Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid

Xue-Xian Zhang1,2,3, Andrew K. Lilley1, Mark J. Bailey1 and Paul B. Rainey2,3

1 Centre for Ecology and Hydrology NERC, Mansfield Road, Oxford OX1 3SR, UK
2 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
3 School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Correspondence
Xue-Xian Zhang
xx.zhang{at}auckland.ac.nz


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (ornpl) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of ornpl generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (ornE.coli). Plasmid-derived ornpl was capable of partially complementing the P. putida orn mutant, demonstrating functionality of ornpl. Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of ornpl from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the ornpl was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.


Abbreviations: DAP, diaminopimelic acid; Orn, oligoribonuclease; CFC, cetrimide, fucidin and cephalosporin; IVET, in vivo expression technology

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AJ617292.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The genus Pseudomonas includes several species of plant-colonizing bacteria (Palleroni, 1984). Strains of some species can cause disease (e.g. Pseudomonas syringae), whereas others, for example strains of Pseudomonas fluorescens, can promote plant growth (reviewed by Bloemberg & Lugtenberg, 2001).

The genomes of plant-associated Pseudomonas species frequently contain a significant component of extra-chromosomal DNA in the form of one or more plasmids (Vivian et al., 2001; Thomas, 2000); the transfer of these plasmids within and between bacterial populations is thought to facilitate adaptation to novel environments (Hartl et al., 1984). Despite their ecological importance, knowledge of the traits encoded on naturally occurring plasmids is limited. Such plasmids rarely encode selectable phenotypes, and the identification of plasmid-encoded genes that contribute to the ecological performance of bacteria in natural environments is challenging.

Analysis of the phytosphere microflora of sugar beet at a field site in Oxford (UK) in the mid-1990s revealed an abundance of self-transmissible mercury-resistance plasmids that move freely within the resident Pseudomonas community (Lilley et al., 1996; Lilley & Bailey, 1997a). These plasmids have molecular sizes ranging from 60 to 425 kb and have been grouped into five distinct categories (groups I to V) by RFLP analysis. In field-grown sugar beet these plasmids are most commonly encountered in bacteria isolated from mature plants (Lilley & Bailey, 1997a). One particular group I plasmid, pQBR103, has been the subject of extensive analysis. Interestingly, carriage of this plasmid reduces fitness in bacteria colonizing sugar beet seedlings, but not bacteria colonizing mature plants (Lilley & Bailey, 1997b). These data suggest a significant genotype-by-environment interaction and the existence of pQBR103-encoded traits that confer some selectable advantage to bacteria colonizing mature sugar beet plants.

In a parallel study (unpublished) we used a promoter trapping strategy (IVET) to isolate pQBR103-encoded genes of possible relevance to plant colonization. The promoter-trap, based on a promoterless copy of 'dapB, a gene encoding 2,3-dihydrodipicolinate reductase and required for the biosynthesis of diaminopimelic acid (DAP) and lysine (Gal et al., 2003), led to the isolation of 37 plant-induced fusions. These fusions are transcriptionally silent in minimal medium, but active in the phytosphere of sugar beet. The plant-induced genes are of particular interest because they are predicted to contribute to the fitness of the host bacteria in planta – a prediction recently confirmed for a locus encoding an acetylated cellulose polymer (Gal et al., 2003; Spiers et al., 2003). Sequence analysis of the 37 fusions revealed few with any similarity to sequences deposited in public DNA or protein sequence databases. However, one fusion, pIVETD5, is to an ORF (orf6) adjacent to a gene (orn) predicted to encode oligoribonuclease, which is an essential gene in Escherichia coli (Ghosh & Deutscher, 1999). Orn hydrolyses small oligoribonucleotides and is involved in the final step of mRNA degradation (Zhang et al., 1998). Although putative Orn proteins have been identified in the sequenced Pseudomonas genomes, none of their functions have been characterized. Here we report the comparative and functional analysis of orn genes carried on an indigenous Pseudomonas plasmid and the chromosome of P. putida KT2440 and show that the plasmid-derived orn gene is plant inducible and widespread among group I plasmids.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growing conditions.
P. fluorescens SBW25 was isolated from field-grown sugar beet at the University of Oxford farm, Wytham, Oxford, UK (Bailey et al., 1995). Plasmid pQBR103 is representative of a group of large plasmids conferring resistance to mercuric chloride and acquired by genetically marked P. fluorescens (SBW25EeZY6KX) during the course of field release experiments at Wytham (Lilley & Bailey, 1997a). All the indigenous plasmids (prefixed with pQBR) used in this study were exogenously isolated from plant-colonizing Pseudomonas (Lilley et al., 1996) and are listed in Table 1. Bacterial strains and other recombinant plasmids used in this study are listed in Table 2.


