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Applied and Environmental Microbiology, January 2000, p. 54-63, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequence Analysis of the Cryptic Plasmid pMG101 from Rhodopseudomonas palustris and Construction of Stable Cloning Vectors

Masayuki Inui, Jung Hyeob Roh, Kenneth Zahn, and Hideaki Yukawa*

Research Institute of Innovative Technology for the Earth, Soraku, Kyoto 619-0292, Japan

Received 30 August 1999/Accepted 22 October 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha -proteobacteria.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Purple nonsulfur bacteria (PNSB) are an assemblage of phenotypically diverse species. Under anaerobic conditions in the light, all species grow photoheterotrophically when supplied with various organic substrates or photoautotrophically with CO2 as a sole carbon source. Under microaerobic to aerobic conditions in the dark, many representatives can grow chemoheterotrophically, and some grow chemoautotrophically (40).

To develop a new CO2-fixing bioprocess, we have been performing biochemical and genetic analyses of intermediary metabolism, including CO2 fixation, underlying the complex modes of growth in the PNSB, using Rhodopseudomonas palustris as a model microorganism (4, 23, 24). For this purpose, development of a versatile host-vector system would be helpful.

In R. palustris and other PNSB, broad-host-range vectors have been used to provide the tools for gene transfer. The most widely used vectors are derivatives of RK2 such as pRK415 (25) and pLAFR1 (14). Cloning vector pRK415 has been utilized for genetic analyses of several R. palustris genes (8, 17), and cosmid vector pLAFR1 has been used to make a library of R. palustris DNA (8). However, these plasmids were unstable in R. palustris under nonselective conditions (M. Inui, unpublished data), as also observed in R. sphaeroides (9) and in Rhodospirillum rubrum (42).

Other vectors derived from the broad-host-range plasmid RSF1010 such as pDSK519 (25) can also replicate in PNSB, including R. palustris (Inui, unpublished data). But this vector was also unstable under nonselective conditions in R. palustris and R. sphaeroides (Inui, unpublished data). Thus, these broad-host-range plasmids show segregational instability in R. palustris as well as in other PNSB.

R. palustris species in general contain no endogeneous plasmids, but it has been reported that several other PNSB may contain one or more such plasmids. R. sphaeroides may carry up to six cryptic plasmids ranging in size from 42 to 140 kb (13). Rhodobacter capsulatus strains also have either one or two plasmids, with a size range similar to that found in R. sphaeroides (21, 57). All strains of R. rubrum tested appear to include a single 55-kb plasmid (30). So far there has been no report on use of these large endogenous plasmids to construct vectors for PNSB.

On the other hand, shuttle vectors for the marine photosynthetic bacterium Rhodobacter marinus have been constructed by connecting an endogenous plasmid to an Escherichia coli cloning vector, because broad-host-range vectors like those mentioned above are ineffective in this organism (5, 32, 33). These plasmids have not been demonstrated to function in other PNSB.

To establish a versatile vector system to facilitate genetic analysis in R. palustris, we have screened PNSB for endogenous plasmids of relatively small size. Here we report on the isolation, replication range, and stability of plasmid pMG101 and on sequence analysis of the replication region. Using this information, we have been able to construct stable R. palustris-E. coli shuttle vectors.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are listed in Table 1.

                              
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  		TABLE 1.   Bacterial strains and plasmids used in this study

Culture conditions. E. coli strains were grown aerobically at 37°C in Luria-Bertani medium (44). PNSB were cultivated aerobically at 30°C in van Niel's complex medium (53). Rhizobium and Bradyrhizobium species were grown aerobically at 30°C in 805 medium (1 g of yeast extract, 5 g of mannitol, 0.7 g of K2HPO4, 0.1 g of KH2PO4, and 1 g of MgSO4 · 7H2O per liter, adjusted pH 7.0 to 7.2). When appropriate, media were supplemented with antibiotics. For E. coli, ampicillin and kanamycin were each used at a final concentration of 50 µg ml-1; for R. palustris, the final concentration of kanamycin was 200 µg ml-1; for other PNSB and Rhizobium and Bradyrhizobium species, the final concentration of kanamycin was 50 µg ml-1.

