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Applied and Environmental Microbiology, June 2005, p. 3369-3372, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3369-3372.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Large-Scale Engineering of the Corynebacterium glutamicum Genome

Nobuaki Suzuki,1 Satoshi Okayama,1,2 Hiroshi Nonaka,1 Yota Tsuge,1,2 Masayuki Inui,1 and Hideaki Yukawa1,2*

Microbiology Research Group, Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan,1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan2

Received 28 October 2004/ Accepted 21 December 2004


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ABSTRACT
 
The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.


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INTRODUCTION
 
The complete genome sequences of greater than 185 microorganisms have been determined, and they are becoming an important resource for the comprehensive understanding of cellular life. Among strains of Corynebacterium glutamicum, bacteria widely used for the industrial production of amino acids, nucleic acids, and organic acids (11, 15), two strains, R (3,314,179 bp) (our unpublished data) and ATCC 13032 (3,309,401 bp [6] or 3,282,708 bp [10]), have been sequenced. Based on whole genome sequences, strain reconstruction studies for improved industrial application have been initiated (21).

By using the genome information of C. glutamicum, we recently found many strain-specific regions existing as "islands" in the common backbone (24). Gene loss and horizontal gene transfer are major genetic processes of genome evolution. These strain-specific islands (SSIs) were possibly shaped on the genome of the ancestral common strain of two C. glutamicum strains by the integration and deletion of many genes. The existence of similar islands was reported for Escherichia coli as K-, O-, or C-islands (13). Genes encoding antibiotic resistance factors, bacteriocins, and specific metabolic functions, such as enzymes involved in the degradation of xenobiotic compounds, are often found in these islands with many transposable elements and cryptic prophages (5, 16, 20, 22). In contrast, genes predicted to have basic core functions of cells are frequently annotated on the backbone regions. This information provides us with the working hypothesis that strain-specific islands may be deleted.

Can cells survive without SSIs? We chose to delete each of 11 SSIs that are larger than 10 kb among thousands of C. glutamicum R strain-specific islands. As a result, every strain successfully lost each targeted island, but all 11 mutants showed growth equivalent to the wild strain, indicating that a total genomic region of 250 kb carrying 233 predicted open reading frames was nonessential for cell survival under normal laboratory conditions. The combination of comparative genomics and large segment deletion was thus demonstrated to be a powerful tool for analyzing genomic and gene functions, and this approach will also be useful in creating improved cells for bioindustry.


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Scheme for deletion of the C. glutamicum R genome.
 
SSIs of the C. glutamicum R strain larger than 10 kb are depicted in Fig. 1. These islands exist in the highly conserved common backbone whose conservation rate is 97.3% between strains R and 13032 (24). The conserved backbone hypothetically includes genes coding for the core functions of C. glutamicum. In contrast, SSIs showed very low homology to other parts of the two C. glutamicum genomes, and their functions may be dispensable. We selected these SSIs as excision targets and deleted these islands by using the Cre/loxP recombination system. Cre/loxP is a simple two-component system currently recognized as a powerful DNA recombination tool. Cre recombinase can catalyze the reciprocal site-specific recombination of DNA at 34-bp loxP sites. When two loxP sites are in the same orientation on a linear DNA molecule, Cre-mediated intramolecular recombination resolves with the excision of the loxP-flanking region (14). It does not require any host cofactor or accessory protein (4). Successful genetic manipulation of the bacterial genomes of Escherichia coli (26), Lactococcus lactis (1), and C. glutamicum (24) has been accomplished with this recombination system.



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  		FIG. 1. Illustration of SSIs larger than 10 kb in the C. glutamicum R genome. The lengths are the following: for 1, 15.6 kb; for 2, 11.1 kb; for 3, 56.2 kb; for 4, 22.6 kb; for 5, 32.8 kb; for 6, 16.2 kb; for 7, 16.5 kb; for 8, 45.1 kb; for 9, 16.3 kb; for 10, 14.6 kb; and for 11, 12.0 kb.

