The dnaA Gene of Rhizobium meliloti Lies within an Unusual Gene ...

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WILLIAM MARGOLIN,1,2* DAVID BRAMHILL,1† AND SHARON R. LONG1. Department of Biological ...... Ivey, D. M. 1990. Nucleotide sequence of a gene from ...
JOURNAL OF BACTERIOLOGY, May 1995, p. 2892–2900 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 177, No. 10

The dnaA Gene of Rhizobium meliloti Lies within an Unusual Gene Arrangement WILLIAM MARGOLIN,1,2* DAVID BRAMHILL,1†

AND

SHARON R. LONG1

Department of Biological Sciences, Stanford University, Stanford, California 94305-5020,1 and Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 770302 Received 17 November 1994/Accepted 10 March 1995

Rhizobium meliloti exists either as a free-living soil organism or as a differentiated endosymbiont bacteroid form within the nodules of its host plant, alfalfa (Medicago sativa), where it fixes atmospheric N2. Differentiation is accompanied by major changes in DNA replication and cell division. In addition, R. meliloti harbors three unique large circular chromosome-like elements whose replication coordination may be complex. As part of a study of DNA replication control in R. meliloti, we isolated a dnaA homolog. The deduced open reading frame predicts a protein of 57 kDa that is 36% identical to the DnaA protein of Escherichia coli, and the predicted protein was confirmed by immunoblot analysis. In a comparison with the other known DnaA proteins, this protein showed the highest similarity to that of Caulobacter crescentus and was divergent in some domains that are highly conserved in other unrelated species. The dnaA genes of a diverse group of bacteria are adjacent to a common set of genes. Surprisingly, analysis of the DNA sequence flanking dnaA revealed none of these genes, except for an rpsT homolog, also found upstream of dnaA in C. crescentus. Instead, upstream of rpsT lie homologs of fpg, encoding a DNA glycosylase, and fadB1, encoding an enoyl-coenzyme A hydratase with a strikingly high (53 to 55%) level of predicted amino acid identity to two mammalian mitochondrial homologs. Downstream of dnaA, there are two open reading frames that are probably expressed but are not highly similar to any genes in the databases. These results show that R. meliloti dnaA is located within a novel gene arrangement. Rhizobium meliloti bacteria differentiate into symbiotic nitrogen-fixing bacteroids within the root nodules of alfalfa (13, 26). This process, which converts growing free-living bacteria into intracellular forms, is accompanied by significant changes in cell physiology and metabolism (44, 46). One of the more striking changes that occur is the cessation of DNA replication and cell division. To initiate our studies of genes involved in cell cycle regulation, we previously isolated two unique ftsZ genes, and we are now studying their roles in R. meliloti cell division (29). Since proper DNA replication is normally a prerequisite for cell septation, we are interested in identifying key DNA replication genes that may serve as control points. In addition, the tripartite genome of R. meliloti, which consists of a main chromosome and two large megaplasmids, is a unique model system for studying the coordination and segregation of multiple prokaryotic chromosomes. Thus, it is crucial to understand the functions of key replication genes in order to elucidate both normal DNA replication and segregation events in free-living R. meliloti cells and their alterations during differentiation. The dnaA gene is of central importance in prokaryotic DNA replication. In Escherichia coli, dnaA is an essential gene whose product is required for melting of the DNA duplex to initiate the replication fork (for reviews, see references 2, 6, 18, 30, and 43). DnaA forms initial complexes with DNA by binding to conserved nonamer sequences called DnaA boxes (10, 20, 50). Duplex opening then occurs by the interaction of DnaA with nearby 13-mer sequences to form an open complex (5). Bind-