DNA manipulations. Plasmid DNA was isolated by the alkaline lysis procedure (44). Restriction endonucleases and T4 ligase were obtained from Takara and used according to the manufacturer's instructions. E. coli strains were transformed by the CaCl2 method (44). PNSB, Rhizobium, and Bradyrhizobium species were transformed by electroporation (9), with the following modification to obtain optimum transformation efficiency. A Bio-Rad Gene Pulser apparatus was used, with a pulse controller and 0.1-cm-gap electroporation cuvette. A resistance of 200 ohms on the pulse controller and settings of 1.75 kV and 25 µF were used. These settings generate a field strength of 17.5 kV/cm. Optimal electroporation frequencies are obtained with one pulse per sample. With this method, we routinely achieve approximately 5 × 105 to 10 × 105 drug-resistant cells of R. palustris per µg of plasmid DNA. Restriction fragments were isolated, when required, from agarose gels (1%, wt/vol) with a Prep-a-Gene matrix (Bio-Rad, Richmond, Calif.) according to the manufacturer's instructions.

Isolation of an endogenous plasmid from PNSB. As elsewhere reported (15), PNSB were isolated from enrichment cultures under anaerobic light conditions, using 1-propanol as a carbon source. Isolates were aerobically grown, and plasmid DNA was isolated as described above.

DNA sequencing. To determine the complete nucleotide sequence of the replicator region of plasmid pMG101, overlapping fragments were subcloned in pUC118, and DNA was sequenced on both strands in an automated 373A DNA sequencer (Applied Biosystems/Perkin-Elmer, Foster City, Calif.). DNA sequence data were analyzed with the INHERIT (Applied Biosystems/Perkin-Elmer) and Genetyx (Software Development, Tokyo, Japan) programs.

Isolation and analysis of 16S rDNA. Two oligonucleotide primers, fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and rD1 (5'-AAGGAGGTGATCCAGCC-3'), were used to PCR amplify nearly full-length 16S ribosomal DNA (rDNA) (55). DNA sequences of PCR-amplified 16S rDNA from strains S55, USDA 4362 (BTAi1), and USDA 4377 were determined as described above. The rDNA sequences of strain S55 and the reference strains covering a total of 1,229 nucleotide positions were aligned with the E. coli sequence by using the CLUSTAL V program (20). A phylogeny was reconstructed by the neighbor-joining method (43) from Knuc values (27). Variable domains and unidentified base positions were not taken into consideration in the analysis.

Segregational plasmid stability. R. palustris cells containing pMG101 derivatives were first grown in selective liquid medium (van Niel's medium containing kanamycin). Cells in the late exponential growth phase were diluted 1,000-fold in van Niel's medium without the antibiotic and grown at 35°C. At 2-day intervals, cultures were diluted 1,000-fold. At each dilution step, cell density was measured to estimate the number of generations, and the cells were plated on nonselective plates. From these plates, 200 colonies each were transferred to selective and nonselective plates to determine the frequency of plasmid loss based on the percentage of kanamycin-sensitive colonies. Similarly, plasmid stability in Bradyrhizobium japonicum and phototrophic Bradyrhizobium species was analyzed after growth in 805 medium with or without kanamycin.

RNA isolation and primer extension. Template RNA was extracted from an R. palustris No. 7 culture in late exponential phase as previously described (23). Primers were synthesized with 5'-end positions 300, 320, and 354 for the parA gene and 1390, 1410, and 1526 for the repA gene, complementary to the mRNA sequence. Primer extension was performed as described elsewhere (23).

Construction of E. coli-Rhodopseudomonas shuttle cloning vectors. Plasmid pHSG298X was made by PCR amplification of plasmid pHSG298, using primers containing an XhoI site. The primers P1 (5'-CGCTCGAGGGAGCCACGGTTGA-3') and P2 (5'-CGCTCGAGCAACACCTTCTTCACGA-3') were used, and the PCR product was ligated to itself and transformed into E. coli JM109. The resulting plasmid has a unique XhoI site between the pHSG298 origin and kanamycin resistance gene. Plasmid pMG103 was constructed by cloning a 3.0-kb SalI-XhoI fragment from plasmid pMG101 into the XhoI site of plasmid pHSG298X. For the construction of plasmid pMG105, a 3.8-kb ApaLI-ClaI fragment containing the pMG101 origin from plasmid pMG103 was ligated with the 1.8-kb ApaLI-ClaI fragment containing the beta -galactosidase (lacZ) gene, which has the opposite orientation of the polylinker site versus that of plasmid pMG103.

Nucleotide sequence accession numbers. The DDBJ/EMBL/GenBank accession numbers for the sequences reported in this paper are AB031076 (the SalI-XhoI 3.0-kb fragment of pMG101), D84187 (rDNA for strain S55), D86354 (rDNA for USDA 4362), D86355 (rDNA for USDA 4377), AB031077 (plasmid pMG103), and AB031078 (plasmid pMG105).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Isolation of plasmid pMG101 and characteristics of the host strain. To develop a vector system for PNSB, we screened various strains for small (<20-kb) plasmids. Among 400 strains isolated from sewage, rice fields, and other places, one strain (S55) contained an endogenous plasmid about 15 kb in size. This plasmid was designated pMG101. The loss of pMG101 from strain S55 did not induce any detectable phenotypic difference (data not shown).