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Table 1. Distribution of ornpl among environmental plasmids isolated from the sugar beet phytosphere

 

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Table 2. Bacterial strains and plasmids

 
Pseudomonas and E. coli strains were routinely grown in Luria–Bertani medium (LB) at 28 °C and 37 °C, respectively. Minimal M9 medium (Sambrook et al., 1989) was also used for the propagation of Pseudomonas. Pseudomonas transconjugants containing pQBR plasmids were grown in LB supplemented with HgCl2 (27 µg ml–1). When necessary, antibiotics were supplemented at the following concentrations (µg ml–1): rifampicin, 50; tetracycline, 10; kanamycin, 100; nalidixic acid, 50; ampicillin, 50; spectinomycin, 100. The IVET fusion strains based on P. fluorescens SBW25{Delta}dapB were cultivated in minimal M9 medium with addition of lysine (60 µg ml–1) and DAP (800 µg ml–1). Half-strength CFC (cetrimide, fucidin and cephalosporin; Oxoid) was used to select P. fluorescens recovered from the plant.

DNA manipulations.
General recombinant DNA techniques were performed according to standard protocols (Sambrook et al., 1989). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB). DNA was sequenced using Big-Dye terminators (Applied Biosystems) on an automated DNA sequencer, model 310 (Perkin Elmer) following the manufacturer's instructions. PCR reactions were performed using Taq DNA polymerase (Qiagen) with an annealing temperature of 58 °C according to the manufacturer's protocol. The template DNA was either purified genomic DNA or whole-cell suspensions. All oligonucleotide primers were obtained from MWG Biotech. Plasmid conjugation was carried out using the filter plate mating method as previously described (Lilley et al., 1996) except that a growing temperature of 28 °C was used.

Sequencing and deletion analysis of ornpl.
Plasmid pIVETD5 is one of the original IVET clones isolated from pQBR103 containing genes that show elevated levels of expression on plant surfaces (unpublished). The insert fragment of pIVETD5 was initially sequenced from both ends using primers to the 5' end of the dapB gene (Pdap) and the 5' end of the bla gene (Pbla) in the pIVETD vector (Gal et al., 2003). The whole 2·3 kb insert was then sequenced on both strands by primer walking, whereby subsequent primers were designed based on the newly derived sequence data. A random shotgun genomic library of pQBR103, which was composed of 2000 single colonies containing 1–3 kb DNA fragments at the SmaI site of pUC18, was sequenced (unpublished data). Sequence alignment of pIVETD5 with the shotgun sequence database of pQBR103 identified five clones that contained overlapping DNA fragments from the ornpl region (Fig. 1a). Sequence alignment was performed using the Sequencher software (Gene Codes Corporation). The sequence gaps were then filled in by primer walking using the five pUC18 clones as sequencing templates. Details of the oligonucleotides used in DNA sequencing are available on request. A 6773 bp sequence was finally obtained and it has been deposited in GenBank (accession number AJ617292).



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Fig. 1. Genetic map of orn and flanking DNA. (a) Map of the 6·773 kb sequenced ornpl region of pQBR103. The ornpl gene is shown as a filled arrow while other ORFs (orf) that have no homologues in the databases are shown as empty arrows. Lines indicate the corresponding fragments cloned into fusion plasmids. Lines with arrows show the fusion positions and the direction of transcription of the promoterless 'dapB'lacZ genes in IVET clones pIVETD5 and pIVETD9. (b) Solid arrows indicate orn and its flanking yjeQ and yjeS genes identified in E. coli and the corresponding homologues in P. fluorescens (SBW25 and PfO-1), P. syringae DC3000 (PS), P. aeruginosa PAO1 (PA) and P. putida KT2440 (PP). Open squares refer to tRNA genes downstream of ornE.coli. Discontinuous arrows indicate a conserved hypothetical protein, which locates elsewhere in P. aeruginosa (PA1037). Open triangles indicate the locations of the PCR primers ornF3/ornR3, which were used to amplify the 2·0 kb orn region of P. putida, and the insertion position of the omega cassette into ornP.putida.

