"Green-like" and "Red-like" RubisCO cbbL Genes in Rhodobacter azotoformans

Yoshihito Uchino*, and Akira Yokota{dagger}

* Institute for Fermentation, Osaka, Japan
{dagger} Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits. One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group. This is the first report that one organism contains both green-like and red-like RubisCO genes. Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene. In the cbbL phylogenic analysis, R. spaeroides and R. azotoformans 1 (red-like) formed a cluster within the red-like group, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed a cluster within the green-like group. This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor. On the other hand, molecular phylogeny of the bacteria indicates that R. veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R. azotoformans and R. sphaeroides, which have red-like cbbL genes, are the latest evolved. Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R. azotoformans and R. sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R. sphaeroides later lost the green-like cbbL gene.

Key Words: form I RubisCO large subunit • green-like and red-like cbbL • Rhodobacter azotoformans • horizontal gene transfer


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO EC.4.1.1.39) is a key enzyme in CO2 fixation in the Calvin Benson Bassham (CBB) reductive pentose phosphate pathway. It is the most abundant enzyme on earth. RubisCO exists in two distinct forms. Form I RubisCO is composed of eight large subunits and eight small subunits (L8S8), which are encoded by the cbbL and cbbS genes, respectively. Form II RubisCO is composed of large subunits only (Lx), and is encoded by the cbbM gene.

In the phylogenetic tree of form I RubisCO large-subunit protein (CbbL), taxa are broadly divided into two major groups, termed "green-like" and "red-like" (Delwiche and Palmer 1996). The green-like group includes Cyanobacteria, the plastids of Glaucophyta, green algae, Euglenophyta, plants, and {alpha}-, ß-, and {gamma}-Proteobacteria. The red-like group includes the plastids of red algae, brown algae, Cryptophyta, and {alpha}- and ß-Proteobacteria. The phylogeny based on this molecule, however, is inconsistent with those based on other molecules. Delwiche and Palmer examined possible causes of this inconsistency and concluded that the RubisCO has probably undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.

Subsequently, Paoli et al. (1998) showed clear proof of a horizontal gene transfer event in the evolution of form I RubisCO. They determined the sequences of form I cbb operon of Rhodobacter capsulatus and indicated that CbbL of R. capsulatus lies within a green-like radiation of the RubisCO phylogenetic tree, well separated from CbbL of the related organism Rhodobacter sphaeroides, which is located within a red-like radiation. R. sphaeroides and R. capsulatus have been grouped in the same taxon in the phylogenies using various molecules (16S rRNA, 5S rRNA, 23S rRNA, cytochrome c, photosynthetic reaction center L and M proteins, and the form II RubisCO). Paoli et al. (1998) attributed the acquisition of form I cbb operon by R. capsulatus to horizontal gene transfer from a bacteria containing a green-like RubisCO, as the cbbL tree is strongly in conflict with phylogenetic information based on other molecules. They showed that form I RubisCO of R. capsulatus was heterogeneous among other purple nonsulfur bacteria by immunological studies with an antibody against form I RubisCO of R. sphaeroides. They also showed that R. capsulatus cbbRI, which encodes a LysR-type transcriptional regulator, resembled cbbR of bacteria that had a green-like cbbL gene, and the order of genes cbbS-cbbQ of R. capsulatus was limited to bacteria that had green-like cbbL genes. They asserted that R. capsulatus cbbRI, cbbL, cbbS, and cbbQ genes were acquired together by one horizontal gene transfer event.

In this paper, we report that Rhodobacter azotoformans contains both green-like and red-like cbbL genes. This is the first such organism identified, and the finding has important implications for the evolutional history of the form I RubisCO. We also analyzed the phylogeny based on cbbL sequences involving all species of Rhodobacter and examined the evolution of form I cbb operon in this genus. Although it has been suggested that Rhodobacter species derive from a common ancestor, the kinds of cbbL among this group are heterogeneous.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Bacterial Strains and Culture Conditions
The bacterial strains used in this study were Rhodobacter azotoformans IFO 16436T, Rhodobacter blasticus IFO 16437T, Rhodobacter capsulatus IFO 16435T, Rhodobacter sphaeroides IFO 12203T, and Rhodobacter veldkampii IFO 16458T. All strains except R. veldkampii IFO 16458T were cultured aerobically at 27°C in the medium containing 1% polypeptone, 0.2% yeast extract, and 0.1% MgSO4. R. veldkampii IFO 16458T was grown phototrophically at 27°C in an SA medium (Kawasaki et al. 1992) containing sulfide solution. In order to obtain pure cultures, isolated colonies were carefully picked up.