Strain S55 was gram negative and nonsporulating, and it formed rods with rounded ends. Cell multiplication occurred by budding. Cultures were red to brown-red under anaerobic light conditions and pale pink to white under aerobic dark conditions. The in vivo spectrum of cell suspensions grown anaerobically in the light exhibited absorption maxima at 375, 590, 805, and 860 nm, which is characteristic for bacteriochlorophyll a, and at 465, 498, and 530 nm, indicating the presence of carotenoids of the normal spirilloxanthin series (40). These results indicate that strain S55 is a species of the genus Rhodopseudomonas.

To confirm the phylogenetic position of strain S55, we determined the rDNA sequence of 1,481 nucleotides of strain S55. The phylogenetic position of strain S55, as well as that of two species of phototrophic Bradyrhizobium, was analyzed with data for sequences longer than those used for previous analyses of these strains (60, 62) (Fig. 1). Strain S55 formed a tight cluster with R. palustris, B. japonicum, and phototrophic Bradyrhizobium species and was less similar to other PNSB and Rhizobium species. A bootstrap analysis confirmed the monophyly of the strain S55 cluster in 100% of trees generated (11). These results confirmed that strain S55 is a strain of R. palustris and is phylogenetically close to B. japonicum and phototrophic Bradyrhizobium species. Our data are in agreement with previous reports that R. palustris, phototrophic Bradyrhizobium species which produce bacteriochlorophyll (12), and Bradyrhizobium japonicum show high sequence similarity and form a branch on 16S rRNA phylogenetic trees (60, 62).


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  		FIG. 1.   Unrooted distance matrix tree showing phylogenetic relationships based on 16S rDNA sequences of selected strains of proteobacteria. The topology of the phylogenetic tree was evaluated by bootstrap analysis with 1,000 replicates. Numbers indicate percentage bootstrap values of 1,000 replicates. USDA 4362 and USDA 4377 are phototrophic Bradyrhizobium species. Abbreviations where relevant and, in parentheses, accession numbers: strain S55 (D84187), R. palustris, Rhodopseudomonas palustris (D25312); B. japonicum, Bradyrhizobium japonicum (D11345); R. roseus, Rhodoplanes roseus (D25313); R. acidophila, Rhodopseudomonas acidophila (M34128); R. viridis, Rhodopseudomonas viridis (D25314); R. rubrum, Rhodospirillum rubrum (D30778); R. sphaeroides, Rhodobacter sphaeroides (D16425); R. capsulatus, Rhodobacter capsulatus (D16428); R. salexigenes, Rhodospirillum salexigenes (M59070); R. tennis, Rhodocyclus tennis (D16208); R. purpureus, Rhodocyclus purpureus (M34132); R. gelatinosus, Rhodocyclus gelatinosus (D16214); E. coli, Escherichia coli (V00348); R. etli, Rhizobium etli (U28916); R. leguminosarum, Rhizobium leguminosarum (U73208).

Replication range and stability of plasmid pMG101. Plasmid pMG101 is the first plasmid to be identified in R. palustris and is smaller than known endogenous plasmids in other PNSB. Restriction mapping of pMG101 was carried out (Fig. 2), and the shuttle vector pMG101Km was constructed. pMG101Km can replicate in R. palustris (strain No. 7 [15], ATCC 17001, and ATCC 17000), B. japonicum ATCC 10324, and phototrophic Bradyrhizobium species (USDA 4362 and USDA 4377). It cannot replicate in R. sphaeroides ATCC 17023 or in nonphototorophic Rhizobium species, R. etli IFO 15573 and R. leguminosarum IFO 14778. Species-specific replication of pMG101Km clearly reflects the phylogenetic relationships of the Rhodopseudomonas group (Fig. 1).


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  		FIG. 2.   Localization of the replication and stability regions. Construction of pMG101 derivatives is described in Table 1. The heavy horizontal lines indicate regions of plasmid pMG101 that were inserted into pHSG298. Replication was considered positive (+) when transformants were present and plasmid DNA was detected after alkaline lysis and gel electrophoresis and negative (-) when no transformants were obtained in three experiments. Plasmid stability in R. palustris was analyzed as described in Materials and Methods.

