* Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki, Japan
Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka, Japan
Fermentation & Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
¶ National Institute of Technology and Evaluation, Shibuya, Tokyo, Japan
Correspondence: E-mail: tgojobor{at}genes.nig.ac.jp.
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Abstract |
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Key Words: Corynebacterium evolution comparative genomics amino acid biosynthesis horizontal gene transfer
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Introduction |
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We have previously sequenced and annotated the genome of Corynebacterium efficiens (Nishio et al. 2003). This bacterium is a close relative of Corynebacterium glutamicum, which has been widely used in the industrial production of glutamate, lysine, and other amino acids by fermentation. The two species are recognized as glutamic acidproducing Corynebacteria (Fudou et al. 2002). The optimal temperature for glutamate production by C. glutamicum is approximately 30°C, and the microorganism does not grow or produce glutamate at temperatures of 40°C or above. On the other hand, C. efficiens can grow and produce glutamate at temperatures greater than 40°C. On the basis of genome comparisons between these two species, three kinds of amino acid substitutions were suggested to be responsible for the thermostability of C. efficiens and the increase of 10% in genome GC content in C. efficiens (Nishio et al. 2003). In addition, the comparative genome-sequence analysis suggested that the absence of a RecBCD pathway may have been responsible for suppressing genome shuffling in Corynebacterium (Nakamura et al., 2003). One of our research interests is the extent to which the genetic control of amino acid biosynthesis differs between these closely related species. It is well known that C. glutamicum overproduces glutamic acid under a variety of conditions such as biotin limitation (Kimura 2003). We are interested in the evolutionary events responsible for the acquisition of this feature. Furthermore, C. glutamicum also overproduces lysine, arginine, threonine, isoleucine, valine, serine, tryptophan, phenylalanine, and histidine (supplementary table 1; Ikeda 2003). It is, therefore, of great interest to investigate the evolutionary processes involved in the acquisition of these productive capabilities. Corynebacterium diphtheriae is a well-known pathogen (Collins and Cummins 1986, Graevenitz and Krech 1991) whose genome has been sequenced by the Sanger Center (Cerdeno-Tarraga et al. 2003). Although the main focus of interest in the study of C. diphtheriae has been its pathogenicity, we were interested in understanding the evolutionary process of functional differentiation between the amino acid producing species and this pathogenic strain.
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Materials and Methods |
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Phylogenetic Analysis
The Blast (Altschul et al. 1997) and FASTA (Pearson 2000) programs were used for database searches, and ClustalW (Thompson et al. 1997) for multiple alignments. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method with P distance or Kimura's distance (Saitou and Nei 1987). Estimates of synonymous (Ks) and nonsynonymous (Ka) per sites and standard deviations were calculated using Li's method (Li 1993) implemented in DAMBE (Xia and Xie 2001). We also used the Nei and Gojobori method (Nei and Gojobori 1986), but it gave virtually the same results.
Comparison of Gene Contents in Corynebacterium
Multiple alignments and phylogenic trees were constructed of the high-GC gram-positive bacteria, C. efficiens, C. glutamicum, C. diphtheriae, M. tuberculosis, M. leprae, and S. coelicolor, using all highly conserved proteins involved in amino acid biosynthesis. Criteria for highly conserved sequences were defined using the FASTA program. The query sequences used in the FASTA program searches were from C. glutamicum or C. efficiens. The Z scores of the FASTA program, identities of overlapping regions, and detected sequence lengths were used to establish the highly conserved sequences. All alignments were checked manually.