Sequencing of cbbL Genes
For amplification and sequencing of cbbL genes, DNA was isolated by standard phenol/chloroform extraction. Fragments of the partial cbbL genes were amplified by PCR using conserved primers RubiLF1 (5'-ACCTGGACSRYGGTCTGGAC-3') and RubiLR1(5'-GTRCCRCCRCCRAAYTGNAG-3'). Temperature was controlled according to the following thermal profile: after initial denaturation at 94°C for 2 min, a total of 30 cycles of amplification was performed with template DNA denaturation at 98°C for 45 sec, primer annealing at 60°C for 45 sec and primer extension at 72°C for 1 min, and final extension at 72°C for 10 min. Amplified fragments were cloned by using an Invitrogen TA CloningTM kit. Extraction and purification of the plasmids were carried out using a Qiagen QIAprep Spin Plasmid Kit. The cloned fragments were sequenced using a Thermo SequenaseTM Cycle Sequencing Kit (Amersham-Pharmacia Biotech), 2.5mM dNTP mixture with 7-deaza-dGTP (Amersham-Pharmacia Biotech), IRD700-labeled Primer (5'-TGTAAAACGACGGCCAGT-3'), IRD800-labeled Primer (5'-CAGGAAACAGCTATGACC-3'), and a Li-Cor 4200 Sequencer according to the manufacturer's instructions. The identity of the obtained sequences was confirmed by Blast search.

Determination of cbbL Type by PCR
To determine which type of cbbL each species of Rhodobacter had, PCR was performed using primer sets of RubiLF1-GREENcbbLR(5'-GAGTCATTGCCGAAGATCG-3') for green-like cbbL genes and RubiLF1-REDcbbLR(5'-GCAGACATTGTAATAGCCCT-3') for red-like cbbL genes, and amplified fragments were electrophoresed on 0.8% agarose. The fragments were sequenced as stated above, and their identity was confirmed by Blast search.

Determination of cbbL Type by Hybridization
To determine which type of cbbL each species of Rhodobacter had, dot blot hybridization was performed. Probes were synthesized by PCR using a PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals, Germany), primer sets (green, RubiLF1-GREENcbbLR; red, RubiLF1-REDcbbLR), and chromosomal DNA (green, R. capsulatus; red, R. sphaeroides) as templates. The genomic DNA samples (2.5 µg) from each species of Rhodobacter were directly pipetted onto positively charged nylon membranes (Boehringer Mannheim, Germany). Hybridization and detection were performed using a DIG Luminescent Detection Kit (Boehringer Mannheim, Germany) according to the manufacturer's protocol.

To determine the numbers of copies of green-like and red-like cbbL genes in R. azotoformans, Southern hybridization was performed. The genomic DNA was digested with the restriction enzymes Hind III and Pst I. The digested DNA and {lambda}-EcoT14 I DNA standard marker were electrophoresed on 1% agarose gel and transferred onto positively charged nylon membrane. Synthesis of probes, hybridization, and detection were performed as described above.

Sequencing of pufL and pufM Genes
The pufL and pufM genes of R. azotoformans IFO 16436T and R. veldkampii IFO 16458T were amplified using primers reported previously (Uchino et al., unpublished data). The protocol for sequencing was as stated above.

Phylogenetic Analysis
Phylogenetic analyses were performed based on the nucleotide sequences of 16S rRNA and 23S rRNA, and both nucleotide and putative amino acid sequences of cbbL, gyrB, and pufL.