To determine the stability of plasmid pMG101, R. palustris, B. japonicum, and phototrophic Bradyrhizobium species cultures containing pMG101Km were grown for 30 generations under nonselective conditions. The plasmid was stably maintained in R. palustris (100% maintenance), less stable in phototrophic Bradyrhizobium species (50% maintenance), and least stable in B. japonicum (0% maintenance). However, pMG101Km was stably maintained in all of these hosts when kanamycin was added. Plasmid replication is thought to be determined by the interaction of host- and plasmid-encoded factors with the replication region of the plasmid (54). The differences in stability might reflect differences in host factors in each strain.

Localization of the replication and stability region. To identify the replication and stability region of pMG101, we constructed a series of pMG101 derivatives by subcloning restriction fragments into plasmid pHSG298 (Fig. 2), which contains a kanamycin resistance gene, and these plasmids were transformed into R. palustris. Plasmid pHSG298, which contains a ColE1 origin, was not able to replicate in R. palustris (data not shown). The plasmid pMG101 derivatives pMG101A, pMG101C, pMG101E, pMG101G, and pMG102 could replicate in R. palustris. However, when pMG101B, pMG101D, and pMG101F were transformed in R. palustris, no kanamycin-resistant colonies were obtained.

To determine the location of the stability region of pMG101, R. palustris cells harboring the pMG101 derivatives were grown for 50 generations without selection, and retention of kanamycin resistance was evaluated by transferring at least 200 isolates to selective and nonselective media (Fig. 2). Plasmids pMG101A, pMG101C, and pMG102 were stably maintained in R. palustris; in contrast, plasmids pMG101E and pMG101G were gradually lost, and the percentage of plasmid-containing cells decreased to 10 to 11% after 50 generations. Moreover, no plasmid loss was observed in R. palustris containing pMG102 for over 100 generations without selective pressure (data not shown). The 3.0-kb SalI-XhoI fragment therefore appears to include both replication and stability functions of pMG101, and digestion at a PstI restriction site within this fragment destabilizes the plasmid.

DNA sequence of the 3.0-kb SalI-XhoI fragment of pMG101. To clarify the plasmid partitioning and replication functions of pMG101, the complete nucleotide sequence of the 3.0-kb SalI-XhoI fragment was determined. The G+C content of this fragment was 57.9%, which is below the range (from 64.8 to 66.3%) previously reported for the genome of R. palustris (22). Computer analysis revealed the presence of two potential open reading frames (ORFs), ORF1 and ORF2.

ORF1 (positions 236 to 889) consists of 654 nucleotides, corresponding to 217 amino acids and a predicted molecular weight of 22,450. The deduced amino acid sequence shows weak but significant sequence similarities to sequences of the parA gene products of plasmid pTAR from Agrobacterium tumefaciens (28.5%) (16 [accession no. X05121]), pFAJ2600 from Rhodococcus erythropolis (27.7%) (7 [accession no. AF015088]), and a putative Par-like ORF from the genome of Helicobacter pylori (25.8%) (50 [accession no. AE000608]). ORF1 was therefore designated the parA gene. The result of the best alignment with ParA proteins from pMG101 and pTAR is shown in Fig. 3A. The motif close to the N-terminal region of A-type Sop/Par partitioning proteins (56) constitutes a modified nucleoside triphosphate (NTP)-binding motif, KGGXXK(S/T) (29), which is also present in the ParA protein of pMG101. However, comparative amino acid sequence alignment shows that other conserved regions are not found among these four ParA proteins (data not shown). Upstream of the pMG101 parA gene, there are nine 8-bp imperfect direct repeats (DR I) and a pair of 6-bp perfect and a pair of 10-bp imperfect inverted repeats (IR) at positions of 124 to 229 (Fig. 4A and 5A). The first and second direct repeats overlap with the first and second IRs, respectively. The third to eighth direct repeats, which are separated by 8 bp from the second direct repeat, are clustered together without spacers, and the ninth direct repeat exists 7-bp upstream of the proposed parA initiation codon.


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  		FIG. 3.   Sequence alignments of the putative ParA and RepA proteins. (A) Comparison of amino acid sequences of ParA proteins from plasmids pMG101 and pTAR. A modified NTP-binding motif is indicated by a plus sign above the sequence. (B) Comparison of amino acid sequences of RepA proteins from plasmids pMG101 and pRM21. Identical residues are marked with asterisks below the sequence alignments.