 
In order to construct an ornpl deletion mutant, shotgun plasmid p9d5 (Fig. 1a) was digested with XhoI to remove the 378 bp ornpl fragment and transformed into E. coli DH5{alpha} after ligation. The resulting plasmid p9d5X contains a 1·97 kb insert composed of two DNA fragments of similar lengths flanking the ornpl deletion. This 1·97 kb insert was then PCR-amplified using general M13 forward and reverse primers incorporating BglII sites and cloned into the integration vector pIVETD. The insertion direction was determined by restriction analysis. The recombinant plasmid containing the putative ORFs including the undeleted 200 bp of ornpl in the same transcriptional direction as the promoterless 'dapB was designated pIVETD10. To remove the 'dapB gene pIVETD10 was digested with SpeI and transformed into E. coli DH5{alpha}{lambda}pir. The resulting plasmid, pIVET10, was used to deliver the ornpl mutation. It was mobilized into P. fluorescens SBW25(pQBR103) by conjugation with the help of pRK2013 (Tra+). Integration into pQBR103 by a single homologous recombination event was selected for on LB agar supplemented with HgCl2, tetracycline and X-Gal. Allelic-exchange mutants were selected as previously described (Gal et al., 2003) by growing the blue-coloured transconjugant for two successive 24 hour periods in LB broth without antibiotic selection. The bacterial cells were plated on LB agar containing X-Gal. White tetracycline-sensitive colonies were checked for loss of ornpl by PCR using primers 103orn-F/R (Table 3).


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Table 3. PCR primers

 
Construction of the ornpl : : 'dapB fusions and plant colonization assay.
To construct an ornpl : : 'dapB fusion, plasmid pIVETD10 was digested by XhoI, which removed a 900 bp fragment downstream of the ornpl gene. The cleaved plasmid was religated and transformed into E. coli DH5{alpha}{lambda}pir to generate pIVETD9. It was then conjugated into SBW25{Delta}dapB(pQBR103) by triparental mating with the help of pRK2013. Integration into the ornpl locus was confirmed by PCR using primers Pdap (Gal et al., 2003) and p10b5-F, which is located upstream of ornpl but outside the cloned region of pIVETD9.

Competitive colonization assays of the IVET fusions against the competitor strain of SBW25(pQBR103) were performed as previously described by Rainey (1999). Bacteria were inoculated onto coated sugar beet (Beta vulgaris var. Amethyst) seeds at a ratio of 1 : 1 (~103 cells per seed). The seeds were germinated and cultivated in 5 ml scintillation vials using non-sterile vermiculite as a growth substrate. After 14 days, bacteria were counted on selective plates (M9 with lysine, DAP, CFC, tetracycline and X-Gal) and non-selective plates (M9 with CFC) following recovery from the shoot (the photosynthetic parts) and rhizosphere (roots with attached vermiculite).

Inactivation of ornP.putida and heterologous complementation.
Using the genome sequence of P. putida KT2440, a pair of primers ornF3/ornR3 (Table 3) were designed to amplify a 2·0 kb DNA fragment containing ornP.putida (Fig. 1b). The PCR product was first cloned into the PCR cloning vector pCR2.1 (Invitrogen) and sequenced to check for errors. The 2·0 kb ornP.putida fragment was liberated by EcoRI digestion and cloned into pUC18, where a 2·0 kb omega cassette with tetracycline resistance ({Omega}Tc) was inserted in the middle of ornP.putida at the XhoI site. The omega cassette was obtained by PCR and the entire 4·0 kb insert was subcloned into an integration plasmid vector pIVET11, which was prepared from pIVETD by removing the 'dapB gene and the gene encoding tetracycline resistance. The resultant plasmid, designated pIVET11-1, was mobilized into P. putida UWC1, a spontaneous rifampicin-resistant derivative of KT2440, by the standard procedure of conjugation with the help of pRK2013. Transconjugants were selected on M9 agar supplemented with rifampicin, tetracycline and X-Gal. Allelic-exchange mutants were identified on the basis of white colour on X-Gal-containing media and sensitivity to pipercillin (150 µg ml–1; carried on pIVET11-1). Mutants were confirmed by PCR analysis using primers orn-F/R (Table 3).

