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Applied and Environmental Microbiology, May 2006, p. 3750-3755, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3750-3755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum and Construction of a Single-Gene Disruptant Mutant Library

Nobuaki Suzuki,¹ Naoko Okai,¹ Hiroshi Nonaka,¹ Yota Tsuge,^1,2 Masayuki Inui,¹ and Hideaki Yukawa^1,2*

Microbiology Research Group, Research Institute of Innovative Technology for the Earth, 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 15 November 2005/ Accepted 17 February 2006

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 
A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 
The decoding of bacterial genome sequences is progressing at a remarkably rapid pace. The complete genome sequences of nearly 300 bacteria have already been registered and form an important resource for the comprehensive understanding of cellular life. However, at least half of the open reading frames (ORFs) listed for each sequenced species are annotated as having either a hypothetical or an unknown function. Despite recent advances in bacterial genomics, including developments such as high-throughput techniques like microarrays, the process of gene characterization is still rather slow. Many cellular processes remain poorly understood. Even with Escherichia coli, the bacterium most studied at the molecular level, the functions of 20% of its 4,285 annotated genes are still not known (19).

A popular approach to identify the functions of genes is to construct gene disruption mutants and look for consequent phenotype changes. For Bacillus subtilis, construction of comprehensive gene knockouts on a genomic scale is complete (12). For E. coli and Saccharomyces cerevisiae, a library of single-gene deletions using a PCR-based mutagenesis approach is nearly complete (http://shigen.lab.nig.ac.jp/ecoli/pec/index.jsp) (3). However, though this approach is useful in identifying the roles of various important genes, it requires numerous deletion experiments, the creation of target vectors, and complex PCR experiments.

In contrast, the alternative strategy of transposon-insertion mutagenesis can easily generate large mutant pools. This approach is cost-effective and applicable to a wide variety of bacteria. Its drawbacks lie first in the difficulty in identifying transposon insertion sites, necessitating complex procedures, and second in the dependence of the position of the transposon insertion on the transposon characteristics.

phi29 DNA polymerase from the B. subtilis phage 29 has a unique capacity for strand displacement. It replicates DNA strands on the denatured linear DNA, displacing the downstream DNA fragment (14). By using this polymerase, a rolling-circle DNA amplification method which amplifies DNA templates 10,000-fold in a few hours was recently developed (2). It can obviate many genomic DNA extraction steps. To determine the transposon insertion location, the more efficient thermal asymmetric interlaced PCR (TAIL-PCR) can be used instead of cloning or inverted PCR (13). It can amplify unknown DNA sequences adjacent to known sequences such as transposons (11).

In this study an artificial transposon, miniTn31831, was constructed using insertion sequence IS31831 with 24-bp imperfect indirect terminal inverted repeats, which belongs to the ISL3 family (15, 21, 22). It exhibits no obvious target sequence specificity and has a transposition efficiency of approximately 4 x 10⁴ mutants per µg DNA (21). A Tn5-based minitransposon is also randomly inserted into the host's genomic DNA (4, 6). It belongs to the IS4 family and has been shown to insert via transposition in the chromosomes not only of its native host, E. coli, but also of Salmonella enterica serovar Typhimurium, Proteus vulgaris, Corynebacterium diphtheriae, and S. cerevisiae (5, 15, 17). DNA sequences favored for insertion by miniTn31831 are different from those favored by Tn5-based minitransposons (7, 22). Since the variety of insertion sites depends on transposon characteristics, generating mutants in every targeted gene would be very difficult. However, this drawback can be overcome by using different types of transposons.

By using the combination of two kinds of transposons, direct genomic DNA amplification by phi29 polymerase, and TAIL-PCR, we developed a novel, high-throughput transposon mutagenesis technique. Using this method, which bypasses some limitations of transposon mutagenesis, a comprehensive single-gene disruption mutant library covering nearly 80% of the 2,990 ORFs of Corynebacterium glutamicum R was generated. Thirty of the 2,332 disrupted genes showed high homology to essential genes of E. coli or B. subtilis but are nonessential in C. glutamicum. This approach could be useful in understanding and improving cell features for bioindustry, given the wide use of C. glutamicum for biochemical production (10, 16).