ing of ATP is essential for DnaA to form the open complex and initiate replication (5, 42). DnaA also negatively regulates transcription of itself and several other genes by binding to DnaA boxes near their promoters (7, 43). Thus, DnaA regulates both transcription and replication. It is at present the best candidate for the timer that signals cells to begin DNA synthesis (25). The R. meliloti dnaA homolog is therefore potentially involved in DNA replication arrest during bacteroid differentiation. Homologs of dnaA have been cloned in a wide variety of eubacteria and have been shown to be quite well conserved (43). In addition, dnaA is consistently found directly adjacent to several genes such as dnaN, rpmH, and rnpA in a wide variety of species (35, 47). In Borrelia burgdorferi, the order of the genes with respect to dnaA is different but the same genes are still adjacent to dnaA (36). The chromosomal replication origin, oriC, is either adjacent to or within a map unit of dnaA in most bacterial species. As a result, many of the dnaA homologs have been cloned by ‘‘walking’’ from cloned replication origins. These physical methods for cloning dnaA have been employed in large part because it has not been possible to complement E. coli dnaA mutations with cloned nonenteric dnaA homologs. In this study, we isolated the R. meliloti dnaA homolog, determined its relatedness to other bacterial dnaA genes, and demonstrated that R. meliloti lacks the gene arrangement surrounding dnaA that is shared among other bacteria that have been examined to date.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, TX 77030. Phone: (713) 794-1744. Fax: (713) 7941782. Electronic mail address: [email protected]. † Present address: Department of Biochemistry, Merck Research Laboratories, Rahway, NJ 07065.

Bacterial strains, plasmids, and bacteriophages. E. coli strain XL1-Blue (Stratagene) was used as a host for the following plasmids. Plasmid pWM165 contains the dnaA PCR product cloned into the EcoRV site of pBluescript SK2. l phage E contains the 7-kb BamHI fragment carrying the dnaA region. pWM179 and pWM180 contain the 1-kb NotI fragment representing the 39 end of dnaA cloned into the NotI site of pBluescript SK1, with dnaA in the same orientation as Plac (pWM179) or in the opposite orientation (pWM180). The

MATERIALS AND METHODS

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VOL. 177, 1995 5.7-kb EcoRI fragment of l 5 containing all of dnaA was cloned into the EcoRI site of pBluescript SK1 to create pWM181. The 2.7-kb EcoRV fragment from pWM181, which includes a polylinker EcoRV site, was cloned into the EcoRV site of pBluescript SK2 such that dnaA is oriented opposite to Plac (pWM182) or in the same direction as Plac (pWM183). These two plasmids were used as substrates for further subcloning, and exonuclease deletions constructed in these plasmids were used for sequencing. The 1-kb BamHI-EcoRI fragment upstream of dnaA and the 2.5-kb NotI-BamHI fragment downstream of dnaA were cloned into pBluescript SK1 which was digested with the compatible enzymes to create pWM226 and pWM225, respectively. The bacteriophage library containing BamHI-digested R. meliloti DNA was constructed in l GEM-11. Media, chemicals, and enzymes. E. coli and R. meliloti strains were grown in Luria-Bertani broth (32) or on Luria-Bertani agar at 37 and 308C, respectively. Ampicillin was used as necessary at 50 mg/ml for the growth of plasmid-containing E. coli strains. Restriction enzymes, T4 DNA ligase, and Klenow polymerase were from New England Biolabs or Promega. Radiochemicals were from Amersham. DNA preparation and manipulation. Total genomic DNA from R. meliloti 1021 was prepared as described previously (32). Minipreparations of plasmid DNA from E. coli were made by a modified boiling plasmid method, in which no salt was added to the boiled DNA before isopropanol precipitation (40). Minipreparations of phage l DNA were made from high-titer liquid lysates as described previously (31). PCR. We employed a strategy similar to that for amplification of ftsZ (28) to successfully amplify a segment of R. meliloti dnaA. Locations for primers were chosen on the basis of their degree of amino acid conservation across diverse species, relative lack of codon degeneracy (particularly at the 39 ends of the primers), and adequate separation (at least 100 bp) between the primers. To ensure that no mismatches occurred, the primers were not biased toward the high R. meliloti G1C content. The following pair of primers were used: 59CGS GARCTBGARGGNG39 (to prime synthesis of the nontemplate strand) and 59GTRTGRTCNCGNCCNCCGAA39 (to prime synthesis of the template strand) (where R is A or G; S is G or C; B is G, T, or C; and N is G, A, T, or C). PCR was performed in a Hybaid thermal cycler in a 50-ml reaction mixture exactly as described previously (28) except that a 608C annealing temperature was used. The predominant PCR product observed was 0.3 kb, which was the size predicted from the primer locations. The product was purified by the freezesqueeze method (45), the ends were filled in with Klenow polymerase plus deoxynucleoside triphosphates, and the product was cloned into EcoRV-cleaved pBluescript SK2. Plasmids from four transformants containing inserts of approximately 300 bp were subjected to DNA sequencing. One isolate, pWM165, revealed similarity to dnaA and was chosen as the source of the 100% homologous probe. Screening the genomic library. The cloned insert in pWM165 was radioactively labeled with [a-32P]dATP by random hexamer labeling (40) and used to probe a l library of BamHI-digested R. meliloti genomic DNA as described previously (28). After positive plaques were repurified and rescreened, phage DNA from several positive plaques was isolated and digested with BamHI. All yielded an approximately 6-kb BamHI fragment that contained an internal 1-kb NotI fragment found to hybridize to the PCR product on an R. meliloti genomic Southern blot. One DNA preparation was chosen for subcloning. DNA sequencing and analysis. For the bulk of the sequence analysis, a series of exonuclease III deletions was constructed in plasmids pWM182 and pWM183 as described previously (40). For the remainder of the sequencing, either subclones were constructed or custom primers were used. Single-stranded DNA was used for manual sequencing with the Sequenase 2.0 kit (U.S. Biochemicals), and double-stranded DNA was used for automated sequencing with an Applied Biosystems sequencing machine (Core facility, Microbiology Department, University of Texas Medical School). Single-stranded DNA was prepared from plasmid derivatives of pUC118, pUC119, or pBluescript with the M13K07 helper phage as described previously (39). Double-stranded DNA was prepared with the Qiagen plasmid minikit. DNA and predicted protein sequences were mapped and analyzed with the University of Wisconsin Genetics Computer Group programs (11). Searches of the databases were conducted with TFASTA (24), TBLASTN, and BLASTP (1). Multiple sequence alignments and sequence similarity trees were made with PILEUP. Nucleotide sequence accession number. The nucleotide sequence of dnaA and flanking regions reported in this paper has been deposited in the GenBank database and assigned accession number L39265.