Complementation of the P. putida ornP.putida mutant was performed by cloning the orn genes from P. putida, pQBR103 and E. coli into pME6010 (Heeb et al., 2000), a plasmid vector that can replicate in Pseudomonas. Primers used to amplify the coding regions of the ornP.putida, ornpl, and ornE.coli are listed in Table 3. To ensure efficient expression of the cloned orn genes, a Pseudomonas-specific ribosome-binding site (RBS) ‘GAGGA’ was introduced into the primers at the position of –10 from the ORF start codon. Restriction sites of KpnI and HindIII were incorporated into the PCR primers to allow for cloning of the products into the corresponding sites of pME6010. An omega cassette with spectinomycin resistance was then inserted downstream of the cloned orn gene in vector pME6010 at the HindIII site. This vector is designed such that genes inserted in the multi-cloning site are transcribed under the control of a constitutive promoter, Pk (Heeb et al., 2000). Plasmids were mobilized into the ornP.putida mutant PBR447 by conjugation. Growth was assessed by culturing each strain in both LB broth and minimal M9 broth. Overnight cultures were diluted in the same medium to an initial OD600 of ~0·02. The OD600 values were measured using a GENESYS 20 spectrophotometer (Thermo Electron).

Phylogenetic analysis.
Amino acid sequences were aligned using CLUSTAL_X (Thompson et al., 1997) and a phylogenetic tree constructed using the neighbour-joining method (Saitou & Nei, 1987). The tree was displayed in TreeView (Page, 1996). The orn genes and the flanking sequences of the following species were obtained from genome databases (in parentheses): E. coli (http://www.genome.wisc.edu), P. fluorescens SBW25 (http://www.sanger.ac.uk), P. fluorescens PfO-1 (http://www.jgi.doe.gov), P. syringae DC3000 and P. putida KT2440 (http://www.tigr.org/) and P. aeruginosa PAO1 (http://www.pseudomonas.com/). Other Orn sequences were collected from NCBI database (Mycobacterium tuberculosis, G7227904; Streptomyces griseus, AB036424; Caenorhabditis elegans, NP_505606; Xylella fastidiosa, NP_778746; Xanthomonas axonopodis, NP_642365; Ralstonia solanacearum, NP_519063; Neisseria meningitidis, NP_283406; Drosophila melanogaster, NP_651184; Arabidopsis thaliana, Q9ZVE0; Homo sapiens, NP_056338; Schizosaccharomyces pombe, CAB37438; Ectocarpus siliculosus virus, NP_077624).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The orn gene carried on plasmid pQBR103 is plant inducible
DNA sequence analysis of pQBR103 IVET clone pIVETD5 revealed the presence of a putative oligoribonuclease gene (ornpl) (Fig. 1a). Additional sequencing of pQBR103 DNA flanking the 2·3 kb insert cloned in pIVETD5 revealed nine predicted ORFs, none of which, with the exception of ornpl, showed any significant similarity to genes or proteins within EMBL or GenBank databases. Seven of the putative ORFs are orientated in the same direction as ornpl, suggesting the possibility that they are transcribed from a single plant-inducible promoter (Fig. 1a). Given that the original 'dapB transcriptional fusion in pIVETD5 is to the sixth of these seven genes and that ornpl is the third, it was possible that ornpl was not plant-inducible and that the plant responsive element was downstream of ornpl.

To determine whether ornpl is specifically induced in the plant environment, a transcriptional fusion was generated between ornpl and a promoterless copy of 'dapB. We then determined, once the fusion was integrated into the genome of P. fluorescens SBW25{Delta}dapB(pQBR103), whether this strain could grow in the plant environment. Growth of P. fluorescens SBW25{Delta}dapB can only occur if transcription of the promoterless 'dapB gene occurs in the plant environment (Gal et al., 2003). In this instance this would require the promoter driving expression of ornpl to be active in the plant environment.

The ornpl : : 'dapB fusion strain PBR442 [P. fluorescens SBW25{Delta}dapB(pQBR103 ornpl : : pIVETD9)], is unable to grow on minimal M9 medium in the absence of DAP and lysine showing that the promoter driving ornpl expression is inactive on this medium. In order to test whether the promoter is active in the plant environment, PBR442 was inoculated onto sugar beet seeds. In addition, further seeds were inoculated with: (1) the original orf6 : : 'dapB IVET fusion strain PBR424; (2) an IVET strain with a fusion between promoterless 'dapB and a constitutive promoter (PBR391; positive control) and (3) two IVET strains with fusions between fragments of DNA with no detectable promoter activity (PBR393, PBR394; negative controls). Results are shown in Fig. 2. Both PBR442 and PBR424 increased in frequency over the course of 2 weeks when inoculated onto seeds at ~2x103 cells per seed, as did the positive control strain PBR391. In contrast, the negative controls failed to increase in frequency. The ability of PBR442 to grow in both the rhizosphere and phyllosphere, but not on minimal M9 medium, demonstrates that the promoter driving expression of ornpl is induced in the plant environment.