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  		FIG. 4.   Structural and transcriptional analysis of the pMG101 parA and the repA genes. (A) Sequence features around and including the parA mRNA 5' end. The nine 8-bp imperfect direct repeats (DR I) are shown by boxes. Two pairs of IRs are indicated by rightward and leftward arrows below the sequence. Regions at the canonical promoter distances of -35 and -10 are indicated by bold underlining. A rightward arrow marked +1 shows the start point of the parA transcript. Single-letter amino acid codes for the carboxyl terminus of a possible ORF and the amino terminus of the ParA protein are presented below the sequence. (B) Sequence features around and including the repA mRNA 5' end. The partial DR II and partial DR III are shown by boxes. Putative -35 and -10 promoter sequences are indicated by bold underlining. A rightward arrow marked +1 shows the start point of the repA transcript. The first base of the start codon is indicated at position +172, and the single-letter amino acid code for the amino-terminal residues of the RepA protein is presented below the sequence. (C) Sequence features around and downstream from the carboxyl terminus of the repA gene product. The single-letter amino acid code for the carboxyl terminus of the RepA protein is shown below the sequence. DR II and DR III sequences are indicated by boxes. AT- and a GC-rich regions are presented by dashed and double underlines, respectively.


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  		FIG. 5.   Sequence alignments of the nine 8-bp direct repeats (DR I) (A), the six 17-bp imperfect direct repeats (DR II) and a partial homologous sequence (B), and the five 7-bp imperfect direct repeats (DR III) and a partial homologous sequence (C). The consensus sequences are shown below the sequence alignments. Two pairs of IRs, which are overlapped with DRs, are noted by rightward and leftward arrows above DR sequences in panel A.

ORF2 is present downstream of the putative parA gene (positions 1473 to 2369), transcribed in the same direction, and composed of 897 nucleotides, corresponding to 298 amino acids with an estimated molecular weight of 33,844. The deduced amino acid sequence indicates weak but significant sequence similarities to sequences of the putative repA gene products of plasmid pRM21 from Rhodothermus marinus (26.3%) (accession no. U10426), pCL1 from Chlorobium limicola (25.2%) (accession no. U77780), and pSa from Shigella flexneri (23.3%) (39 [accession no. U30471]). ORF2 was therefore designated the repA gene. The best alignment with RepA proteins from pMG101 and pRM21 is shown in Fig. 3B. However, among the four RepA proteins, specific conserved regions were not observed (data not shown). Downstream of the putative repA gene, six 17-bp imperfect direct repeats (DR II) and five 7-bp imperfect direct repeats (DR III) were detected at positions 2392 to 2643 (Fig. 4C, 5B, and 5C).

Downstream of the direct repeat region is a relatively AT-rich (65.7%) sequence between positions 2611 and 2680, and downstream of the AT-rich segment is found a GC-rich (69.2%) sequence between positions 2681 and 2732 (Fig. 4C).

Transcriptional analysis of parA and repA genes. We thought that an understanding of the transcription patterns within the minimal replicon would aid in efforts to understand replication control and also to genetically engineer the plasmid for use as a vector. To localize the parA and repA promoters, the transcriptional start sites of the parA and repA genes were determined by using primer extension with three 20-mer oligonucleotides having different 5'-end positions (see Materials and Methods).

Primer extension analysis of the parA gene revealed one major extension product with all three primers corresponding to the A of the AUG start codon of the parA gene. The result with the primer at 320 is shown in Fig. 6A. Putative -10 and -35 promoter sequences, similar to the E. coli sigma 70 promoter consensus (19) separated by a typical 17-nucleotide interval, were identified upstream of the transcriptional start point, and the putative promoter sequence is overlapped with DR I sequences (Fig. 4A).


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  		FIG. 6.   Mapping of the transcriptional start sites of the parA (A) and repA (B) genes by primer extension analysis. High-resolution electrophoresis of 5'-labeled cDNA products are shown aligned with a sequencing ladder generated from DNA from the same region and with the same primer. Dideoxy sequencing marker lanes are indicated with CTAG, and cDNA synthesis from the RNA samples is shown in the next lane (PE). Migration positions of the primer extension products are indicated with arrows. Portions of the DNA sequence are indicated at the left; asterisks indicate the putative transcriptional start site.

Reverse transcription of the repA mRNA yielded two major bands with all three primers, corresponding to 5' ends at 172 and 173 bases upstream of the translation initiation codon. The result with the primer at 1390 is shown in Fig. 6B. Upstream of the transcriptional start site, putative -10 and -35 promoter sequences, separated by a typical 17-nucleotide interval, were observed, and a partial copy of DR II at positions 1275 to 1280 was found between the -10 and the -35 promoter sequences (Fig. 4B and 5B). In addition, a partial copy of DR III at positions 1429 to 1435 also occurs between the promoter sequence and the translation initiation codon (Fig. 4B and 5C).

Deletion mapping and gene disruption analysis. To define more precisely the replication and stability functions of pMG101, various fragments were amplified by PCR, cloned into the E. coli vector pHSG298, and then tested for the ability to promote plasmid replication and stability in R. palustris (Fig. 7). Plasmid pMG102 was used as a positive control in these experiments.