RESULTS AND DISCUSSION Cloning the R. meliloti dnaA gene. We initially tried to clone R. meliloti dnaA by hybridization techniques, using E. coli dnaA as a probe. This was unsuccessful, probably in part because of the significantly different G1C biases of the two species. We next tried probing with dnaA of Pseudomonas putida, which has a G1C content closer to the high (65%) G1C content of R. meliloti. We were still unable to detect a strong hybridization

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FIG. 1. Restriction enzyme cleavage site map of the BamHI fragment containing dnaA. The map shows the 7-kb BamHI fragment containing dnaA, from clone E from the l library containing BamHI-cleaved R. meliloti DNA. Restriction sites: B, BamHI; E, EcoRI; N, NotI; H, HindIII; V, EcoRV. The black box represents the PCR product probe. The hatched boxes show the open reading frames in the sequenced region found to have typical codon usage for R. meliloti. All six open reading frames (see text for details) are read from left to right. Dashes represent the continuation of the reading frames outside the sequenced region. Plasmid derivatives of pBluescript SK2 with inserts containing various segments of this region are denoted by horizontal lines at the bottom.

signal. Other methods, such as probing with G1C-biased oligonucleotides specific to domains highly conserved among the DnaA proteins known at the time, were equally unsuccessful. One of the potential reasons for this was that the highly conserved domains were too short to allow strong detection of the signal. However, two of these regions were just long enough for PCR primers to anneal and to be used to amplify a segment of dnaA which then was used as a homologous probe to isolate the intact gene (Fig. 1). To confirm that the PCR product was from R. meliloti, the PCR product was used to probe a genomic blot of R. meliloti DNA. Only one specific restriction fragment hybridized strongly in several restriction digests, including a 1-kb NotI fragment and a 6-kb EcoRI fragment (data not shown). This result confirms that the probe was derived from R. meliloti DNA and that a similar dnaA homolog probably is not present elsewhere in the genome, unlike other duplicated genes such as groEL (38) and ftsZ (29). The probe was used to screen a phage l library of BamHI-digested R. meliloti DNA, as described previously (28). When DNA from purified positive plaques was digested with NotI, a 1-kb fragment was found, suggesting that these clones contained the genomic DNA flanking the PCR product. The 6-kb EcoRI fragment containing the NotI fragment was cloned to further characterize the dnaA region (Fig. 1). The DNA sequence of the dnaA gene and the region surrounding it is shown in Fig. 2. An open reading frame encoding a predicted protein 37% identical to E. coli DnaA extends from one of several potential methionine start codons (at positions 2340, 2409, 2430, 2436, and 2511) after the in-frame stop codon at nucleotide position 2331 to the stop codon at position 3951. In addition, since several bacterial dnaA genes initiate with GTG codons, we searched for GTG, CTG, and TTG codons downstream from potential ribosome binding sequences that might be potential initiation points. Although there were several TTG codons (positions 2415 and 2442) and a CTG codon at 2526, they were not preceded by purine runs and therefore probably are not translation initiation sites. The ATG at 2511 is a possible start codon for a 480-amino-acid protein, except that there are no G residues between positions 23 and 212 relative to the ATG codon, making it a relatively poor potential ribosome binding site. We tentatively decided upon the ATG codon at position 2430 as the probable start codon, since