The following 16S rRNA, 23S rRNA, pufL, cbbL, and gyrB sequence data were obtained from the GenBank:

  1. 16S rRNA sequences. Rhodobacter azotoformans (accession number D70846), Rhodobacter blasticus (accession number D13478), Rhodobacter capsulatus (accession number D16428), Rhodobacter sphaeroides (accession number D16425), Rhodobacter veldkampii (accession number D16421), and Paracoccus denitrificans (accession number X69159);
  2. 23S rRNA sequences. R. azotoformans (accession number AB050733), R. blasticus (accession number AB050734), R. veldkampii (accession number AB050735), R. capsulatus (accession number X06485), R. sphaeroides (accession number X53853), and P. denitrificans (accession number X87287);
  3. pufL sequences. Rhodobacter blasticus (accession number D50649), Rhodobacter capsulatus (accession number Z11165), Rhodobacter sphaeroides (accession number AF195122), and Rhodospirillum rubrum (accession number J03731);
  4. cbbL sequences. Allochromatium vinosum 1 (accession number M26396), Allochromatium vinosum 2 (accession number D90204), Bradyrhizobium japonicum (accession number AF041820), Hydrogenophaga pseudoflava (accession number U55037), Hydrogenophilus thermoluteolus (accession number D30764), Hydrogenovibrio marinus 1 (accession number D43621), Hydrogenovibrio marinus 2 (accession number D43622), Nitrobacter vulgaris (accession number L22885), Nitrobacter winogradskyi ATCC 14123 (accession number AF109914), Nitrobacter winogradskyi IFO 14297 (accession number AF109915), Ralstonia eutropha (accession number M17744), Ralstonia eutropha pHG1 (accession number U20585), R. capsulatus IFO 16581 (=SB1003) (accession number L82000), R. sphaeroides (accession number M64624), Sinorhizobium meliloti (accession number AF211846), Thiobacillus denitrificans (accession number L42940), Acidithiobacillus ferrooxidans 1 (accession number AF129925), Acidithiobacillus ferrooxidans 2 (accession number AF307091), Thiobacillus intermedius (accession number AF046933), Xanthobacter flavus (accession number P23011), and manganese-oxidizing bacterium SI85-9A1 (accession number L32182);
  5. gyrB sequences. R. azotoformans (accession number AB065365), R. blasticus (accession number AB065367), R. capsulatus (accession number AB014940), R. sphaeroides (accession number AB014944), R. veldkampii (accession number AB065366), and Paracoccus aminophilus (accession number AB014972).

Multiple sequence alignments were performed using ClustalW 1.8 (Macintosh) (Thompson et al. 1994). Finally, the alignments were refined by eyes, and the positions with gaps and undetermined and ambiguous sequences were removed for subsequent analysis.

Phylogenic trees were reconstructed using three different algorithms: Neighbor-Joining (NJ [Saitou and Nei 1987]), maximum-parsimony (MP [Fitch 1971]) using PAUP* version 4.0b8 (Mac) (Swofford 2000), and the maximum-likelihood (ML [Felsenstein 1981]) using NucML (for nucleotide) and ProtML ( for amino acid) in the MOLPHY version 2.3 package (UNIX) (Adachi and Hasegawa 1996).

In the NJ analysis, the distances were estimated by the Kimura two-parameters model (Kimura 1980), the Tamura and Nei model (Tamura and Nei 1993) and the HKY 85 model (Hasegawa, Kishino, and Yano 1985) for nucleotide data, and p-distance for amino acid data. In the MP analysis, all characters were unordered and of equal weight, and all searches employed 100 replicates of the random-sequence addition heuristic search algorithm with tree-bisection-reconstruction (TBR) branch swapping. The ML trees based on the HKY85 model (for nucleotide) and the JTT model (for amino acids [Jones, Taylor, and Thornton 1992]) were searched with the local rearrangement method (Adachi and Hasegawa 1996). The NJ and MP trees were used as seed topologies.

In the ML analysis, the local bootstrap probabilities (LBPs) were calculated as the statistical confidences of the tree branches (Hasegawa and Kishino 1994). In the analysis based on cbbL sequences, the ML trees based on nucleotide and amino acid sequences were constructed with 25 OTUs (operational taxonomic units), including the data obtained in this research and the green-like and red-like cbbL data registered in the GenBank.

In the analysis based on 16S rRNA, 23S rRNA, gyrB, and pufL sequences, the trees constructed by the NJ, MP, and ML methods based on nucleotide and amino acid sequences each employed six OTUs, comprising five species of Rhodobacter and one outgroup. The outgroup for 16S rRNA and 23S rRNA analysis was Paracoccus denitrificans, the outgroup for gyrB analysis was Paracoccus aminophilus, and the outgroup for gyrB was Rhodospirillum rubrum. A consensus tree was constructed from the several topologies obtained in the analysis based on the four molecules.

