above the detection limit of 10-9 per recipient (van Elsas et al., 1998
). In contrast, exogenous isolation of plasmids from rhizosphere soil of field-grown mature wheat, maize and sugarbeet repeatedly failed under the same conditions. This indicated a lower incidence of functional pIPO2-like plasmids in the respective soil or rhizospheric bacterial communities compared to the numbers associated with young wheat plants.
In the present work, a direct molecular screen for pIPO2 sequences in total microbial community DNA, obtained from replicate samples from a range of different environmental habitats, was performed (Table 1). The previously developed PCR primer system I shown to be specific for pIPO2 (van Elsas et al., 1998
), as well as primer system II (which amplified part of the repA gene homologue), was used. Primer system II was, on theoretical grounds, specific for plasmid pIPO2, as BLAST searches against the whole GenBank/EMBL database did not reveal any significant match with any sequence, including those of plasmids pSB102, pXF51, R388, RP4 and R751. In addition, laboratory tests showed that the broad-host-range plasmids R388, RN3, RP4, R751, RSF1010, pIE723 and pIE639, as well as a range of 10 E. coli narrow-host-range plasmids, did not produce amplicons, whereas plasmid pIPO2 DNA consistently produced the expected PCR fragment. pIPO2 DNA added to each environmental DNA extract consistently caused amplification in direct correlation with the concentration of added target DNA, i.e. progressive dilution of added target DNA in the environmental extract yielded progressively fainter signals which ultimately became extinct. This established a detection threshold for each habitat, which was calculated to be about 5x103 plasmids per g of soil or per ml of water.
The detection of pIPO2 prevalence in the different habitats was consistent in primer systems I and II (Table 1). Evidence for the occurrence of pIPO2 was obtained for seven of the 10 rhizospheres tested and for two of the 11 bulk soils studied, whereas such evidence was not found for seawater, wastewater, manure and compost (added to soil) (Table 1
). Thus, the only habitats with consistent evidence for pIPO2-specific sequences were the rhizospheres of young wheat, rice and maize, and those of oats, grass, tomato and cauliflower, as well as soils previously used for cultivation of crop plants such as the Lovinkhoeve (samples from five time points, but not in three other samples) and Wildekamp arable land (summer 2000 sample, oats grown in the field) samples. On the basis of these results, we postulate that pIPO2 either has a preference for host organisms that thrive in the rhizospheres of a variety of crop plants or confers a property on its host that promotes survival in these environments. Conversely, pIPO2 appears not to survive in hosts that prefer bulk soils or it does not promote survival of its host in bulk soils.
A number of microbial groups are known to be subject to changes in relative dominance in the rhizosphere of crop plants such as wheat, maize, tomato and potato. For instance, Miller et al. (1989) showed that fluorescent pseudomonads are avid responders to young roots of wheat and maize, enhancing their abundance. Upon plant ageing, these organisms were shown to decline. This principle can most likely be extended to many crop plants, including the ones used in this study (wheat, maize, tomato and cauliflower). Smalla et al. (2001)
recently showed that the dynamics of microbial populations associated with field-grown strawberry, oilseed rape and potato was strongly dependent on plant type and on season, i.e. most likely due to plant developmental stage. By and large, and possibly a bit speculatively, one can assume that a range of bacterial groups, which includes the Gram-negative and Gram-positive rhizosphere-strategists (such as pseudomonads, stenotrophomonads, several enterics, bacilli and paenibacilli), are able to respond to the enhanced availability of nutrients in root exudates by enhancing their prevalence. Such a response can be different per bacterial group and per plant, and is most likely highly dependent on the types of compounds present in the root exudates. Thus, we suggest that the detection of plasmids at different stages of seedling development probably reflects changes in the composition of the associated microbial community.
Conclusion and prospects for further work
The broad-host-range plasmid type represented by pIPO2 was found to be a compact genetic structure containing mainly conjugation and inheritance functions. A region containing adjacent ORFs that encode small proteins with unknown function was identified (ORF32ORF45). This region is homologous to a similar region found in plasmid pSB102 (Schneiker et al., 2001 ). The potential phenotype encoded by the ORFs of this region might be identified by studies that address host/plasmid functioning in the natural habitat, i.e. including the plant as a major driving factor. Since most other pIPO2 functions are related to plasmid replication, maintenance and conjugative transfer functions, the plasmid at present appears to be cryptic (van Elsas et al., 1998
). Therefore, expression of transfer-related genes might cause an enhanced metabolic load without a balancing advantage, explaining the apparently reduced survival in bulk soils of bacteria carrying pIPO2.