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FIG. 2. Nucleotide sequence of dnaA and flanking open reading frames, and amino acid sequence of predicted protein products. The DNA sequence from the BamHI site past the NotI site downstream of dnaA is shown, along with the single-letter amino acid codes below the six major open reading frames, all reading rightward. Reading frame designations are on the right; nucleotide numbers are on the left. The probable initiator methionine for dnaA is shown. Double underlining indicates potential DnaA boxes with 100% homology to the degenerate DnaA box consensus sequence (41); two of the three sequences are on the bottom strand. Asterisks denote stop codons. Putative ribosome binding site sequences are indicated by dots above the sequences. Inverted repeats which are portions of potential Rhoindependent terminator structures are represented with a pair of dashed arrows above the DNA sequence. Other inverted repeats are indicated by solid arrows above the DNA sequence. Significant direct repeats are underlined once.

it is preceded by the best potential ribosome binding sequence, GGAAGG, 7 bp upstream (Fig. 2). The predicted protein initiating from that codon would be 507 residues in length and have a mass of 57 kDa, larger than the 467-residue 53kDa E. coli DnaA. The codon preferences of R. meliloti dnaA agree well with those of other R. meliloti genes, with 29 rare codons in the entire reading frame (as determined with the University of Wisconsin Genetics Computer Group program CODONPREF [11]; rare-codon threshold set to 0.1). The codon preference is higher than 1.0 for the entire sequence except the first 200 bp, where it is approximately 1.0. This last finding supports the argument for an initiation site ATG at 2511, which is where the above-average codon preference begins. Verification of the true amino terminus of R. meliloti DnaA must await protein sequence analysis. An inverted repeat between positions 3980 and 3995, followed by a

run of T residues, is probably a Rho-independent termination site. Confirmation of R. meliloti DnaA protein by immunoblot and comparison with other eubacterial DnaA proteins. To confirm the DNA sequence prediction, we probed an immunoblot containing R. meliloti and E. coli total proteins with anti-E. coli DnaA polyclonal antiserum (Fig. 3). Only one major band was seen in the R. meliloti protein extract, consistent with the single band on the Southern blot and the idea that the dnaA gene is not duplicated. R. meliloti DnaA migrates more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than does E. coli DnaA, consistent with its predicted 57-kDa size compared with 53 kDa for E. coli DnaA. We also confirmed that the cloned dnaA gene on pWM181 directed the synthesis of a 57-kDa protein by in vitro transcription-translation in an R. meliloti S-30 extract (27).

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FIG. 3. Immunoblot of E. coli and R. meliloti DnaA proteins. Total protein from E. coli and R. meliloti was extracted, denatured, reduced with 2-mercaptoethanol, separated by SDS-PAGE, and blotted onto a nitrocellulose membrane. The membrane was blocked with phosphate-buffered saline–Nonidet P-40 and probed with antiserum raised against E. coli DnaA protein (gift of K. Sekimizu). The arrow indicates the R. meliloti DnaA protein band.