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Fig. 2. Induction of the plasmid-derived orn gene in the rhizosphere (filled columns) and shoot (open columns) of sugar beet seedlings. PBR393 and PBR394 are two preselected Lac strains (negative controls) with 'dapB'lacZ genes fused to pQBR103 DNA without detectable promoter activity. PBR391 is an IVET strain (positive control) with a fusion between 'dapB'lacZ and a constitutive promoter. PBR424 and PBR442 are orf6 : : 'dapB'lacZ and ornpl : : 'dapB'lacZ fusion strains, respectively. All fusion strains were grown in direct competition with the wild-type SBW25 (pQBR103). After 14 days, the competitor strain was present at similar levels to the positive control across all treatments (data not shown). Data are means and standard errors from five plants.

 
Comparative and phylogenetic analysis of plasmid-derived orn gene
The deduced amino acid sequence of the plasmid-derived orn (ornpl) gene showed 34 % identity and 51 % similarity with the well-defined E. coli oligoribonuclease, which belongs to the DEDDh subfamily of 3' to 5' exoribonucleases (Zuo & Deutscher, 2001). Sequence alignment of chromosome-derived Orn from P. fluorescens SBW25, P. putida KT2440 (hosts of pQBR103), and Orn from E. coli, Streptomyces and human, whose functions have been investigated biochemically, is shown in Fig. 3. The plasmid-derived Orn possesses four characteristic invariant acidic amino acids distributed in three separate motifs; the fifth highly conserved acidic residue between motifs II and III, and the catalytically important histidine (Fig. 3), suggest that the plasmid-borne orn is a functional gene.



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Fig. 3. Alignment of the deduced amino acid sequence of pQBR103-derived orn (pQBR) with oligoribonuclease of E. coli (Ec) and homologues from Streptomyces griseus (Str), Homo sapiens (Hs), P. fluorescens SBW25 (PF) and P. putida KT2440 (PP). Identical sequences are indicated by an asterisk (*) and amino acid residues of pQBR103-derived Orn matching one and more than one amino acids of other four proteins are shown as a dot (.) and a colon (:), respectively. The three conserved motifs of the 3' to 5' exoribonuclease DEDD family are indicated by boxes (Zuo & Deutscher, 2001). The four highly conserved negatively charged residues (D-E-D-D) within the three motifs and the fifth (D) between motifs II and III, as well as the catalytically important histidine (H), are highlighted in bold.

 
The phylogenetic relationships of plasmid-derived Orn and its representative homologues in prokaryotes, eukaryotes and viruses are shown in Fig. 4. Orn from proteobacteria, which in our analysis included four representatives from the genus Pseudomonas, clustered within a single group, whereas the plasmid-derived Orn formed a separate cluster. The G+C content of the plasmid-derived Orn gene is 53·06 mol%, which is similar to the estimated mean G+C content (53·16 mol%) of plasmid pQBR103 but much lower than that of Pseudomonas chromosomes (58–67 mol%). As shown in Fig. 1(b), the E. coli orn gene (ornE.coli) is flanked by two genes encoding hypothetical proteins (YjeQ and YjeS). This organization is highly conserved among Pseudomonas species (Fig. 1b). However, ornpl is flanked by sequences that have no homologue in the public gene databases. These data suggest that the plasmid-derived Orn gene was acquired by horizontal gene transfer from an unknown organism: the donor appears not to be a member of the Proteobacteria.



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Fig. 4. Phylogenetic tree showing the evolutionary relationships of plasmid-derived Orn and selected homologues representing different origins including P. fluorescens SBW25 (PF), P. putida KT2440 (PP), P. aeruginosa PAO1 (PA) and P syringae DC3000 (PS). Accession numbers of the Orn homologues are listed in Methods. Percentage bootstrap values larger than 50 obtained after 1000 iterations are shown on branches. The scale bar refers to the number of substitutions per site.