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 
A schematic diagram of the generation of the mutant library and identification of insertion locations is illustrated in Fig. 1. Each miniTn31831 transposon (pMV23 plasmid) and Tn5-based minitransposon (EZ::TnKan2 transposome system; Epicenter, WI) was inserted into C. glutamicum R by electroporation (6, 21). Cells were subsequently spread plated on A medium containing 50 µg/ml kanamycin and incubated for 1 to 2 days at 33°C. Transposon insertion mutants appeared at insertion efficiencies of 2.0 x 10⁵ and 3.0 x 10⁴ CFU/µg, respectively, for miniTn31831 and the Tn5-based minitransposon. Totals of 10,259 miniTn31831-generated and 7,281 Tn5-based minitransposon-generated mutants were spotted on square plates and used in subsequent analyses.

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FIG. 1. Illustration of transposon mutagenesis and determination of insertion locations. Mutagenized cells were selected by plating on A medium [2 g/liter yeast extract, 7 g/liter Casamino Acids, 2 g/liter (NH2)2CO, 7 g/liter (NH4)2SO4, 0.5 g/liter KH2PO4, 0.5 g/liter K2HPO4, 0.5 g/liter MgSO4 · 7H2O, 6 mg/liter Fe2SO4 · 7H2O, 4.2 mg/liter Mn2SO4 · H2O, 0.2 mg/liter biotin, 0.2 mg/liter thiamine, 4% glucose] containing kanamycin (50 µg/ml). After colony pickup, mutants were arrayed into 96-well plates and analyzed.

In order to identify transposon insertion sites, genomic DNA was prepared and direct genomic sequencing and TAIL-PCR were performed. Genomic DNAs of mutant cells were directly amplified using phi29 polymerase. By this method, a complicated procedure to extract genomic DNA was obviated. Initially, a small number of cells (10⁷ to 10⁸) was suspended in 4.5 µl reaction buffer for phi29 polymerase, but not much DNA fragment was amplified. Next, cells were suspended in 100 µl of Tris-EDTA buffer, and 0.5 µl of cell suspension was mixed with 4.5 µl reaction buffer. Samples were then incubated at 95°C for 5 min, chilled on ice for several minutes, and reacted with phi29 DNA polymerase for 30 h at 30°C. After the reaction, amplification of genomic DNAs was observed in most samples. A 2-µl aliquot of each amplified DNA was used in direct genomic sequencing (8) or TAIL-PCR (13). The phi29 polymerase and reaction buffer used were part of the GenomiPhi DNA amplification kit (Amersham Biosciences, NJ). The amplification reaction was done according to the manufacturer's protocol.

Direct genomic DNA sequencing was first performed using the standard ABI procedure for direct genomic DNA sequencing. However, clear data were obtained from only 30% of the samples, and the analyzed DNA sequence was less than 100 bp. In order to improve the data quality, the procedure was modified as follows. One microliter of sequencing primer (10 µM), 4 µl of BigDye Terminator (Applied Biosystems, CA), 0.5 µl of SequenceRx enhancer solution A (Invitrogen, CA), and 2.5 µl of H₂O were mixed with 2 µl of amplified genomic DNA. Cycle sequencing was performed at 98°C for 4 min and then at 96°C for 60 s, 50°C for 15 s, and 60°C for 4 min for a total of 65 cycles. As a result, 200 to 400 bp was read from 50% of samples on average. However, further improvement of the sequencing data quality was difficult.