Given its similarity to the novel broad-host-range conjugative plasmid pSB102 and to the X. fastidiosa plasmid pXF51, we propose that pIPO2 is an example of a novel class of plasmids that are prevalent in hosts that associate in symbiotic or in other ways with plants. The direct molecular evidence that pIPO2 is prevalent in the rhizosphere of several crop plants is consistent with this hypothesis and pinpoints the original host of pIPO2 as an organism that associates primarily with plants and takes advantage (currently unknown) of the presence of pIPO2. The most obvious possibility is that the clustered and probably coordinately expressed orfs of unknown function (orfs 3845) encode a metabolic pathway or apparatus that confers an advantageous phenotype to its host, which is perhaps specific to plant-associated communities. One aspect of future work will be to define what this phenotype is.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balzer, D., Pansegrau, W. & Lanka, E. (1994). Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of Plasmid RP4. J Bacteriol 176, 4285-4295.[Abstract]
Bayer, M., Eferl, R., Zellnig, G., Teferle, K., Dijkstra, A., Koraimann, G. & Högenauer, G. (1995). Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection. J Bacteriol 177, 4279-4288.[Abstract]
Bignell, C. & Thomas, C. M. (2001). The bacterial ParAParB partitioning proteins. J Biotechnol 91, 1-34.[Medline]
Christie, P. J. & Vogel, J. P. (2000). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8, 354-360.[Medline]
Cobbe, N. & Heck, M. M. S. (2000). SMCs in the world of chromosome biology from prokaryotes to higher eukaryotes. J Struct Biol 129, 123-143.[Medline]
Dang, T. A., Zhou, X. R., Graf, B. & Christie, P. J. (1999). Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter. Mol Microbiol 32, 1239-1253.[Medline]
Das, A. & Xie, Y. H. (2000). The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another. J Bacteriol 182, 758-763.
Daugelaviius, R., Bamford, J. K., Grahn, A. M., Lanka, E. & Bamford, D. H. (1997). The IncP plasmid-encoded cell-envelope-associated DNA transfer complex increases cell permeability. J Bacteriol 179, 5195-5202.[Abstract]
Deibler, R. W., Rahmati, S. & Zechiedrich, E. L. (2001). Topoisomerase IV, alone, unknots DNA in E. coli. Genes Dev 15, 748-761.
de la Cruz, F. & Davies, J. (2000). Horizontal gene transfer and the origin of species: lessons from bacteria. Trends Microbiol 8, 128-133.[Medline]
de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172, 6568-6572.[Medline]
Dodd, I. B. & Egan, J. B. (1990). Improved detection of helixturnhelix DNA-binding motifs in protein sequences. Nucleic Acids Res 18, 5019-5026.[Abstract]
Duarte, G. F., Rosado, A. S., Seldin, L., De Araujo, W. & van Elsas, J. D. (2001). Analysis of bacterial community structure in sulfurous-oil-containing soils and detection of species carrying dibenzothiophene desulfurization (dsz) genes. Appl Environ Microbiol 67, 1052-1062.
Egelman, E. H. (2001). Pumping DNA. Nature 409, 573-575.[Medline]
Eisenbrandt, R., Kalkum, M., Lai, E. M., Lurz, R., Kado, C. I. & Lanka, E. (1999). Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. J Biol Chem 274, 22548-22555.
Eisenbrandt, R., Kalkum, M., Lurz, R. & Lanka, E. (2000). Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases. J Bacteriol 182, 6751-6761.
Gerdes, K., Ayora, S., Canosa, I. & 10 other authors (2000). In The Horizontal Gene Pool Bacterial Plasmids and Gene Spread, pp. 4985. Edited by C. M. Thomas. Amsterdam, The Netherlands: Harwood Academic Publishers.
Golub, E. I. & Low, K. B. (1986). Unrelated conjugative plasmids have sequences which are homologous to the leading region of the F factor. J Bacteriol 166, 670-672.[Medline]
Gomis-Rüth, F. X., Moncalián, G., Perez-Luque, R., Gonzalez, A., Cabezón, E., de la Cruz, F. & Coll, M. (2001). The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Nature 409, 637-641.[Medline]
Grahn, A. M., Haase, J., Bamford, D. H. & Lanka, E. (2000). Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems. J Bacteriol 182, 1564-1574.