 
The plasmid-derived orn gene is functional
Functionality of ornpl could in principle be demonstrated by showing that ornpl was capable of complementing a knockout mutant of the chromosomal orn. We began by constructing an ornpl mutant (pQBR103{Delta}orn, see Methods). Growth of the mutant in the genetic background of SBW25 was assessed on minimal (M9) and complex (LB) media, but no obvious defects were observed. To determine whether a phenotype might manifest in the rhizosphere and phyllosphere, the fitness of SBW25(pQBR103{Delta}orn) relative to SBW25(pQBR103) was determined by competitive colonization assays on sugar beet seedlings. No significant difference was observed between mutant and wild-type in this environment (data not shown).

We next sought to inactivate the chromosomal copy of orn. We chose to do this work in P. putida KT2440 rather than P. fluorescens SBW25 because genome sequence from SBW25 was not available at the time. In addition, KT2440 is a permissive host of pQBR103 and similar plasmids (Lilley et al., 1996). On the basis of the analysis of E. coli orn mutants we expected the ornP.putida mutant to be lethal. To our surprise, insertion of an omega (tetracycline resistance) cassette into the middle of the ornP.putida gene of KT2440 (KT2440 ornP.putida : : {Omega}Tc) was not lethal, but did result in a mutant (designated PBR447) that had an obvious growth defect. This defect was noticeable in LB broth, but more pronounced in minimal M9 medium (Fig. 5), in which PBR447 failed to reach the same cell density as the wild-type strain even when the incubation time was prolonged to 48 h. In LB agar plate culture, PBR447 formed small colonies with a dimpled centre distinguishable from the convex colony formed by the wild-type strain. Interestingly, no such morphological differences were evident on minimal M9 medium agar plates, although colonies of the mutant were significantly smaller (Fig. 5).



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Fig. 5. Growth of P. putida KT2440 ({circ}) and KT2440 (pQBR103) ({bullet}), PBR447 (KT2440ornP.putida : : {Omega}Tc; {triangleup}) and PBR447 (pQBR103) ({blacktriangleup}) in liquid medium of LB (a) and M9 (c). Data are means and standard errors of five replicates. The colonies shown in (b) are typical of P. putida KT2440 (Orn+) and PBR447 (Orn) growing in LB or M9 agar (1·8 %) after 2 days incubation at 28 °C. The two strains were plated in the same Petri dish.

 
Lack of lethality due to inactivation of ornP.putida allowed investigation of the functionality of the plasmid-derived orn gene through genetic complementation. We first asked whether presence of the entire pQBR103 was capable of complementing PBR447. The plasmid was introduced into PBR447 and its ability to restore growth and colony morphology was assessed. Fig. 5 shows that pQBR103 failed to complement the lesion. This result was not surprising and is consistent with our knowledge that ornpl is not active on minimal medium.

Since the factor(s) that induces ornpl expression in the plant environment is not known, the putative coding region of ornpl was cloned (by PCR) into the broad-host-range plasmid pME6010, placing ornpl under the control of a constitutive kanamycin-resistance promoter (Pk). An artificial RBS was incorporated into the forward primer to ensure efficient translation of ornpl; a spectinomycin-resistant omega cassette was inserted downstream of ornpl to ensure transcriptional termination and to provide a selectable marker for plasmid conjugation. In addition (and in parallel) the coding region of ornp.putida was cloned into pME6010. The two plasmids – one carrying ornpl (pME6010 : : ornpl{Omega}) and the other ornP.putida (pME6010 : : ornP.putida{Omega}) – were independently introduced into the ornP.putida mutant PBR447 and growth of the transconjugants was compared in M9 broth. The growth defect of the ornP.putida mutant was fully restored upon introduction of the chromosomal copy of orn (pME6010 : : ornP.putida{Omega}) (Fig. 6a). This demonstrates that the slow growth of PBR447 was caused by the ornP.putida mutation. The plasmid-derived orn gene (pME6010 : : ornpl{Omega}) was also capable of complementing the defect in PBR447, but not to the same extent as ornP.putida. Partial complementation of PBR447 by the plasmid-derived copy of ornpl is not unexpected given the divergent phylogenetic origins of this gene, but nonetheless, the ability of this gene to increase growth of PBR447 provides evidence of its functionality.