The R. meliloti DnaA protein sequence is clearly similar to those of other bacterial DnaA proteins (Fig. 4). Conservation is strongest in the ATP binding domain (GLGKTHL) and in several carboxy-terminal regions, particularly those chosen for PCR primer synthesis (RELEGA and GGRDHTTVLHA, the latter of which has been proposed to be the DnaA box binding domain on the basis of its conservation [41]). However, the sequence is divergent in many regions from those of other gram-negative bacteria and is even more divergent at many positions from the enteric bacterial sequences than is the sequence of the gram-positive species Streptomyces coelicolor. This is surprising, considering that R. meliloti and the enteric bacteria, unlike Streptomyces species, are all members of the proteobacterial group. The R. meliloti sequence is most closely related to that of Caulobacter crescentus (49), a member of the a subgroup of proteobacteria which also has a genome containing approximately 65% G1C residues (37). In addition to sharing with C. crescentus certain amino acid divergences from the enteric bacteria, R. meliloti DnaA shares with it several short insertion regions, including one at the amino terminus that is consistent with the assigned initiation codon at 2430 or 2436 rather than 2511. The similarity between the predicted DnaA proteins of R. meliloti and C. crescentus, as well as the divergence from other gram-negative organisms’ DnaA proteins, is highlighted in a dendrogram based on sequence relatedness (Fig. 5). Absence of downstream conserved genes and upstream DnaA boxes. DNA sequence analysis extending 700 bp downstream from dnaA revealed two open reading frames that we have designated orfX and orfY (Fig. 2). They were so assigned because they have high percentages of preferred R. meliloti codons and putative ribosome binding sites and thus are likely to be expressed in R. meliloti. They had no significant similarities to any genes in the database, including dnaN, dnaK, recF, and gyrB. The dnaN gene is normally found immediately downstream of dnaA, except in C. crescentus, where dnaK is downstream and dnaN is instead located much further downstream (49). It is possible that further DNA sequencing downstream from dnaA will reveal a dnaK homolog. If orfX is expressed, its promoter probably lies immediately downstream from the putative dnaA gene terminator. No easily recognizable 210 promoter sequences occur in this region.

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Since other dnaA homologs lie downstream from sequences with similarity to DnaA boxes, we searched for DnaA boxes in the vicinity of R. meliloti dnaA. A sequence matching the degenerate DnaA box consensus (41) lies between residues 2431 and 2439, overlapping the methionine codons at 2430 and 2436. Two other potential DnaA boxes that match the degenerate consensus lie at 3413 to 3421 and 3580 to 3588, within the proposed reading frame. None of these sequences matches the stringent DnaA box sequence of E. coli (17). The potential function, if any, of these sequences in binding DnaA or in the possible regulation of expression or replication origin activity is unclear. No other DnaA box sequences were detected within or beyond 1 kb upstream of the gene. Similarity to Caulobacter rpsT gene upstream of dnaA. The first open reading frame upstream from dnaA is homologous to rpsT, which in E. coli encodes the gene for ribosomal protein S20 (22) (Fig. 6). The R. meliloti homolog is 39% identical to the E. coli gene and 58% identical to the published portion of the C. crescentus homolog, which is also found immediately upstream of the dnaA homolog of C. crescentus. The rpsT gene does not map near dnaA or the origin of replication in any other species examined to date. Instead, other bacterial dnaA genes are linked to a gene for a large-subunit ribosomal protein, L34 (rpmH), and rnpA, which encodes the ribonuclease P protein component. As in C. crescentus, there is an intergenic region of several hundred base pairs between R. meliloti rpsT and dnaA. However, the R. meliloti intergenic region differs significantly from the corresponding C. crescentus region, in that the former lacks DnaA box sequences and contains several long runs of AT base pairs. Such AT stretches are unusual in both Caulobacter and Rhizobium species, both of which have genomes with high G1C contents. In addition, the region contains several direct repeats, including a cluster immediately upstream from dnaA (Fig. 2). The AT stretches and repeats and the proximity to dnaA suggested the possibility that this was R. meliloti oriC, despite the presence of only one distally located DnaA box. However, we were not able to demonstrate autonomous replication activity in R. meliloti with clones containing this region, suggesting that the unusual repeats and AT runs may contain regulatory sites for dnaA expression but not for replication. Therefore, oriC may be several kilobase pairs upstream of dnaA, as it is in C. crescentus. This is consistent with the orientation of dnaA and the three upstream (see below) and two downstream reading frames described in this paper, since it has been shown that transcription of most bacterial genes is oriented in the same direction as replication fork movement (8, 48). The region containing dnaA appears to map to the R. meliloti main chromosome on the basis of the lack of hybridization to megaplasmid DNA (27). Further characterization of the region upstream of dnaA is necessary to determine if oriC is present in the vicinity. The similarity between the R. meliloti and C. crescentus dnaA genes and the presence of common upstream rpsT genes suggest that these two related species have a local gene organization distinct from that of the diverse population of other eubacteria for which dnaA has been characterized. This is surprising, since the evolutionary distance between enteric and gram-positive species, which have similar regional gene organizations, is much greater than the distance between either group and subgroup 2 of the a-proteobacteria (37). This observation suggests that this gene organization may be shared among several of the a-proteobacteria in addition to Caulobacter and Rhizobium species. Similarity of fadB1 to mitochondrial genes encoding enoylCoA hydratase. The lack of conservation of gene organization