In order to excise SSIs, three plasmids were constructed (Fig. 2). pCRA411 and pCRA412 are suicide vectors that introduce the loxP site into the chromosome via homologous recombination, and pCRA406 is a replicative Cre expression vector for C. glutamicum (24). First, approximately 1-kb short segments carrying the 5' and 3' flanking regions of a target SSI were amplified by PCR and integrated into pCRA411 or pCRA412 with the spectinomycin (Sp) or kanamycin (Km) resistance gene, respectively. Both pCRA411 and pCRA412 were integrated into C. glutamicum by electroporation (24). Cells that integrated two loxP sites into the genome were selected with corresponding antibiotics (Km, 50 µg/ml; Sp, 200 µg/ml) and confirmed by direct cell PCR. Second, the resultant cells were transformed by pCRA406 and selected by chloramphenicol (5 µg/ml). Cre recombinase was constitutively expressed under the lac promoter (3).



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  		FIG. 2. Schematic representation of Cre/loxP-mediated deletion of the C. glutamicum R genome. Kmr, Spr, and cat indicate kanamycin, spectinomycin, and chloramphenicol resistance genes, respectively. GR1 and 2 are short segments of the C. glutamicum R genome. These segments were amplified by PCR and integrated into plasmids for homologous recombination. Black arrows (P1 and P2) under the chromosome represent PCR primers.

In this experiment, successful deletion strains lost both Sp and Km resistance genes along with each SSI. We selected cells which lost antibiotic resistance and confirmed the deletion of SSIs by PCR using primers P1 and P2 (Fig. 2). Furthermore, PCR products of primers P1 and P2 were extracted from gels, and their DNA was sequenced. Cultivation of C. glutamicum was performed at 33°C in complex medium (7).


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Result of large segment deletions.
 
Deletions of SSIs 1 and 3 were described earlier (24). In this study, the remaining nine SSIs were excised. In each of the deletion experiments, many colonies grew as a result of the transformation of Cre expression vector. For each SSI deletion, 96 of these colonies were transferred to a new plate and monitored for Km or Sp resistance. All of them showed Km and Sp sensitivity. One colony from each SSI deletion experiment was selected and used in subsequent experiments. The antibiotic resistances of the resultant deletion strains are shown in Fig. 3A and B. In order to confirm the deletion of the SSI, PCR was performed, and the 2-kb DNA fragments were successfully amplified with primers P1 and P2. No fragments were observed from the parental strain (Fig. 3C). The sequences of PCR products were determined with P1 and P2, and GR1 was directly connected to GR2 via a loxP site (Fig. 2). These results confirmed the excision of SSIs by the Cre/loxP-mediated recombination reaction. Deletion strains were designated RD1 to RD11, corresponding to SSIs 1 to 11.



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  		FIG. 3. The result of Cre expression in order to delete the SSIs. The upper and lower parts of each plate are from after and before transformation of pCRA406. The numbers on the plate indicate the SSI used for deletion experiments. Cells were plated on complex medium containing (A) kanamycin and spectinomycin or (B) no antibiotics and incubated for 24 h at 33°C. (C) Verification of SSI deletion was conducted by PCR. Direct cell PCR was performed, and about 2-kb DNA fragments were successfully amplified with P1 and P2 primers with the Cre+ strain. No fragments were observed from the strains before transformation of pCRA406. M indicates the marker.


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Growth rates of deletion strains.
 
Wild-type and deletion strains (RD1 to RD11) were cultivated in minimal medium (7). Cells were initially cultured in 2.5 ml complex medium, centrifuged, and washed twice with 5 ml minimal medium. They were then diluted in approximately 1.0 ml of minimal medium and their optical densities adjusted to 10.9 to 11.7. Eight hundred microliters of each was inoculated in 100 ml minimal medium. The initial optical density at 610 nm (OD610) of the cultures was 0.10 to 0.13. Despite the loss of numerous genes, no significant differences in growth rate or final OD610 were observed between the deletion strains and the wild type (Table 1).