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Results |
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The phylogenetic tree of AroQ was constructed in the same manner as that of IlvD and its topology also differed from the 16S rRNAbased phylogenetic tree (figs. 1, 2C). C. efficiens CE1739, C. diphtheriae DIP1342, Corynebacterium, pseudotuberculosis, M. leprae ML0519, and M. tuberculosis Rv2537c form a cluster in the phylogenetic tree. AroQ in C. efficiens CE1739, C. diphtheriae DIP1342, M. leprae ML0519, and M. tuberculosis Rv2537c is part of the aroCKBQ operon. Another AroQ cluster was composed of an additional aroQ in C. efficiens CE0442, C. glutamicum Cgl0423, and S. coelicolor SCO1961. The additional aroQ in C. efficiens CE0442 and aroQ in C. glutamicum Cgl0423 lie next to aroE on the chromosome, whereas in S. coelicolor SCO1961 there is no nearby aromatic amino acid biosynthesis gene. These results suggest that the evolution of the aroQ gene in high-GC gram-positive bacteria is related to operon organization, and it is curious that C. efficiens retained two aroQ genes within conserved operon structures.
The phylogenetic tree of GlnA showed that the paralogous GlnA of C. efficiens CE2116 was positioned outside that of C. diphtheriae DIP1644 (fig. 2D). This result suggests that glnA of C. efficiens CE2116 was not acquired by gene duplication within its own evolutionary linage (unless it is a pseudogene), but rather by gene duplication in the common ancestor of Corynebacterium, or by horizontal gene transfer. To find a more likely explanation, we compared the genome structures of the three Corynebacteria (fig. 3). In C. efficiens and C. diphtheriae, there were additional genes next to orthologous GlnA when compared to C. glutamicum. These additional genes are from CE2105 to CE2116 in C. efficiens and DIP1644 to DIP1661 in C. diphtheriae, as shown in Fig. 3. These genes were dissimilar at both the DNA and amino acid levels, implying that they were acquired independently in each species. The C. diphtheriaespecific genes are annotated as putative phage-related and antibiotic resistancerelated pathogenicity island and showed unusual GC content and dinucleotide signature (Cerdeno-Tarraga et al. 2003). This result suggested that the C. diphtheriaespecific genes were acquired by horizontal gene transfer. On the other hand, the paralogous ocd gene (CE2115) encoding ornithine cyclodeaminase and the paralog glnA (CE2116) (fig. 3) were C. efficiensspecific genes. The paralogous ocd gene (CE2115) was located next to the paralog glnA (CE2116) in C. efficiens. The phylogenetic tree of Ocd showed that the paralogous Ocd of C. efficiens (CE2115) was positioned outside the orthologous corynebacterial Ocd (CE1700, Cgl1582) (fig. 2E). Moreover, C. diphtheriae has lost the ocd gene. The orthologous ocd gene was not located near the orthologous glnA in C. efficiens CE2104 and C. glutamicum Cgl2214, suggesting that the paralogous glnA (CE2116) and paralogous ocd (CE2115) genes of C. efficiens were not acquired by gene duplication in the common ancestor of Corynebacterium. A possible explanation was the lack of a RecBCD pathway (Nakamura et al. 2003), genome rearrangement could not take place, and duplicated genes must remain close to where they originate. Another possible explanation was that it was a pseudogene. An analysis of the number of nonsynonymous versus synonymous substitutions showed a larger number of nonsynonymous substitutions in the paralogous glnA of C. efficiens (CE2116) than in the orthologous corynebacterial glnA (CE2104, Cgl2214, DIP1644); however, the number was not as high as in Mycobacterium, and GC-content analysis showed that there was no difference in the second-position GC content (tables 2, 3). If the paralogous glnA gene was a pseudogene on which there were no functional constraints, a significant difference would be observed in the second-position GC content of paralogous gene when comparing with that of orthologous gene. Evidently, the paralogous glnA of C. efficiens (CE2116) is not a pseudogene, but was acquired by horizontal gene transfer.
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One of the biologically important characteristics in C. glutamicum is that it has been known to be a biotin-requirement organism (Kimura 2003). The biotin requirement is also observed in C. efficiens. These bacteria lack the complete biotin biosynthesis pathway from pimelate to biotin. Glutamic acid overproduction in C. glutamicum is caused by the shortage of biotin (Kimura 2003). It is of interest to note that C. diphtheriae may not be a biotin-requiring organism because it possesses the complete biotin biosynthesis pathway. From this reason, it is strongly speculated that C. diphtheriae does not possess the glutamic acid overproduction mechanism induced by the biotin limitation. Moreover, in C. diphtheriae, DIP1381 encoding 6-carboxyhexanoateCoA ligase as the first enzyme in biotin biosynthesis, may have been acquired by horizontal gene transfer in C. diphtheriae (table 1 and supplementary table 2). This is because any other high-GC gram-positive bacteria except C. diphtheriae did not possess orthologous genes of DIP1381.