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FIG. 4. Multiple alignment of selected bacterial DnaA proteins. This alignment was obtained by using the PILEUP program of the University of Wisconsin Genetics Computer Group (11). DnaA protein sequences include those of E. coli (Eco) (19), Buchneria aphidicola (Bap) (23), P. putida (Ppu) (14), Micrococcus luteus (Mlu) (15), S. coelicolor (Sco) (9), Bacillus subtilis (Bsu) (34), C. crescentus (Ccr) (49), R. meliloti (Rme), B. burgdorferi (Bbu) (36), and Myco-plasma capricolum (Mca) (16). The R. meliloti protein residues are numbered. Residues which are chemically similar in all proteins in the alignment are indicated below by asterisks.

continues further upstream from rpsT. The next upstream gene, which has typical R. meliloti codon usage, has been designated fadB1. It encodes a homolog of the mammalian mitochondrial enoyl-coenzyme A (CoA) hydratase (EC 4.2.1.17), which catalyzes a key step in beta oxidation of fatty acids (33). The similarity between the E. coli fadB gene, which encodes a bifunctional enzyme that includes the hydratase function, and eukaryotic homologs was previously reported (12). A bacterial monofunctional homolog of the mammalian mitochondrial

enoyl-CoA hydratase was recently found in Rhodobacter capsulatus, another member of subgroup 2 of the a-proteobacteria (3). What is particularly striking is that the predicted R. meliloti enoyl-CoA hydratase amino acid sequence is 55% identical to rat mitochondrial enoyl-CoA hydratase and 53% identical to the human mitochondrial enzyme (Fig. 7). This is comparable to the degree of identity between the mitochondrial enzymes of nematodes (Caenorhabditis elegans) and rats (56%) but is strik-

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FIG. 5. Dendrogram of DnaA proteins. A tree of evolutionary relatedness among bacterial DnaA proteins was obtained with the PILEUP program used for the alignment in Fig. 4. Pmi, Proteus mirabilis. (See the legend to Fig. 4 for other strain abbreviations.)

ingly higher than the degree of identity between other bacterial homologs and the rat protein (31%) or between other bacterial homologs and the R. meliloti protein (34%). As expected from these data, a protein sequence similarity tree among the homologs from R. meliloti, Rhodobacter capsulatus, E. coli, and mammalian mitochondria places R. meliloti and mammalian mitochondrial copies on the same root (data not shown). There are several possible explanations for the particularly high level of homology observed between R. meliloti fadB1 and the eukaryotic enoyl-CoA hydratases. First, the progenitors of mitochondria are probably close relatives of the family Rhizobiaceae, so it is reasonable to speculate that fadB1 DNA from an ancestral endosymbiont could have been transferred to eukaryotic cells. Conversely, fadB1 DNA may have been transferred from an ancestral animal to a progenitor of the Rhizobiaceae. Another possibility is that the sequence similarities may have evolved independently in response to similar physiological demands. This is not unreasonable, since bacteroids within root cells may experience an environment very similar to