The total numbers of substitutions in the specified topologies were counted with MacClade 3.08a. Levels of synonymous (dS) and nonsynonymous (dN) nucleotide diversity were calculated with the YN00 program in the PAML package version 3.0a (Mac) (Yang 2000) using the method of Yang and Nielsen (2000). Codon usages were shown also using the YN00 program in the PAML package (Yang 2000).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Two Different cbbL Genes from R. azotoformans
Two types of sequences were obtained from cloned cbbL samples of R. azotoformans. One sequence was homologous to the cbbL of R. capsulatus in the GenBank database, and the other sequence was homologous to that of R. sphaeroides in the database. These sequences were deposited in the DNA Data Bank of Japan (DDBJ) (table 1).


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Table 1 Accession Numbers of the Partial Sequence Data Determined in This Study.

 
Determination of cbbL Type by PCR
In the PCR using selective primer sets for green-like cbbL, fragments of about 1 kbp were amplified from the samples using R. azotoformans, R. blasticus, R. capsulatus, and R. veldkampii DNA as templates (fig. 1). In the PCR using selective primer sets for red-like cbbL, fragments of about 800 bp were amplified from the samples using R. azotoformans and R. sphaeroides DNA as templates. These results indicate that R. blasticus, R. capsulatus, and R. veldkampii had only green-like cbbL genes, R. sphaeroides had only a red-like cbbL gene, and R. azotoformans had both green-like and red-like cbbL genes. The sequences of the amplified fragments were determined and deposited in the DDBJ databank (table 1).



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FIG. 1. PCR analysis for determination of type of cbbL genes in each species of Rhodobacter. Primer sets selectively amplifying green-like cbbL genes (RubiLF1-GREENcbbLR) (lanes 1 to 5) and red-like genes (RubiLF1-REDcbbLR) (lanes 6 to 10) were used. Lane 11 shows a 100-bp DNA ladder marker. Lanes 1 and 6 show samples using R. capsulatus DNA as templates. Lanes 2 and 7 are R. sphaeroides. Lanes 3 and 8 are R. azotoformans. Lanes 4 and 9 are R. blasticus. Lanes 5 and 10 are R. veldkampii. Lanes 1, 3, 4, and 5 show about 1,000-bp fragments amplified and lanes 7 and 8 show about 800-bp fragments amplified

 
Determination of cbbL Type by Hybridization
In the dot blotting, the probes for green-like cbbL genes hybridized samples of R. azotoformans, R. blasticus, R. capsulatus, and R. veldkampii, and the probes for red-like cbbL genes hybridized samples of R. azotoformans and R. sphaeroides (fig. 2). These results indicate that R. blasticus, R. capsulatus, and R. veldkampii had only green-like cbbL genes, R. sphaeroides had only a red-like cbbL gene, and R. azotoformans had both green-like and red-like cbbL genes.



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FIG. 2. Dot blot analysis for determination of type of cbbL genes in each species of Rhodobacter. Probes selectively hybridizing green-like cbbL genes (lane 1) and red-like cbbL genes (lanes 2) were used. Lane 1 shows that probes for green-like cbbL genes hybridized samples of R. azotoformans, R. blasticus, R. capsulatus, and R. veldkampii positively. Lane 2 shows that probes for red-like cbbL genes hybridized samples of R. azotoformans and R. sphaeroides positively

 
The result of the Southern hybridization is shown in figure 3. In lane 1 (probes for green-like cbbL), one signal appeared at the position of about 20 kb. In lane 2 (probes for red-like cbbL), one signal appeared at the position of about 10 to 15 kb. These results suggested that R. azotoformans had a green-like cbbL gene and a red-like cbbL gene.



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FIG. 3. Southern blot analysis for determination of the numbers of green-like and red-like cbbL genes in R. azotoformans. The genomic DNA digested with the restriction enzymes Hind III and Pst I was electrophoresed on 1% agarose gel and transferred onto positively charged nylon membrane. Probes selectively hybridizing green-like cbbL genes (lane 1) and red-like cbbL genes (lanes 2) were used. In lane 1, one signal appeared at the position of about 20 kb. In lane 2, one signal appeared at the position of about 10 to 15 kb

 
Sequencing of pufL and pufM Genes
The partial sequences of the pufL and M genes of R. azotoformans IFO 16436T and R. veldkampii IFO 16458T were determined and deposited in the DDBJ databank (table 1).