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  		FIG. 7.   Deletion mapping and gene disruption analysis of the 3.0-kb SalI-XhoI fragment containing the pMG101 replication and stability region. The restriction cleavage map of this fragment is shown at the top. Arrows depict the parA and repA genes and the C-terminal region of a possible ORF. DR I, DR II, and DR III are indicated by open leftward, closed leftward, and open rightward arrowheads, respectively. The partial DR II and DR III are represent by DR II* (shaded leftward arrowhead) and DR III* (dotted rightward arrowhead). Putative -10 and -35 promoter sequences of the parA and repA genes are shown by small closed boxes. AT- and GC-rich sequences are indicated by shaded and bold underlines, respectively. The scale is graduated in base pairs from the left (SalI site) to the right (XhoI site), corresponding to the nucleotide sequence of the 3.0-kb SalI-XhoI fragment. pMG102 deletion and frameshift mutant derivatives are listed at the left; each fragment is represented by a horizontal bold line. Deletion positions in base pairs are shown at the end of each horizontal bold line. Frameshift positions are indicated by downward arrowheads. Construction of these plasmids is described in Table 1. Assay of replication in R. palustris was as described in the legend to Fig. 2. ± indicates that transformation efficiency is low and that it takes a longer incubation time to obtain transformants than is the case for the control plasmid pMG102. Plasmid stability in R. palustris was evaluated as described in Materials and Methods.

Plasmid pMG102-1, containing the 2,954-bp fragment (positions 100 to 3053) which lacked a possible upstream ORF within the 3.0-kb SalI-XhoI fragment, can replicate and be maintained in R. palustris as well as a control plasmid, pMG102. Plasmid pMG102-2, containing the 2,844-bp fragment (positions 210 to 3053) which lacked eight of nine DR I sequences and a putative -35 promoter sequence of the parA gene, was able to replicate but was not maintained (25% plasmid maintenance) in R. palustris growing under nonselective conditions (Fig. 4 and 7). It is likely that DR I sequences and a putative parA promoter region are required for pMG102 stability. In addition, the parA product is likely to be essential for pMG102 stability, because a frameshift mutation created by insertion of 4 bp into the MunI site within the parA sequence in plasmid pMG102-10 resulted in instability in R. palustris under nonselective growth conditions (Fig. 7).

Plasmid pMG102-3, containing the 2,054-bp fragment (positions 1000 to 3053), can replicate in R. palustris, but plasmid pMG102-4, which contains the 1,954-bp fragment (positions 1100 to 3053), was not able to replicate, suggesting that the 100-bp sequence at positions 1000 to 1100 is essential for pMG102 replication. However, DNA sequence analysis did not detect any known sequence motifs, specific DNA structures, or homologous nucleotide or amino acid coding sequences in this region.

Analysis of the deletion mutant plasmids pMG102-5, pMG102-6, and pMG102-7 indicated that a region containing six DR II, four DR III, and an AT-rich sequence was sufficient for pMG102 replication (Fig. 7). Plasmid pMG102-9, which included all DR II, DR III, AT-rich, and GC-rich sequences, replicated and was maintained stably in R. palustris as well as a control plasmid, pMG102. Plasmid pMG102-8, containing the 2,700-bp fragment (positions 1 to 2700), which lacked a part of the GC-rich sequence and downstream sequences from the 3.0-kb SalI-XhoI fragment, can replicate in R. palustris, but transformation efficiency is 1,000 times lower and an incubation time of 5 days is required to obtain transformants, compared with 2 days for the pMG102 control. Moreover, plasmid pMG102-8 showed segregational instability in R. palustris under nonselective growth conditions. These data suggest that the GC-rich sequence plays an important role in the replication or stability of plasmid pMG102. Furthermore, a frameshift mutation created by insertion of 2 bp into the NspV site in the repA sequence in plasmid pMG102-11 blocked replication in R. palustris, suggesting that the repA gene product is essential for pMG102 replication (Fig. 7).

Construction of E. coli-R. palustris shuttle cloning vectors. To further improve the versatility of the E. coli-R. palustris shuttle cloning vector, plasmids pMG103 and pMG105, which are 5,680 bp in size and contain a kanamycin resistance marker, were constructed as described in Materials and Methods. The shuttle vectors have a polylinker containing unique EcoRI, SacI, KpnI, BamHI, SalI, XbaI, Sse8387I, and SphI sites, and insertional inactivation of the lacZ gene can be used to identify cloned inserts by blue/white colony screening in the E. coli host strain JM109 when plated on isopropyl-beta -D-thiogalactopyranoside - 5 - bromo - 4 - chloro - 3 - indolyl - beta -D-galactopyranoside (IPTG-X-Gal) agar plates (Fig. 8). Both of these plasmids were stably maintained in R. palustris for over 100 generations without selective pressure.