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  		TABLE 1. The features of SSIs and cells with SSI deletionsa


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Features of strain-specific regions.
 
A total of 11 SSIs were deleted from the C. glutamicum R genome. Their sizes, GC contents, and predicted open reading frame numbers are listed in Table 1. The average GC contents of SSIs 3 and 4 clearly differed from that of other regions (for SSI 3, SSI 4, and the whole genome, 60.7, 45.7, and 54.1%, respectively), indicating horizontal gene transfer. SSI 3 and SSI 5 carried many transposable elements, and SSI 8 and SSI 10 had the sequences of corynephage {phi}304L (19) and {phi}16 attP and attB (18). Genes encoding phenylacetic acid degradation enzymes were found as an operon on SSI 6. Many genes coding for xenobiotic compounds exist as extrachromosomal genetic elements and are often integrated into the host chromosome (9, 25). Interestingly, however, a similar operon was found in the Corynebacterium efficiens genome but not in that of the closest strain of C. glutamicum, ATCC 13032. These results indicate that the SSIs of C. glutamicum R were possibly shaped by an evolutionary process of horizontal gene transfer from other bacteria, and the deletion of these genes hypothetically has little effect on the survival of C. glutamicum cells under laboratory conditions. No significant features were found in the remaining SSIs, SSIs 1, 2, 7, 9, and 11.


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Conclusion.
 
In this study, by using comparative analysis of C. glutamicum genomes, we predicted 11 regions that were nonessential for cell survival and through experiments confirmed that they are not needed for cell growth under normal laboratory conditions. DNA sequences also indicated that these regions were recently shaped through an evolutionary process and may not have included the core functions of C. glutamicum.

The deletion of strain-specific islands is the first step in reducing the genome size of bacteria. Genomes probably contain many genes that are nonessential for cell survival (2, 12, 17), and the creation of host cells in which unnecessary genes are deleted for bioprocesses would give potential advantages for bioindustry. Genes which express by-products during production drive up the product purification costs. Recently, we proposed the concept of minimum genome factories (8). This can be defined as recombinant strains whose metabolism is engineered to acquire desired products. It typically involves an exhaustive reduction of the genome to the optimal minimum subset, as defined by the targeted application. The utilization of such large segment deletion methods will greatly contribute in creating minimum genome factories.

C. glutamicum is one of the most widely used bacteria for bioindustry. Though the improvement of C. glutamicum is important for the enhanced production of biochemicals, numerous gene functions remain unknown. Even with E. coli, the most thoroughly studied bacterium on the molecular level, functions of 20% of its 4,285 annotated genes are still not revealed (23). Thus, the large segment deletion approach will be useful in facilitating the investigation of unknown gene functions.


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Nucleotide sequence accession numbers.
 
DNA sequences of SSIs and genes predicted on these regions were registered with DDBJ (http://www.ddbj.nig.ac.jp/Welcome-e.html). The accession numbers are as follows: for SSI 1, AB185495; for SSI 2, AB193029; for SSI 3, AB193030; for SSI 4, AB193031; for SSI 5, AB193032; for SSI 6, AB193033; for SSI 7, AB193034; for SSI 8, AB193035; for SSI 9, AB193036; for SSI 10, AB193037; and for SSI 11, AB193038.


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ACKNOWLEDGMENTS
 
This study was carried out as a part of the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by Ministry of Economy, Trade & Industry (METI) and entrusted by New Energy and Industrial Technology Development Organization (NEDO).


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology Research Group, Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizu-Cho, Soraku-Gun, Kyoto 619-0292, Japan. Phone: 81-774-75-2308. Fax: 81-774-75-2321. E-mail: mmg-lab{at}rite.or.jp. Back


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Applied and Environmental Microbiology, June 2005, p. 3369-3372, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3369-3372.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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