Phylogenetic Analysis of cbbL
The nucleotide and amino acid sequences of cbbL were aligned, the positions with gaps and the undetermined and ambiguous sequences were removed, and the remaining 945 nucleotide sites and 315 amino acid residues were used for these phylogenetic analyses. Trees obtained by the MP method based on nucleotide and amino acid sequences were used as starting topologies of ML analysis. The topologies of the resultant ML trees were same as those of the MP trees (figs. 4 and 5). In both trees, taxa were broadly divided into two groups, green-like and red-like, with 100% statistical reliability. Within the red-like group, R. spaeroides and R. azotoformans 1 (red-like) formed one cluster with high bootstrap values (100% and 95% based on nucleotide and amino acids, respectively), and within the green-like group, R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed one cluster with 46% and 45% bootstrap values.



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FIG. 4. The maximum-likelihood tree based on cbbL nucleotide sequences (945 sites). Search was performed using the maximum-parsimony tree as seed topology under the HKY85 model. The numbers on the branches in both trees indicate the local bootstrap probabilities (LBPs) as percentages. Taxa were roughly divided into green-like and red-like groups with 100% statistical reliabilities. R. spaeroides and R. azotoformans 1 formed one cluster within the red-like classification groups, and R. capsulatus, R. azotoformans 2 (green-like), R. blasticus, and R. veldkampii formed one cluster within the green-like groups. The topologies within the green-like groups are different from those of tree based on amino acid sequences

 


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FIG. 5. The maximum-likelihood tree based on cbbL amino acid sequences (315 residues). Search was performed using the maximum-parsimony tree as seed topology under the JTT model. The numbers on the branches in both trees indicate the local bootstrap probabilities (LBPs) as percentages. The topologies within the green-like groups are different from those of tree based on nucleotide sequences

 
The trees based on nucleotides and amino acids differed in the topologies within green-like Rhodobacter groups and the lengths of branches. In the tree based on nucleotides, R. blasticus was located in a basal position within the green-like Rhodobacter group, R. capsulatus and R. veldkampii formed a cluster at 82% bootstrap value, and the branches of Rhodobacter species were of similar length. In the tree based on amino acids, R. blasticus was also located in a basal position within the green-like Rhodobacter group, and R. azotoformans 2 and R. capsulatus form a cluster at 98% bootstrap value, with a very short branch between them. Moreover, the topologies within green-like Rhodobacter groups differ from those of the trees based on other molecules, namely, 16S rRNA, 23S rRNA, gyrB, and pufL (fig. 6).



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FIG. 6. Two topology patterns obtained by NJ, MP, and ML analyses based on the nucleotide and amino acid sequences of 16S rRNA, 23S rRNA, gyrB, and pufL. R. veldkampii is located in a basal position, and R. sphaeroides and R. azotoformans form a cluster

 
16S rRNA, 23S rRNA, gyrB, and pufL Phylogeny
The nucleotide and amino acid sequences of 16S rRNA, 23S rRNA, gyrB, and pufL were aligned, the positions with gaps and the undetermined and ambiguous sequences were removed, and the remaining 1,302 nucleotide sites, 2,732 sites, 675 sites (225 amino acid residues), and 588 sites (196 residues) were used, respectively, for these phylogenetic analyses.

In the analysis based on 16S rRNA nucleotide sequences, the different algorithms and different evolutional models all yielded topologies of pattern 1 (fig. 6A). In the analysis based on 23S rRNA nucleotide sequences, all methods yielded pattern 2 (fig. 6B).

In the analysis based on gyrB nucleotide sequences, the topology obtained by the NJ and MP methods differed from both pattern 1 and pattern 2 in having the cluster of R. sphaeroides and R. azotoformans in a basal position. However, the ML tree constructed using the NJ and MP trees as starting topologies was of pattern 1 topology. In the analysis based on gyrB amino acid sequences, all methods yielded pattern 1 topology.

In the analysis based on pufL nucleotide sequences, the NJ and MP trees had a topology in which R. veldkampii and R. blasticus in pattern 2 were interchanged, but the ML tree constructed using the NJ and MP trees as starting topologies was of pattern 2 topology. In the analysis based on pufL amino acid sequences, the NJ tree had pattern 2 topology, the MP tree had pattern 1, and the ML tree had pattern 2.