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Discussion |
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The loss of genes in C. diphtheriae may be correlated with the microorganism's loss of amino acidproduction capability. Our analysis suggested that C. diphtheriae has lost many genes present in the common ancestor and that this loss is reflected in its genome size. C. diphtheriae lacks the genes gltBD, ddh, metE, and metB, whose products encode redundant pathways for glutamate, lysine, and methionine biosynthesis in the amino acidproducing species (fig. 4). Surprisingly, C. diphtheriae has also lost all genes of the sulfur incorporation pathway, suggesting that it cannot synthesize cysteine. The addition of cysteine was critical for toxin production and cell growth of C. diphtheriae (Nagarkar et al. 2002), consistent with the absence of the sulfur incorporation pathway.
To estimate the evolutionary events needed for the capacity for amino acid overproduction in the industrially useful Corynebacteria genome, we compared amino acid biosynthesisrelated genes of C. efficiens and C. glutamicum. We found that although amino acid biosynthesis pathways were well conserved, the number of paralogs related to amino acid biosynthesis differed (table 1). Our phylogenetic analysis suggested that the paralogs glnA (CE2116) (Schulz et al., 2001) and ocd (CE2115) of C. efficiens were acquired by horizontal gene transfer. If ocd and glnA paralogous genes were acquired together, then the creation of the ammonia recycle pathway could be achieved in C. efficiens. Gene transfer may, therefore, be one of the important factors in the evolution of the amino acidproducing species.
Choice for particular genes may also have been important in the evolution of bacterial phenotypes. In the phylogenetic tree of AroQ (fig. 1C), one cluster contains only nonpathogenic bacteria, and another pathogenic bacteria other than C. efficiens (the pathogenic cluster). This was the only phylogenetic tree of all the phylogenetic trees for amino acid biosynthesisrelated genes in the high-GC gram-positive bacteria to show that Corynebacteria are separated into two clusters of pathogens and nonpathogens. One possible evolutionary explanation is that gene duplication occurred in the common ancestor of the high-GC gram-positive bacteria and that, as a result of the choice, each species except C. efficiens, lost one of the two aroQ genes, depending on their phenotypic features. Mutation of the common aromatic amino acid biosynthetic gene for the inhibition of the folic acid biosynthesis is one of the strategies for vaccine development against pathogenic bacteria. In fact, it has been observed that the growth in more than 10 pathogens was attenuated by single mutation of aromatic amino acid biosynthesisrelated genes (aro genes) (Simmons et al. 1997). In C. pseudotuberculosis, mutation of aroQ weakened the pathogenicity of the microorganism in the mouse (Simmons et al., 1997). Thus, aroQ may be related to pathogenicity.
To understand the phylogeny of IlvD, there are two possible evolutionary events: ancient gene duplication or horizontal gene transfer (fig. 2B). Our results suggested that ilvD in C. efficiens was acquired by ancient gene duplication rather than by horizontal gene transfer. In the case of TrpB, the phylogenetic tree clearly showed that gene duplication had occurred in the common ancestor and that C. glutamicum may have lost the duplicated ORF (fig. 2A). The paralogous trpB was located near the orthologous trpB in C. efficiens and C. diphtheriae. This location in the Corynebacteria supports the rule that duplicated genes are located next to one another owing to the absence of genome rearrangement resulting from the lack of an RecBCD pathway (Nakamura et al. 2003). It has been proposed that the paralogous trpB in C. diphtheriae is a pseudogene because of the long branch length (Xie et al., 2002). However, persistence of this paralog in C. diphtheriae but not in C. glutamicum seems strange because C. diphtheriae appears to have lost many genes during its evolution and the selective pressure to discard unnecessary genes appears to have been much higher in its case than in the case of C. glutamicum.