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Fig. 6. Growth of P. putida PBR447 (KT2440ornP.putida : : {Omega}Tc) carrying orn genes from pQBR103 (a) and E. coli (b) and a schematic map (c) showing the construct used to express orn. (a, b) All strains were grown in minimal M9 medium: KT2440 ({circ}); PBR447 containing pME6010 : : ornP.putida{Omega} ({square}); PBR447 containing pME6010 : : ornpl{Omega} ({bullet}); PBR447 containing pME6010 : : ornE.coli{Omega} ({blacktriangleup}); PBR447 containing the vector pME6010{Omega} ({triangleup}). Data are means and standard errors of five repeats (error bars lie within the symbols). (c) The filled bar is the multi-cloning site of pME6010 although only the restriction sites used in this study are shown (X, XhoI; K, KpnI; H, HindIII). The orn gene was expressed under the control of a constitutive kanamycin resistance promoter (Pk).

 
Outside the context of E. coli, orn has received relatively little attention. There are no functional studies of orn from Pseudomonas, but evidence that orn from the chromosome of KT2440 is an oligoribonuclease could be obtained by showing that the well-characterized oligoribonuclease gene from E. coli (ornE.coli) is able to complement the defect in PBR447. The ornE.coli gene from E. coli DH5{alpha} was amplified by PCR and cloned into pME6010 exactly as described above. Plasmid pME6010 : : ornE.coli{Omega} was then introduced into the ornP.putida mutant PBR447 and its ability to restore growth of PBR447 was examined. PBR447 (pME6010 : : ornE.coli{Omega}) grew as quickly as the wild-type strain KT2440, showing that the E. coli-derived orn gene can fully complement the ornP.putida mutant. Together these data provide strong support for the claim that both ornP.putida and ornpl are functional oligoribonucleases.

The orn gene is not required for conjugative transfer of pQBR103
The yeast mitochondrial genome harbours an orn gene that has been implicated in escape of mitochondrial DNA to the nucleus (Hanekamp & Thorsness, 1999). This led us to consider the possibility that orn might be associated with conjugative transfer. To determine whether pQBR103{Delta}ornpl is deficient in conjugation, its ability to transfer to wild-type P. putida KT2440 and the ornP.putida mutant of KT2440(PBR447) was assessed. Both pQBR103 and pQBR103{Delta}ornpl were able to transfer at a similar frequency (1·06x10–6–1·6x10–7, transconjugants per recipient) from P. fluorescens SBW25 (donor) to KT2440 (recipient). We were similarly able to obtain transconjugants from matings between SBW25(pQBR103{Delta}ornpl) and PBR447.

Distribution of ornpl among indigenous Pseudomonas plasmids
To determine whether the ornpl gene is widely distributed among indigenous plasmids occurring at the same field site, 12 plasmids representing four different plasmid groups (Lilley & Bailey, 1997a) were selected for a PCR assay of the ornpl gene. Primers were designed to amplify the 620 bp coding region of ornpl and the results are shown in Table 1. The orn gene was found in five plasmids, including pQBR103, which have molecular sizes ranging from 287 kb to 425 kb. The five PCR-amplified ornpl genes were subsequently sequenced and shown to be identical at the DNA level.

The five plasmids carrying orn are all members of the dominant group I plasmids (Table 1), which were defined originally on the basis of restriction fragment patterns (Lilley et al., 1996). Sequence identity of the orn genes suggested the possibility that the variation among these plasmids is primarily the result of spontaneous recombination events. To this end we sequenced 300 bp from the plasmid replicative origin (oriV) of the five plasmids with identical orn sequences as accomplished previously for pQBR11 (Viegas et al., 1997). Sequence results showed that the oriV regions of the five plasmids are identical. Together, these data suggest that the plasmid-derived orn genes were acquired by a single horizontal gene transfer event.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Functional analyses of plasmids from natural environments pose special challenges. Plasmid pQBR103 is representative of a large group of conjugative plasmids present in plant-colonizing Pseudomonas species. pQBR103 was isolated by its ability to confer resistance to mercuric chloride on recipient bacteria, but other than this marker, the functional traits encoded by the plasmid were unknown (Bailey et al., 2001).

Here we have described the identification and functional characterization of a novel oligoribonuclease gene carried on pQBR103. Plasmid-encoded orn (ornpl) was initially isolated using a promoter-trapping strategy that allowed recovery of pQBR103 genes that were transcriptionally active on sugar beet plants, but inactive on minimal M9 medium (unpublished). While ornpl was not directly fused to the gene trapped by the promoterless 'dapB reporter, fusions between ornpl and a promoterless copy of 'dapB demonstrate that the gene is plant inducible.