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that experienced by the mitochondrion. Confirmation and characterization of the R. meliloti fadB1 protein product, as well as comparison of sequences of other hydratases, including those from other mitochondrial and bacterial sources, will be necessary in order to establish more precisely their evolutionary relationships. Similarity of an open reading frame upstream of fadB1 to a gene encoding a bacterial DNA repair protein. Immediately upstream of fadB1 is a partial (39) sequence of a homolog of E. coli and Bacillus firmus fpg, which encodes a formamidopyrimidine-DNA glycosylase (EC 3.2.2.23) involved in the repair of oxidatively damaged DNA (4, 21). Also known as mutM, the gene is located in E. coli at 81 min, fairly close to dnaA and oriC (84 min) and fadB (87 min) but not adjacent. This is the only gene that we have found in this region of the R. meliloti chromosome other than dnaA that is obviously related to DNA metabolism. The sequenced portion of the R. meliloti fpg homolog is 47% identical to E. coli fpg at the amino acid sequence level. Possible roles of the dnaA region and DnaA protein in R. meliloti. Our results indicate that the region surrounding dnaA in R. meliloti is unusual. First, genes such as rnpA, rpmH, and dnaN that are normally adjacent to dnaA in a wide variety of bacterial species are not adjacent to R. meliloti dnaA. Second, unlike in most bacterial species, the R. meliloti chromosomal replication origin does not appear to lie immediately upstream of dnaA. Despite these differences, the region has some similarity to one in a related species, C. crescentus, in which the chromosomal replication origin is several kilobase pairs upstream. It has been proposed that the conserved gene arrangement including dnaA found in most bacteria represents an ancestral bacterial replication origin, and its high degree of conservation is postulated to be due to the crucial juxtaposition of regulatory elements for DNA replication genes and other genes involved in macromolecular synthesis (47). Although the reasons for this juxtaposition are not known and replication timing in R. meliloti has not been characterized, our findings indicate that successful chromosomal DNA replication in R. meliloti does not require the conserved gene arrangement around dnaA. As discussed above, the dnaA region of Caulobacter species also appears to lack this conserved gene arrangement. Comparative analysis of the dnaA regions in R. meliloti and species of other related genera such as Caulobacter, Rhodobacter, and Brucella should help to elucidate whether the R. meliloti gene arrangement represents a distinct class of replication origin regions. The somewhat divergent amino acid sequence of R. meliloti DnaA may reflect its potentially unique role in coordinating replication of three large chromosome-like replicons, which include the main chromosome and two megaplasmids. We have begun to address this problem by cloning a replication origin from one of the megaplasmids, pSym-b, and have found several potential DnaA boxes within the minimal origin sequence (29). It remains to be seen whether DnaA is involved in initiating and coordinating replication from this as well as

FIG. 6. Alignment of rpsT homologs. The predicted protein products from the rpsT genes of E. coli (Ecorpst) (22), R. meliloti (Rmerpst), and the partial sequence from C. crescentus (Ccrrpst) (49) are shown. Vertical lines between the sequences represent chemically similar residues, while asterisks below the E. coli sequence and those above the C. crescentus sequence represent residues that are identical to those in the R. meliloti sequence.

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FIG. 7. Alignment of enoyl-CoA hydratase homologs. Predicted amino acid sequences from cDNA or DNA sequences were obtained from the SWISSPROT protein database and include human mitochondrial (HUMMECH) (Hum), rat mitochondrial (RNECH) (Rat), C. elegans mitochondrial (ECHM_CAEEL) (Cel), R. meliloti (this work) (Rme), Rhodobacter capsulatus (ECHH_RHOCA) (Rca), and E. coli (ECOFADBA) (Eco) sequences. The complete sequences are shown for all except the E. coli fadB product, of which only the amino-terminal hydratase domain is shown. The first 29 amino acids of the mitochondrial proteins are precursor sequences. Asterisks below the alignment denote identities between the R. meliloti and Rhodobacter capsulatus homologs. Letters below the alignment represent amino acids which are identical between the R. meliloti homolog and the rat and/or human mitochondrial homolog and are capitalized when all three mitochondrial homologs and the R. meliloti protein are identical.

other replication origins on pSym-b, the chromosome, and the other megaplasmid, pSym-a. Further studies of dnaA and other replication factors should contribute to our understanding of how multiple prokaryotic chromosomes replicate. In addition, these studies should eventually allow us to target potential recipients of the plant signal that leads to arrest of DNA replication and cell division during R. meliloti differentiation.

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ACKNOWLEDGMENTS We thank G. Zweiger for communication of sequence information prior to publication and thank members of S. R. Long’s laboratory for helpful discussions. We are grateful for the technical assistance of M. Kumar and J. Yang in making plasmid constructions for sequence analysis and to K. Sekimizu and J. Kaguni for antibody preparations against E. coli DnaA. This work was supported by USDA grant 88-37262-3978 and National Institutes of Health grant 2R01-GM30962. W.M. was additionally supported by a National Science Foundation Postdoctoral Fellowship in Plant Biology.

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