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  		FIG. 8.   Physical and genetic maps of pMG103 and pMG105. Notation: lacZ, beta -galactosidase alpha -peptide gene from pHSG298 or pHSG299; Kmr, kanamycin resistance gene; pMG101 ori, the 3.0-kb SalI-XhoI fragment for pMG101 origin of replication, which includes parA and repA genes; pHSG298 ori and pHSG299 ori, origins of replication from pHSG298 and pHSG299, respectively. Arrows indicate direction of transcription. Unique restriction sites are shown.

To estimate the copy number of pMG103 per chromosome of R. palustris, we constructed plasmid pMG103-pckA, containing the R. palustris No. 7 pckA gene. This gene is present in only a single copy on the chromosome (24). Southern blotting of R. palustris total DNA harboring plasmid pMG103-pckA, digested with SmaI, BamHI, and PstI, was performed. Hybridization with the pckA gene probe detected a 2.5-kb SmaI-PstI fragment representing the pckA gene encoded on the R. palustris No. 7 chromosome and a 2.2-kb BamHI-PstI fragment representing the pckA gene from plasmid pMG103-pckA. The ratio of two bands was evaluated from densitometry of the relative intensities of the hybridization signals (data not shown), and pMG103 copy number was estimated to be between three to five per R. palustris chromosome.

In previous reports, R. palustris cells containing the vector pMG102 bearing a cloned R. palustris pckA or ppc gene exhibited enzyme activities four times greater than that of the wild-type strain (23, 24). These data agree with the proposed copy number of plasmid pMG101 based on hybridization.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have isolated a 15-kb cryptic plasmid, pMG101, from a natural isolate of PNSB, identified the partition and replication region in a 3.0-kb SalI-XhoI subfragment, determined its DNA sequence and transcriptional start sites, and used this information to construct stable R. palustris-E. coli shuttle vectors. These shuttle vectors have already proved useful in studies of gene function in R. palustris (23, 24).

Sequence studies presented here suggest that the closest relationship is between the Par protein of pMG101 and that of pTAR, a plasmid isolated from Agrobacterium which, like R. palustris, is an alpha -proteobacterium. Portions of the adjacent sequence exhibit a repetitive sequence organization like that of the cis-acting par locus of pTAR.

The pTAR plasmid-partitioning system is a member of the sop/par family, which includes sop of plasmid F and par of plasmid P1 (56). Upstream of the pTAR parA gene, the cis-acting partition site contains a set of twelve 7-bp repeat sequences and the promoter region at which ParA binds to regulate its own transcription (16). Upstream of the parA gene of plasmid pMG101, there is an array of nine 8-bp repeats (DR I) that overlap the putative parA promoter (Fig. 4). The data (Fig. 4) suggest that the region upstream of the pMG101 parA gene, which includes the nine repeats, is the cis-acting recognition site at which ParA interacts to bring about partitioning and regulation of its own transcription, as proposed for the partition system of plasmid pTAR.

Structural and expression analyses of the parA gene revealed that the transcriptional start site corresponds precisely to the position of the deduced translation initiation codon for this gene (Fig. 4 and 6). Upstream of the site, we find an E. coli sigma 70-like promoter but no distinct Shine-Dalgarno (SD) sequence. In addition, a 4-bp insertion into the MunI site, which is located just downstream of the deduced initiation codon, destabilizes the plasmid in R. palustris under nonselective conditions. This finding suggests that the insertion created the expected frameshift mutation in the parA gene, which is predicted to be initiated only three codons upstream of this insertion (Fig. 7). Moreover, comparison of the deduced amino acid sequences with sequences of the pMG101 parA and pTAR parA gene products showed similar lengths and significant similarity along the length of the protein as well as a putative NTP-binding motif close to the N-terminal region (Fig. 3).

The pMG101 parA gene is unlikely to be translated by the usual system of SD interaction of the mRNA with 16S rRNA during initiation, because in contrast to most R. palustris genes (23, 24) and those of other PNSB, we find that the parA mRNA does not possess a SD sequence. In several prokaryotes, genes in which transcription and translation are initiated from the same nucleotides have been found (1, 2, 6, 10, 28, 31, 41, 49, 58). It was proposed that in E. coli, these genes lack SD sequences but possess a downstream box (DB) located just downstream of initiation codon which is complementary to a region (anti-downstream box [ADB]) in 16S rRNA, and the DB can function as a translation initiation signal (46).