Greated, A., Titiok, M., Krasowiak, R., Fairclough, R. & Thomas, C. M. (2000). The replication and stable inheritance functions of IncP-9 plasmid pM3. Microbiology 146, 2249-2258.
Hill, K. E., Weightman, A. J. & Fry, J. C. (1992). Gene transfer in the aquatic environment: persistence and mobilization of the catabolic recombinant plasmid pD10 in the epilithon. Appl Environ Microbiol 58, 1292-1300.[Abstract]
Hill, K. E., Marchesi, J. R. & Fry, J. C. (1996). Conjugation and mobilization in the epilithon. In Molecular Microbial Ecology Manual , pp. 1-28. Edited by A. D. L. Akkermans, J. D. van Elsas & F. J. de Bruijn. Dordrecht, The Netherlands:Kluwer Academic Publishers.
Jagura-Burdzy, G. & Thomas, C. M. (1992). kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein. J Mol Biol 225, 651-660.[Medline]
Jovanovic, O. S., Ayres, E. K. & Figurski, D. H. (1992). The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein. J Bacteriol 174, 4842-4846.[Abstract]
Krause, S., Bárcena, M., Pansegrau, W., Lurz, R., Carazo, J. M. & Lanka, E. (2000a). Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc Natl Acad Sci USA 97, 3067-3072.
Krause, S., Pansegrau, W., Lurz, R., de la Cruz, F. & Lanka, E. (2000b). Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases. J Bacteriol 182, 2761-2770.
Kumar, R. B., Xie, Y. H. & Das, A. (2000). Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore. Mol Microbiol 36, 608-617.[Medline]
Kurland, C. G. (2000). Something for everyone horizontal gene transfer in evolution. EMBO Rep 1, 92-95.
Kurtz, S. & Schleiermacher, C. (1999). REPuter fast computation of maximal repeats in complete genomes. Bioinformatics 15, 426-427.
Lawrence, J. C. & Ochman, H. (1998). Molecular archaeology of the Escherichia coli genome. Proc Natl Acad Sci USA 95, 9413-9417.
Lessl, M., Pansegrau, W. & Lanka, E. (1992). Relationship of DNA transfer systems: essential transfer factors of plasmid RP4, Ti, and F share common sequences. Nucleic Acids Res 20, 6099-6100.[Medline]
Lessl, M., Balzer, D., Weyrauch, K. & Lanka, E. (1993). The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation. J Bacteriol 175, 6415-6425.[Abstract]
Li, Z., Hiasa, H., Kumar, U. & DiGate, R. J. (1997). The traE gene of RP4 encodes a homologue of Escherichia coli DNA topoisomerase III. J Biol Chem 272, 19582-19587.
Lilley, A. K. & Bailey, M. J. (1997). The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions. Appl Environ Microbiol 63, 1577-1583.[Abstract]
Lilley, A. K., Fry, J. C., Day, M. J. & Bailey, M. J. (1994). In situ transfer of an exogenously isolated plasmid between Pseudomonas spp. in sugar beet rhizosphere. Microbiology 140, 27-33.
Lin, M., Smalla, K., Heuer, H. & van Elsas, J. D. (2000). Effect of an Alcaligenes faecalis inoculant strain on bacterial communities in flooded soil microcosms planted with rice seedlings. Appl Soil Ecol 15, 211-225.
Macartney, D. P., Williams, D. R., Stafford, T. & Thomas, C. M. (1997). Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751. Microbiology 143, 2167-2177.[Abstract]
Marques, M. V., da Silva, A. M. & Gomes, S. L. (2001). Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa. Plasmid 45, 184-199.[Medline]
Miller, H. J., Henken, G. & van Veen, J. A. (1989). Variation and composition of bacterial populations in the rhizospheres of maize, wheat and grass cultivars. Can J Microbiol 35, 656-660.
Moncalián, G., Cabezón, E., Alkorta, I., Valle, M., Moro, F., Valpuesta, J. M., Goñi, F. M. & de la Cruz, F. (1999). Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation. J Biol Chem 274, 36117-36124.