Second, TAIL-PCR was performed using the procedure of Liu et al. (13) modified as follows. One microliter of amplified genomic DNA, 1 µl ExTaq buffer (Takara, Shiga, Japan), 0.8 µl deoxynucleoside triphosphate mixture (Takara), 0.05 µl ExTaq (Takara), 0.8 µl AP1 primer [5'-NGTCGA(G/C)(A/T)GANA(A/T)GAA] (32 µM), 0.8 µl GSP1 primer (5'-CTCCTTCATTACAGAAACGGC) (3.2 µM), and 5.6 µl H₂O were mixed and reacted. A 1-µl aliquot from a 50-fold dilution of the primary PCR products was added to a secondary PCR mixture (10 µl) containing 1 µl ExTaq buffer, 0.8 µl deoxynucleoside triphosphate mixture, 0.05 µl ExTaq, 0.8 µl AP1 primer (32 µM), and 0.8 µl GSP2 primer (5'-GCTGAGTTGAAGGATCAGATC) (3.2 µM). Thermal cycling of primary and secondary PCRs was done as described by Liu et al. (13). After amplification, 2-µl aliquots of secondary PCR products were used for the sequencing. The sequencing procedure was as follows. Two microliters of DNA, 0.5 µl of sequencing primer (3.2 µM), 1 µl of BigDye Terminator (Applied Biosystems), 3 µl of 5x sequencing buffer (Applied Biosystems), and 3.5 µl of H₂O were mixed. Cycle sequencing was performed at 94°C for 1 min and then at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min for 40 cycles. Sequencing was performed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The primers used for sequencing were 5'-AGGTTTCCGTAATTTGAACCACTACATT (miniTn31831) and 5'-ACAACAAAGCTCTCATCAACCGTGG (Tn5-based minitransposon).

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 
Transposon insertion sites in 17,540 mutants were automatically identified using BLAST and Perl (Fig. 2). When all sequencing data were compared to the genome sequence of C. glutamicum R, 11,241 positive hits to a position on the C. glutamicum R genome were identified. The joints between the inserted transposons and genome were listed according to annotated ORF. The insertion locations of 8,042 of these samples matched the insides of ORFs. Transposon insertions within ORFs were confirmed by cell-direct PCR using custom primers of each annotated ORF. Amplified DNA fragments were compared with each corresponding ORF of the wild-type strain on agarose gels. The lengths of miniTn31831 and Tn5-based minitransposons are 1.8 kb and 1.2 kb, respectively. Successful transposon insertion within an ORF caused an increase in the length of amplified DNA. Since amplified DNA fragments of 378 samples were the same size as that of the wild type, they were withdrawn from the gene disruption list. Due to redundancy of transposon insertions within ORFs, the total number of disrupted genes was 2,332, representing 78.1% of the 2,990 predicted ORFs of C. glutamicum R. The average number of hits per ORF was 3.45. The distribution of transposon insertions is illustrated in Fig. 3.

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FIG. 2. Perl script for high-throughput identification of transposon insertion points in the C. glutamicum R genomic DNA. Primers 1, 2, and 3 are GSP1, GSP2, and the sequencing primer, respectively. CDS, coding sequence.

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FIG. 3. Distribution of transposon insertions. The outside and middle circles indicate individual miniTn31831 and Tn5-based minitransposon insertions, respectively.

Most preferred sequences for miniTn31831 and Tn5-based minitransposon insertions were different (5, 7, 22). The distribution patterns of each transposon on the genome were also different (Fig. 3 and 4). miniTn31831 and the Tn5-based minitransposon could disrupt 1,231 and 1,862 genes, respectively. Due to the utilization of different types of transposons, ubiquitous distribution resulting in 2,332 disrupted genes was achieved. Coding sequences (ORFs) constitute 86.3% of the C. glutamicum R genome. Only 55.3% of miniTn31831s, compared to 87.6% of Tn5-based minitransposons, were inserted into C. glutamicum R coding sequences. Usually the A/T ratio of noncoding regions is higher than that of coding regions, and miniTn31831 tends to transpose into AT-rich regions (Fig. 4) (22). Furthermore, the density of transposon insertion between 1 Mbp and 2 Mbp was lower than that of other areas of the genome (Fig. 4). The reason is unknown, but it may relate to genome structure.

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FIG. 4. Number of transposon insertions on the C. glutamicum genome. Solid and dashed lines mark the number of transposon insertions and GC contents, respectively. (A) miniTn31831; (B) Tn5-based minitransposon. The numbers were calculated per 50 kb.