In sum, all analytical methods except those based on gyrB and pufL nucleotide sequences ultimately yielded topologies of patterns 1 and 2, the consensus tree of these two topologies is shown in figure 7. R. veldkampii is located in a basal position, and R. sphaeroides and R. azotoformans form a cluster.



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FIG. 7. A hypothesis of evolutional history of cbb genes within Rhodobacter. Consensus topology of the two patterns (fig. 6) is used as the underlay on which the parsimonious evolution of cbbL is superimposed. It is suggested that the common ancestor of Rhodobacter had only a green-like cbbL gene, that the common ancestor of R. azotoformans and R. sphaeroides subsequently gained a red-like cbbL gene by a horizontal gene transfer, and that the ancestor of R. sphaeroides later lost the green-like cbbL gene. If the horizontal gene transfer occurred in a predecessor of all species of Rhodobacter, our hypothesis is not inconsistent with that of Paoli et al. (1998)

 
Synonymous and Nonsynonymous Nucleotide Substitution Rate
Synonymous and nonsynonymous nucleotide substitution rates (dS and dN) and their ratio ({omega} = dN/dS) among green-like cbbL of Rhodobacter were calculated (table 2). Four genes (R. azotoformans 2, R. blasticus, R. capsulatus, and R. veldkampii) were used in the phylogenetic analysis (945 sites), and their G+C contents were 65.4%, 63.7%, 63.2%, and 61.3%, respectively.


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Table 2 Synonymous and Nonsynonymous Nucleotide Substitution Rate Ratio Among Green-like cbbL of Rhodobacter.

 
The {omega} values of R. veldkampii versus R. azotoformans 2, R. blasticus, and R. capsulatus were 0.024, 0.038, and 0.033. The {omega} values of R. blasticus versus R. azotoformans 2 and R. capsulatus were 0.054 and 0.052, which were about twice the {omega} value of R. veldkampii.

The {omega} value of R. azotoformans 2 versus R. capsulatus was 0.006, very low compared with the above values. This is a result of the low nonsynonymous nucleotide substitution rate (dN) value (0.007); the synonymous nucleotide substitution rate (dS) value (1.0773) is comparable to the values of other combinations.

Thus, it was shown that the synonymous and nonsynonymous nucleotide substitution rates among green-like cbbL genes of Rhodobacter were different.

Codon Usage
The codon usage of the two form I cbbL genes of R. azotoformans is shown in table 3.


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Table 3 The Codon Usage of Two Form I cbbL Genes of R. azotoformans.

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Green-like and Red-like cbb Genes of R. azotoformans
The present analysis has shown that R. azotoformans possesses both green-like and red-like cbbL genes, that R. blasticus, R. capsulatus, and R. veldkampii possess only green-like cbbL genes, and that R. sphaeroides possesses a only red-like cbbL gene. Previous work had indicated that R. capsulatus possess a green-like cbb gene and R. sphaeroides possess a red-like cbb gene (Gibson, Falcone, and Tabita 1991; Paoli et al. 1998). Thus, we investigated the kinds of cbbL of all species of Rhodobacter and for the first time identified an organism contains both green-like and red-like RubisCO genes. Dual form I cbb gene copies have been reported in Acidithiobacillus ferrooxidans, Allochromatium vinosum, Hydrogenovibrio marinus. and Ralstonia eutropha (Viale, Kobayashi, and Akazawa 1989; Kusano et al. 1991; Kusian et al. 1995; Nishihara et al. 1998). However, none of these bacteria has both green-like and red-like cbb genes. A. ferrooxidans, A. vinosum, and H. marinus possess only green-like genes, and R. eutropha possesses only red-like genes.

Kobayashi et al. (1991) stated that similarities between the dual genes of A. vinosum (rbcL-rbcS versus rbcA-rbcB) in codon usage and G+C content indicate the evolution from a common ancestral set of genes after duplication, rather than lateral gene transfer. The codon usage and the G+C contents of the two cbb genes of A. ferrooxidans, H. marinus, and R. eutropha were also basically identical in each species (data not shown). Thus, if Kobayashi's assertion is correct, it is unlikely that lateral gene transfers occurred recently in three organisms. In R. eutropha, the chromosomal and the megaplasmid-borne cbbL genes form a cluster within a red-like radiation and are homologous to each other (figs. 4 and 5), and we agree with the hypothesis of the gene duplication (Kusian et al. 1995; Delwiche and Palmer 1996). In A. ferrooxidans, A. vinosum, and H. marinus, which all belong to the {gamma}-Proteobacteria, Delwiche and Palmer (1996) stated that a duplication of the cbb gene occurred early in {gamma}-proteobacterial evolution, in a common ancestor of Allochromatium and Hydrogenovibrio (although they did not mention A. ferrooxidans, the statement also applies to this bacterium). However, the relations of their cbb genes are ambiguous in our phylogenic trees (figs. 4 and 5).