Evidence that ornpl encodes a functional oligoribonuclease was initially inferred from in silico analyses of the predicted protein. Subsequent experiments showed that ornpl could partially complement an orn mutant (ornP.putida) of P. putida KT2440. At the same time we showed that the orn mutant (ornP.putida) of P. putida KT2440 could be complemented by the orn gene from E. coli. Together these data provide compelling evidence that ornpl is functional.

Oligoribonucleases have received little attention. Nevertheless, studies to date show that Orn is a 3' to 5' exoribonuclease and an important component of the mRNA degradation pathway in bacteria (Ghosh & Deutscher, 1999; Ohnishi et al., 2000); a similar role has been suggested for the conserved human homologue (Nguyen et al., 2000). In E. coli, Orn is one of eight known exoribonucleases and the only one known to be essential for cell viability (Zuo & Deutscher, 2001). Inactivation of the orn gene results in accumulation of small oligoribonucleotides (2–5 residues in length), which are thought to be toxic to E. coli cells (Ghosh & Deutscher, 1999).

Little is known about mRNA degradation in the genus Pseudomonas. However, given the phylogenetic closeness of E. coli and Pseudomonas (both members of the gamma subdivision of the Proteobacteria) and similarity of Orn from E. coli and Pseudomonas (67 % identical), we were surprised to find that inactivation of ornP.putida was not lethal. The growth defect we observed was also noted in an orn mutant of Streptomyces griseus (Ohnishi et al., 2000).

The reason for the difference between phenotypes of orn mutants in E. coli relative to P. putida is unclear. One possibility is that in P. putida there is functional redundancy among oligoribonucleases, but analysis of the KT2440 genome sequence did not reveal obvious Orn homologues. A further possibility is that mRNA degradation pathways are different in the two bacteria. For instance, E. coli contains a gene encoding RNase II, which is a principal 3' to 5' hydrolytic exoribonuclease, but this is absent in Pseudomonas. Further evidence of differences between Pseudomonas and E. coli comes from a recent study of another key exoribonuclease PNPase (polynucleotide phosphorylase) in P. putida (Favaro & Deho, 2003), which when mutated did not exhibit cold sensitivity as expected from parallel studies in E. coli.

The precise role of ornpl and the reason for its presence in the group I Pseudomonas plasmids from Wytham is unclear. The selective forces operating on plasmids, combined with the evidence presented here of functionality and plant-inducibility, suggest that ornpl is likely to have ecological relevance. Precisely what this might be remains unclear, but it is possible that Ornpl functions in a role other than mRNA degradation, a suggestion that has some credibility given the demonstration that the human Orn homologue, Sfn (small fragment nuclease), is capable of hydrolysing both RNA and DNA oligomers (Nguyen et al., 2000).

Finally, our discovery that identical copies of ornpl are present in a subset of group I Pseudomonas plasmids from Wytham and yet share little in common with chromosomal orn from {gamma}-Proteobacteria indicates that ornpl is of foreign origin. The complete lack of allelic diversity in ornpl suggests that ornpl was acquired just once before dissemination by lateral gene transfer. The lack of diversity suggests that the transfer event was recent, but in the absence of complete sequence analysis of these plasmids it is not possible to exclude the possibility that intragenetic recombination combined with plasmid instability generates the restriction fragment differences previously reported among the five ornpl-containing plasmids (Lilley et al., 1996). Indeed, the corresponding lack of allelic diversity in putative oriV from the same five plasmids suggests a single plasmid type capable of converting between different semi-stable states.

Our data do not permit us to identify the ancestral organism from which ornpl originated, although it seems certain that ornpl does not originate from the Proteobacteria. However, even with the usual caveats associated with long-branch attraction (Baxevanis & Ouellette, 2001), our data do suggest that ornpl originates from a eukaryotic source. This is of some interest because it is consistent with data that suggest that orn first evolved in eukaryotes and was then transferred to eubacteria (but not the archaea) by lateral gene transfer (Zuo & Deutscher, 2001). Perhaps ornpl reflects an additional dissemination event from a eukaryote.


   ACKNOWLEDGEMENTS
 
We thank Andrew Spiers, Sarah Turner and Robert Jackson for helpful discussions and Lena Ciric for help with DNA sequencing. This work was supported by NERC (UK).


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Received 16 April 2004; revised 4 June 2004; accepted 9 June 2004.



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