The classification of disrupted ORFs is shown in Table 1. The ratio of disrupted genes involved in translation, ribosomal structure, and biogenesis was only 37.0%. This category contains ribosomal proteins, translation elongation factors, translation initiation factors, etc., and is generally believed to be essential for cell survival. In contrast, 89.1% of genes in the category of carbohydrate transport and metabolism were disrupted. Since in this study the disruptants were isolated on a complex solid medium, inactivation of this category of genes should have little effect on cell survival. To determine the rate of disruption, the 2,990 ORFs were randomly classified into 30 groups and the percentages of disrupted genes calculated. The disruption rate varied from 70% to 85% (data not shown). That 13 groups from Table 1 fall within these percentages suggests that gene function may affect the rate of disruption of the genes.

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TABLE 1. Classification of disrupted genes

Transposon insertions were not observed in 658 ORFs (see Table S1 in the supplemental material). These genes may have been missed either by chance, because of sequence-specific insertion rates, or because the mutation was lethal. These genes without insertions were designated candidate essential genes. They were distributed randomly throughout the C. glutamicum R genome (data not shown). In E. coli and B. subtilis, the number of essential genes is considered to vary from 200 to 300 (http://shigen.lab.nig.ac.jp/ecoli/pec/index.jsp and http://bacillus.genome.jp/). Comparison between the translations of C. glutamicum R predicted ORFs and those of E. coli or B. subtilis essential genes indicates that a total of 251 ORFs from C. glutamicum strain R have a strict ortholog among E. coli or B. subtilis essential genes (see Table S1 in the supplemental material). Since only 221 of these are included in the candidate essential genes, the remaining 30 could be disrupted (see Table S2 in the supplemental material). To confirm that the genes could actually be disrupted, two were completely deleted from the genome by using the Cre/loxP system (20), and both mutants could grow (data not shown).

Among these 30, only 6 genes were essential in both E. coli and B. subtilis. They are homologs of fabD, fabG, accD, PTH, and groEL. One more paralogue of each of PTH, fabG, and groEL was found in the C. glutamicum R genome. accD is one of the four genes involved in the initiation of fatty acid synthesis (1). It is a subunit of acetyl coenzyme A (acetyl-CoA) carboxylase, which catalyzes the reaction between acetyl-CoA and CO₂ to form malonyl-CoA. C. glutamicum carries several putative propionyl-CoA carboxylase genes (9). As propionyl-CoA carboxylase is closely related to acetyl-CoA carboxylase, some of these genes may encode acetyl-CoA- and not propionyl-CoA-dependent carboxylase. The fabD and fabG genes are probably essential in prokaryotes, but Mycobacterium and Corynebacterium are possibly exceptions due to their possessing two different fatty acid synthases (FAS-IA and -IB) (9, 23). Recently one of them was reported to be essential in C. glutamicum (18). In this study, fasA, encoding FAS-IA, was included in the list of candidate essential genes. Since FAS-IA and -IB function as isoenzymes of the fabD and fabG products, inactivation of fasD and fasG was possible.

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 
In this study, a simple, high-throughput transposon-mediated mutagenesis method was developed. Using this method, 2,332 genes covering 78.0% of the predicted C. glutamicum R ORFs were disrupted, and 30 genes which have strict similarity to known essential genes were revealed not to be essential in C. glutamicum. A total of 658 candidate essential genes of C. glutamicum were also identified. In addition, by using this library, we recently isolated 98 auxotrophic mutants, 76 of which required amino acid supplementation to grow on minimal medium (data not shown). As C. glutamicum is one of the most widely used bacteria, these studies should greatly contribute to the investigation of gene functions and creation of improved cells for bioindustry. Comprehensive single-gene disruption libraries using targeted methods are usually constructed by international consortia. A more cost-effective and high-throughput method was developed here. This approach could be applicable to a wide variety of microbes, because both miniTn31831 and the Tn5-based minitransposon randomly transpose without any host cofactors.

 
We thank C. Omumasaba for critical reading of the manuscript. We are also grateful to Y. Ikeda, A. Kato, and S. Minakuchi for technical support.

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

 
* Corresponding author. Mailing address: Microbiology Research Group, Research Institute of Innovative Technology for the Earth, 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.

Supplemental material for this article may be found at http://aem.asm.org/.

Top Abstract Introduction Scheme for transposon insertion... Mapping of insertion sites... Conclusion. References

 

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Applied and Environmental Microbiology, May 2006, p. 3750-3755, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3750-3755.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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