Myers, E. W. & Miller, W. (1988). Optimal alignments in linear space. Comput Appl Biosci 4, 11-17.[Abstract]
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Sys 8, 581-599.
OCallaghan, D., Cazevieille, C., Allardet-Servent, A., Boschiroli, M. L., Bourg, G., Foulongne, V., Frutos, P., Kulakov, Y. & Ramuz, M. (1999). A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol Microbiol 33, 1210-1220.[Medline]
Ochman, H., Lawrence, J. G. & Groisman, E. A. (2000). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299-304.[Medline]
Okumura, M. S. & Kado, C. I. (1992). The region essential for efficient autonomous replication of pSa in Escherichia coli. Mol Gen Genet 235, 55-63.[Medline]
Pansegrau, W. & Lanka, E. (1996). Enzymology of DNA transfer by conjugative mechanisms. Progr Nucleic Acids Res Mol Biol 54, 197-251.[Medline]
Pansegrau, W., Schoumacher, F., Hohn, B. & Lanka, E. (1993). Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation. Proc Natl Acad Sci USA 90, 11538-11542.[Abstract]
Pansegrau, W., Lanka, E., Barth, P. T. & 7 other authors (1994a). Complete nucleotide sequence of Birmingham IncP plasmids: compilation and comparative analysis of sequence data. J Mol Biol 239, 623663.[Medline]
Pansegrau, W., Schröder, W. & Lanka, E. (1994b). Concerted action of three distinct domains in the DNA cleavingjoining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4. J Biol Chem 269, 2782-2789.
Planet, P. J., Kachlany, S. C., DeSalle, R. & Figurski, D. H. (2001). Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci USA 98, 2503-2508.
Preston, K. E., Radomski, C. C. A. & Venezia, A. (2000). Nucleotide sequence of a 7-kb fragment of pACM1 encoding an IncM DNA primase and other proteins associated with conjugation. Plasmid 44, 12-23.[Medline]
Pugsley, A. P. (1993). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50-108.[Abstract]
Pukall, R., Tschäpe, H. & Smalla, K. (1996). Monitoring the spread of broad host and narrow host range plasmids in soil microcosms. FEMS Microbiol Ecol 20, 53-66.
Reese, M. G., Harris, N. L. & Eeckman, F. H. (1996). Large scale sequencing specific neural networks for promoter and splice site recognition. In Biocomputing: Proceedings of the 1996 Pacific Symposium. Edited by L. Hunter & T. E. Klein. Singapore: World Scientific Publishing.
Rosado, A. S., Duarte, G. F., Seldin, L. & van Elsas, J. D. (1998). Genetic diversity of nifH gene sequences in Paenibacillus azotofixans strains and soil samples analyzed by denaturing gradient gel electrophoresis of PCR-amplified gene fragments. Appl Environ Microbiol 64, 2770-2779.
Rost, B., Casadio, R., Fariselli, P. & Sander, C. (1995). Prediction of helical transmembrane segments at 95% accuracy. Protein Sci 4, 521-533.
Sambrook, J, Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P. C. & Baron, C. (1999). Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens. J Bacteriol 181, 7485-7492.
Schmiederer, M. & Anderson, B. (2000). Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region. DNA Cell Biol 19, 141-147.[Medline]
Schneiker, S., Keller, M., Dröge, M., Lanka, E., Pühler, A. & Selbitschka, W. (2001). The genetic organization and evolution of the broad-host-range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa. Nucleic Acids Res 29, 5169-5181.
Shirasu, K., Koukolíková-Nicola, Z., Hohn, B. & Kado, C. I. (1994). An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol 11, 581-588.[Medline]
Sieira, R., Comerci, D. J., Sanchez, D. O. & Ugalde, R. A. (2000). A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 182, 4849-4855.
Simpson, A. J. G., Reinach, F. C., Arruda, & 113 other authors (2000). The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406, 151157.[Medline]
Smalla, K., Cresswell, N., Mendonca-Hagler, L. C., Wolters, A. & van Elsas, J. D. (1993). Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J Appl Bacteriol 74, 78-85.
Smalla, K., Osborn, A. M. & Wellington, E. M. H. (2000). Isolation and characterisation of plasmids. In The Horizontal Gene Pool Bacterial Plasmids and Gene Spread , pp. 207-248. Edited by C. M. Thomas. Amsterdam, The Netherlands:Harwood Academic Publishers.
Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H. & Berg, G. (2001). Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol 67, 4742-4751.
Smit, E., van Elsas, J. D., van Veen, J. A. & de Vos, W. M. (1991). Detection of plasmid transfer from Pseudomonas fluorescens to indigenous bacteria in soil by using bacteriophage R2f for donor counterselection. Appl Environ Microbiol 57, 3482-3488.
Smit, E., Venne, D. & van Elsas, J. D. (1993). Mobilization of a recombinant IncQ plasmid between bacteria on agar and in soil via co-transfer or retrotransfer. Appl Environ Microbiol 59, 2257-2263.[Abstract]
Staden, R. (1996). The Staden sequence analysis package. Mol Biotechnol 5, 233-241.[Medline]
Szpirer, C., Top, E., Couturier, M. & Mergeay, M. (1999). Retrotransfer or gene capture: a feature of conjugative plasmids, with ecological and evolutionary significance. Microbiology 145, 3321-3329.
Thomas, C. M. (editor) (2000a). The Horizontal Gene Pool Bacterial Plasmids and Gene Spread. Amsterdam, The Netherlands: Harwood Academic Publishers.
Thomas, C. M. (2000b). Paradigms of plasmid organisation. Mol Microbiol 37, 485-491.[Medline]
Thorsted, P. B., Macartney, D. P., Akhtar, P. & 9 other authors (1998). Complete sequence of the IncPß plasmid R751: implications for evolution and organization of the IncP backbone. J Mol Biol 282, 969990.[Medline]
Thorstenson, Y. R., Kuldau, G. A. & Zambryski, P. C. (1993). Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure. J Bacteriol 175, 5233-5241.[Abstract]
Top, E., de Smet, I., Verstraete, W., Dijkmans, R. & Mergeay, M. (1994). Exogenous isolation of mobilizing plasmids from polluted soils and sludges. Appl Environ Microbiol 60, 831-839.[Abstract]
van Elsas, J. D. & Smalla, K. (1995). Extraction of microbial community DNA from soils. In Molecular Microbial Ecology Manual, pp. 1.3.3: 111. Edited by A. D. L. Akkermans, J. D. van Elsas & F. J. de Bruijn. Dordrecht, The Netherlands: Kluwer Academic Publishers.
van Elsas, J. D., Trevors, J. T. & Starodub, M.-E. (1988). Bacterial conjugation between pseudomonads in the rhizosphere of wheat. FEMS Microbiol Ecol 53, 299-306.
van Elsas, J. D., McSpadden Gardener, B. B., Wolters, A. C. & Smit, E. (1998). Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in the wheat rhizosphere. Appl Environ Microbiol 64, 880-889.
van Elsas, J. D., Fry, J., Hirsch, P. & Molin, S. (2000a). Ecology of plasmid transfer and spread. In The Horizontal Gene Pool Bacterial Plasmids and Gene Spread , pp. 175-206. Edited by C. M. Thomas. Amsterdam, The Netherlands:Harwood Academic Publishers.
van Elsas, J. D., Kastelein, P., van Bekkum, P., van der Wolf, J. M., de Vries, P. M. & van Overbeek, L. S. (2000b). Survival of Ralstonia solanacearum biovar 2, the causative agent of potato brown rot, in field and microcosm soils in temperate climates. Phytopathology 90, 1358-1366.
van Overbeek, L. S., van Veen, J. A. & van Elsas, J. D. (1995). Induced reporter gene activity, enhanced stress resistance, and competitive ability of a genetically modified Pseudomonas fluorescens strain released into a field plot planted with wheat. Appl Environ Microbiol 63, 1965-1973.[Abstract]
Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982). Distantly related sequences in the - and ß-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1, 945-951.[Medline]
Yeo, H. J., Savvides, S. N., Herr, A. B., Lanka, E. & Waksman, G. (2000). Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell 6, 1461-1472.[Medline]
Zechner, E. L., de la Cruz, F., Eisenbrandt, R. & 8 other authors (2000). Conjugative transfer processes. In The Horizontal Gene Pool Bacterial Plasmids and Gene Spread, pp. 87174. Edited by C. M. Thomas. Amsterdam, The Netherlands: Harwood Academic Publishers.
Received 12 November 2001;
revised 18 December 2001;
accepted 21 January 2002.