Although no homology was observed between the E. coli ADB in 16S rRNA and the corresponding region of R. palustris 16S rRNA, a region just downstream of the parA initiation codon shows significant sequence complementarity to another region at the 5' end of R. palustris 16S rRNA (data not shown). However, the significance of the DB-ADB complementarity in translation of E. coli genes which lack SD sequences has recently been questioned (37). Further studies are necessary to determine whether the leaderless parA transcript of plasmid pMG101 is translated by this or another mechanism.

According to molecular and transcriptional analyses of the replication region of plasmid pMG101, the repA gene was transcribed from 172 and 173 bp upstream of the translation initiation codon, and a putative promoter sequence is found 5' of the transcriptional start site. 3' of the repA gene, two series of direct repeats (DR II and DR III), followed by AT-rich and GC-rich sequences, are found.

Clusters of direct repeats, termed iterons (34), exist in the replication origin regions of several plasmids, including mini-F (36), lambda dv (18, 34), R6K (47), and RK2 (48). Moreover, plasmid pSa, whose RepA protein is similar to that of pMG101 as described above, also contains six 17-bp direct repeats downstream of the repA gene (39). These repeat sequences or iterons are known to be the binding sites for Rep-like proteins (35, 51, 63), and the 17-bp direct repeats (DR II) of pMG101 are likely to represent the binding sites for RepA protein in this plasmid.

AT-rich regions have low thermal stability and are known to be the sites of strand separation at a variety of replication origins (3, 45, 52). Specialized sequences involved in strand separation at origins have been proposed to exist here (3, 45). In the case of the E. coli origin, four AT-rich 13-mers are proposed to be the sites of strand separation, and similar sets of repeats are found in plasmid origins, where they appear to serve the same purpose. The DR III 7-mer repeats are not homologous to these sequences but might function similarly.

GC-rich sequences have high thermal stability and could be important in limiting the extent of strand separation at the adjacent AT-rich regions. This might concentrate the strand separation locally and promote loading of the DNA helicase at this site. A similar sequence organization, consisting of a cluster of direct repeats followed by AT-rich and GC-rich regions, has been observed in the origin regions of RK2 (48) and pSa (39). In each case, deletion of these GC-rich regions strongly affects replication.

Additionally, there are partial copies of DR II and of DR III in the pMG101 repA promoter sequence and between the promoter sequence and the repA initiation codon, respectively (Fig. 4 and 5). It is possible that these partial direct repeats are involved in autoregulation of repA gene expression, as proposed for plasmid R6K (26).

Deletion analysis of the pMG101 replication region indicated that a 100-bp sequence which is located upstream of the repA promoter (at positions 1000 to 1100) was also essential for replication (Fig. 8). The absence of any specific structures or sequences in the 100-bp region gives no clues to its function. However, the existence of auxiliary cis-acting sequences, or enhancers, affecting replication has been demonstrated in several systems (38, 61, 64). Replication enhancers bind a variety of proteins, and the pMG101 sequence may function in the same way (38, 61, 64). Further studies are needed to understand the functions of the genes and the sites and mechanisms controlling pMG101 replication.

The stable E. coli-R. palustris shuttle cloning vectors pMG103 and pMG105 have provided us with a versatile cloning system for PNSB. We have found that pMG101 derivatives can replicate not only in R. palustris but also in the Bradyrhizobium species, and this host range observation clearly reflects the phylogenetic relationships of these species in the alpha -proteobacteria group. This is the first report that an endogenous plasmid from a photosynthetic bacterium replicates in other genera, especially the phenotypically distinct Bradyrhizobium species. These observations support the proposal that photosynthetic bacteria may have been ancestors of the Rhizobiaceae (59). Though the natural transfer of this plasmid to B. japonicum and phototrophic Bradyrhizobium species has not been demonstrated, the possibility that it is involved in horizontal gene transfer between these genera and the possible transmissibility of plasmid pMG101 in this process should be considered.


    ACKNOWLEDGMENTS

We are especially grateful to Peter van Berkum (Agriculture Research Service, USDA) for the gift of strains USDA 4362 and USDA 4377. We thank Yasuyoshi Nakagawa (Institute for Fermentation, Osaka, Japan) for his help in phylogenetic analysis and László Puskás for the construction of plasmid pHSG298X and for valuable discussions.

This work was supported by a grant from the New Energy and Industrial Technology Development Organization.


    FOOTNOTES

* Corresponding author. Mailing address: Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292 Japan. Phone: 81-774-75-2308. Fax: 81-774-75-2321. E-mail: yukawa{at}rite.or.jp.


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Results
Discussion
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