The presence of both green-like and red-like cbbL genes in R. azotoformans provides far clearer evidence of horizontal transfer (figs. 4 and 5). If the origin of dual cbb genes of this species was in duplication, the event of duplication must have occurred in a common ancestor of all green-like and red-like cbb genes. This hypothesis implies long coexistences of green-like and red-like cbb genes and many subsequent independent losses of green-like or red-like cbb genes in several lineages. To date, no organism except R. azotoformans has been identified as having both green-like and red-like cbb genes. The probability of this duplication hypothesis is low. The origin of the dual cbb genes of R. azotoformans is thus thought to be in lateral transfer. The similarities of codon usage (table 3) and G+C contents (green, 65.4%; red, 64.7%) of the two cbb genes of R. azotoformans are not consistent with the lateral transfer. The cause of the similarities may be effects of sufficient time for transferred gene to adjust to the recipient cell's compositional environment, of rapid amelioration of cbbL genes, or of the transfer between cells of similar codon usage and G+C contents (Delwiche and Palmer 1996).

Which gene, the green-like or the red-like, was transferred in R. azotoformans? We performed some analyses in order to clarify history of evolution of the cbb genes of Rhodobacter species.

Phylogeny of cbbL
In the cbbL trees, whether based on the nucleotide sequences or on amino acid sequences (figs. 4 and 5), taxa were broadly divided into green-like and red-like groups. In both trees, the cbb of Rhodobacter species formed clusters: R. spaeroides and R. azotoformans 1 clustered within the red-like group, and R. capsulatus, R. azotoformans 2, R. blasticus, and R. veldkampii clustered within the green-like group. This suggests that the red-like cbbL genes of Rhodobacter species were derived from one ancestor, and the green-like cbbL genes were derived from another ancestor. This allows simplification of the presumed evolutional history of the cbbL genes of Rhodobacter.

The topologies within green-like cbbL genes of Rhodobacter differ between the trees based on nucleotide sequences and amino acid sequences, and they also differ from the phylogenies based on four molecules, 16S rRNA, 23S rRNA, gyrB, and pufL (figs. 4 and 6). One reason for these conflicts is probably the heterogeneity of synonymous and nonsynonymous nucleotide substitution rate ratios among green-like cbbL genes of Rhodobacter (table 2). Table 2 shows that the nonsynonymous nucleotide substitution rate (dS) values are heterogeneous among Rhodobacter and that the dS value of R. azotoformans 2 versus R. capsulatus is especially low. This is reflected in the clustering of R. azotoformans 2 and R. capsulatus and their short branches in the amino acid tree (fig. 5). R. azotoformans 2 and R. capsulatus probably evolved through a restricted numbers of nonsynonymous substitutions. The similarity of the amino acid sequences of green-like cbbL of R. azotoformans and R. capsulatus, which are phylogenetically not close, suggests that the ancestral Rhodobacter amino acid sequences of green-like cbbL are comparatively well conserved in their species. As for R. blasticus and R. veldkampii, they probably evolved without restricting nonsynonymous substitutions. This may be reflected in differences of topologies among some phylogenic trees. The reason for heterogeneity of the nonsynonymous substitution rates among green-like cbbL genes is unverifiable. However, it will be related to the differing needs for CO2 fixation among different species. Rhodobacter species can grow not only photosynthetically but also heterotrophically, and each species inhabits different environments. It is interesting that species that are actively motile by means of flagella, R. azotoformans and R. capsulatus (Hiraishi, Muramatsu, and Ueda 1996), have a low nonsynonymous nucleotide substitution rate of green-like cbb genes, and nonmotile species, R. blasticus and R. veldkampii, have a high nonsynonymous nucleotide substitution rate.