Our findings suggest that almost all the genes required for amino acid production already existed in the common ancestor of Corynebacterium. We also believe that newly acquired genes in glutamic acidproducing Corynebacteria contribute to amino acid overproduction capacity. Actually, ddh, one of the newly acquired genes in the amino acidproducing species, has been known to contribute to lysine production in C. glutamicum. An interesting question is whether the newly acquired and previously unrecognized enzyme phosphoenolpyruvate synthase in C. efficiens and C. glutamicum contributes to the ability of these Corynebacteria to overproduce amino acids. Previous studies of glutamate and lysine production have not highlighted the existence of this enzyme. For example, Park et al. (1997) did not assume this enzyme in the flux calculation for lysine production in C. glutamicum. In Escherichia coli, the same enzyme plays an important role in the production of aromatic compounds (Yi et al. 2002), and, furthermore, pps and its homolog in Thauera aromatica were isolated as phenol-induced proteins (Breinig et al. 2000). In fact, aroG encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, which is on the aromatic amino acid biosynthesis pathway, may have been retained in C. efficiens and C. glutamicum, although it was lost in C. diphtheriae, Mycobacteria, and Streptomyces. Because the gene for benzoate 1,2-dioxygenase reductase, which is related to genes for benzoate degradation (CE2306, Cgl2405), was newly acquired in amino acidproducing species (table 1), phosphoenolpyruvate synthase may cooperate with that gene in these Corynebacteria. Thus, newly acquired genes may also contribute to productivity of amino acids. Small numbers of those genes' homologs are found among known protein sequences. Therefore, they may be have been acquired by horizontal gene transfer.
We have now shown differences in gene contents among Corynebacteria. These differences may provide a clue for elucidating the regulatory mechanisms for amino acid overproduction. Although we do not know the regulatory sequences related to glutamic acid production in C. glutamicum, the comparison of regulatory regions of glutamate overproductionrelated genes among different species may lead to an overview of the regulation for amino acid production mechanism. In C. glutamicum, there may be a strong relationship between the attenuation of 2-oxoglutarate dehydrogenase (ODH) activity and glutamic acid production (Shimizu et al. 2003). One of our interests is the similarity of the regulatory regions among three species of Corynebacteria. The regulatory regions of odhA gene encoding ODH were more strongly conserved between C. efficiens and C. glutamicum than between C. diphtheriae and C. glutamicum or C. efficiens (supplementary fig. 2). On the other hand, enhanced glutamate dehydrogenase (GDH) activity may not contribute to glutamic acid production (Shimizu et al. 2003). The conservation of regulatory regions for gdh genes encoding GDH were almost the same (supplementary fig. 3). These results are consistent with the previous knowledge of glutamic acid production, suggesting the lack of glutamic acid overproduction mechanism in C. diphtheriae. The latter is also supported by the complete biotin biosynthesis pathway of C. diphtheriae. As mentioned earlier, the comparison of regulatory regions among three species of Corynebacteria may be important for studying regulatory systems of amino acid production. In this case, we may have to assume that the important part of regulatory regions is conserved in spite of a difference in the genome GC contents. Note that the genome GC content of C. efficiens was 10% higher than that of C. glutamicum or C. diphtheriae (Nishio et al. 2003).
In this study, we have attempted to analyze the evolutionary process by which the capacity for amino acid overproduction was acquired by glutamic acidproducing Corynebacteria. Gene transfer/duplication/loss events in Corynebacteria may facilitate the formation of amino acid overproduction mechanisms. Retention of ancestral genes and gain of new genes by horizontal gene transfer may have been the major motive forces in establishing the capability of these bacteria for amino acid overproduction, whereas gene loss may have resulted in the loss of that capacity by C. diphtheriae. We have also found some genes that may be responsible for different amino acid productivity between C. efficiens and C. glutamicum by using comparison and detailed analysis of their genome sequences. Experimental analysis will be needed to clarify the contribution of these genes and their regulatory sequences to the overproduction of amino acids.
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Acknowledgements |
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Footnotes |
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