Phylogeny of Rhodobacter as Organisms
In the analyses based on four molecules, 16S rRNA, 23S rRNA, gyrB, and pufL, the obtained topologies were of patterns 1 and 2 (fig. 6). The common features of patterns 1 and 2 are that R. veldkampii is located in a basal position and R. sphaeroides and R. azotoformans form a cluster. It is suggested that the genes of 16S rRNA, 23S rRNA, gyrB, and pufL were transferred vertically at least within the Rhodobacter group, and their phylogenic trees indicate the bacterial phylogenies.

Topologies of patterns 1 and 2 are supported by other properties of Rhodobacter species. During phototrophic growth with sulfide as the electron donor, Rhodobacter species oxidize sulfide to elemental sulfur, and only R. veldkampii can oxidize sulfur further to sulfate after sulfide depletion in batch cultures (Hansen and Imhoff 1985). Only R. veldkampii can utilize thiosulfate as the electron donor, cannot assimilate the sulfate donor, and requires p-aminobenzoate. These properties resemble those of marine photosynthetic Rhodovulum species (Hansen and Imhoff 1985), which have a common ancestor with Rhodobacter species and are phylogenetically located out of the Rhodobacter group (Hiraishi and Ueda 1994).

R. azotoformans is a mesophilic, nonhalophylic, facultative photoheterotroph that has motile ovoid cells, contains vesicular photosynthetic membrane systems together with bacteriochlorophyll a and carotenoids of the spheroidene series, and produces ubiquinone-10 as the sole quinone component. It also requires biotin, niacin, and thiamine as growth factors, photoassimilates a wide variety of organic compounds as carbon sources, and uses sulfide at low concentrations as a photosynthetic bacteria. In these respects, R. azotoformans is most similar to R. sphaeroides, in particular to denitrifying variants of this species (Hiraishi, Muramatsu, and Urata 1995). The capacity for denitrification has been found in only a limited number of anoxygenic phototrophic bacteria (Hiraishi, Muramatsu, and Urata 1995).

In this paper, we regard the consensus topology of patterns 1 and 2 (fig. 6) as indicating the evolutional history of bacteria.

Evolutional History of cbb Genes of Rhodobacter
The consensus topology of patterns 1 and 2 (fig. 6) was used as an underlay, on which the parsimonious evolution of cbbL was expressed (fig. 7). Since R. veldkampii in the basal position has a green-like cbbL gene, it is suggested that the common ancestor of Rhodobacter had a green-like cbbL gene. If the common ancestor of Rhodobacter had both green-like and red-like cbbL genes, many independent losses of red-like genes must subsequently have occurred in each lineage, and the probability of this is low. Since it is suggested that the green-like genes of Rhodobacter derived from one ancestor, the lateral transfer of green-like cbbL genes from other organisms should only have occurred in a common ancestor of Rhodobacter species. Likewise, the red-like cbbL genes of Rhodobacter are also suggested to derive from one ancestor, and this ancestor should have been transferred to Rhodobacter species laterally in one event. Consequently, it is reasonable to propose that the common ancestor of Rhodobacter had only a green-like cbbL gene, that the common ancestor of R. azotoformans and R. sphaeroides obtained a red-like cbbL gene by a horizontal gene transfer, and that the ancestor of R. sphaeroides later lost the green-like cbbL gene (fig. 7).

Paoli et al. (1998) have reported that the form I green-like cbb operon was acquired by R. capsulatus via one horizontal gene transfer from a bacteria containing a green-like RubisCO. This was supported by the finding of Horken and Tabita (1999) that enzymological properties of the form I RubisCO of R. capsulatus resemble certain chemoautotrophic proteobacteria and cyanobacteria. If the horizontal gene transfer occurred in a predecessor of all species of Rhodobacter, our hypothesis is not inconsistent with that of Paoli et al. (1998) (fig. 7). However, the antibodies that Paoli et al. (1998) used for the immunological experiment showing that R. capsulatus may be unique among the nonsulfur purple photosynthetic bacteria were antibodies against R. sphaeroides form I (red-like) RubisCO, which is the minority type among Rhodobacter species.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Kazunori Hatano, Institute for Fermentation, Osaka (IFO), Japan for valuable suggestions and discussions. We thank Yayoi Yamaguchi for her technical support of sequence analysis.


    Footnotes
 
E-mail: uchino{at}nbrc.nite.go.jp. Back

Geoffrey McFadden, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication January 22, 2003.