Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

Mormann S, Lömker A, Rückert C, Gaigalat L, Tauch A, Pühler A, Kalinowski J (2006)
BMC Genomics 7(1): 205.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
Abstract / Bemerkung
Background: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. Conclusion: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.
BMC Genomics
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Mormann S, Lömker A, Rückert C, et al. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics. 2006;7(1): 205.
Mormann, S., Lömker, A., Rückert, C., Gaigalat, L., Tauch, A., Pühler, A., & Kalinowski, J. (2006). Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics, 7(1), 205. https://doi.org/10.1186/1471-2164-7-205
Mormann, S., Lömker, A., Rückert, C., Gaigalat, L., Tauch, A., Pühler, A., and Kalinowski, J. (2006). Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics 7:205.
Mormann, S., et al., 2006. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics, 7(1): 205.
S. Mormann, et al., “Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway”, BMC Genomics, vol. 7, 2006, : 205.
Mormann, S., Lömker, A., Rückert, C., Gaigalat, L., Tauch, A., Pühler, A., Kalinowski, J.: Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway. BMC Genomics. 7, : 205 (2006).
Mormann, Sascha, Lömker, Alexander, Rückert, Christian, Gaigalat, Lars, Tauch, Andreas, Pühler, Alfred, and Kalinowski, Jörn. “Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway”. BMC Genomics 7.1 (2006): 205.
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Identification and structural characterization of a histidinol phosphate phosphatase from Mycobacterium tuberculosis.
Jha B, Kumar D, Sharma A, Dwivedy A, Singh R, Biswal BK., J Biol Chem 293(26), 2018
PMID: 29752410
An evolutionary optimization of a rhodopsin-based phototrophic metabolism in Escherichia coli.
Kim HA, Kim HJ, Park J, Choi AR, Heo K, Jeong H, Jung KH, Seok YJ, Kim P, Lee SJ., Microb Cell Fact 16(1), 2017
PMID: 28619035
Genome of Ca. Pandoraea novymonadis, an Endosymbiotic Bacterium of the Trypanosomatid Novymonas esmeraldas.
Kostygov AY, Butenko A, Nenarokova A, Tashyreva D, Flegontov P, Lukeš J, Yurchenko V., Front Microbiol 8(), 2017
PMID: 29046673
A Novel Method to Predict Genomic Islands Based on Mean Shift Clustering Algorithm.
de Brito DM, Maracaja-Coutinho V, de Farias ST, Batista LV, do Rêgo TG., PLoS One 11(1), 2016
PMID: 26731657
Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum.
Kulis-Horn RK, Persicke M, Kalinowski J., Microb Biotechnol 7(1), 2014
PMID: 23617600
High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.
Reimer LC, Spura J, Schmidt-Hohagen K, Schomburg D., PLoS One 9(2), 2014
PMID: 24504095
Complete genome sequence of Ilumatobacter coccineum YM16-304(T.).
Fujinami S, Takarada H, Kasai H, Sekine M, Omata S, Harada T, Fukai R, Hosoyama A, Horikawa H, Kato Y, Nakazawa H, Fujita N., Stand Genomic Sci 8(3), 2013
PMID: 24501628
Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers.
Alves JM, Klein CC, da Silva FM, Costa-Martins AG, Serrano MG, Buck GA, Vasconcelos AT, Sagot MF, Teixeira MM, Motta MC, Camargo EP., BMC Evol Biol 13(), 2013
PMID: 24015778
Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production.
Licona-Cassani C, Marcellin E, Quek LE, Jacob S, Nielsen LK., Antonie Van Leeuwenhoek 102(3), 2012
PMID: 22847261
Expression and functions of myo-inositol monophosphatase family genes in seed development of Arabidopsis.
Sato Y, Yazawa K, Yoshida S, Tamaoki M, Nakajima N, Iwai H, Ishii T, Satoh S., J Plant Res 124(3), 2011
PMID: 20960216
Histidine biosynthesis.
Ingle RA., Arabidopsis Book 9(), 2011
PMID: 22303266
Complete genome sequence of the soil actinomycete Kocuria rhizophila.
Takarada H, Sekine M, Kosugi H, Matsuo Y, Fujisawa T, Omata S, Kishi E, Shimizu A, Tsukatani N, Tanikawa S, Fujita N, Harayama S., J Bacteriol 190(12), 2008
PMID: 18408034
Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.
Lee JY, Sung BH, Yu BJ, Lee JH, Lee SH, Kim MS, Koob MD, Kim SC., Nucleic Acids Res 36(16), 2008
PMID: 18641039
Construction of signature-tagged mutant library in Mesorhizobium loti as a powerful tool for functional genomics.
Shimoda Y, Mitsui H, Kamimatsuse H, Minamisawa K, Nishiyama E, Ohtsubo Y, Nagata Y, Tsuda M, Shinpo S, Watanabe A, Kohara M, Yamada M, Nakamura Y, Tabata S, Sato S., DNA Res 15(5), 2008
PMID: 18658183

104 References

Daten bereitgestellt von Europe PubMed Central.

Amino acids-technichal production and use
Leuchtenberger W., 1996
Amino acid production processes
Ikeda M., 2003
Industrial strain improvement: mutagenesis and random screening procedures
Rowlands RT., 1984
Manipulation of Corynebacterium glutamicum by gene disruption and replacement.
Schwarzer A, Puhler A., Biotechnology (N.Y.) 9(1), 1991
PMID: 1367217
Pathway analysis and metabolic engineering in Corynebacterium glutamicum.
Sahm H, Eggeling L, de Graaf AA., Biol. Chem. 381(9-10), 2000
PMID: 11076021
Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum.
Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Mockel B, Sahm H, Eikmanns BJ., J. Mol. Microbiol. Biotechnol. 3(2), 2001
PMID: 11321586
Efficient 40 degrees C fermentation of L-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding.
Ohnishi J, Hayashi M, Mitsuhashi S, Ikeda M., Appl. Microbiol. Biotechnol. 62(1), 2003
PMID: 12835923
The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins.
Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Kramer R, Linke B, McHardy AC, Meyer F, Mockel B, Pfefferle W, Puhler A, Rey DA, Ruckert C, Rupp O, Sahm H, Wendisch VF, Wiegrabe I, Tauch A., J. Biotechnol. 104(1-3), 2003
PMID: 12948626
Transposable Element Tools for Microbial Genetics
Berg CM, Berg DE., 1996
Recent advances in large-scale transposon mutagenesis.
Hamer L, DeZwaan TM, Montenegro-Chamorro MV, Frank SA, Hamer JE., Curr Opin Chem Biol 5(1), 2001
PMID: 11166651
Target site selection in transposition.
Craig NL., Annu. Rev. Biochem. 66(), 1997
PMID: 9242914
Transposable elements and the genetic engineering of bacteria
Berg CM, Berg DE, Groisman E., 1989

Berg DE, Howe MM., 1989
Insertion sequences.
Mahillon J, Chandler M., Microbiol. Mol. Biol. Rev. 62(3), 1998
PMID: 9729608
Transposon mutagenesis of coryneform bacteria.
Vertes AA, Asai Y, Inui M, Kobayashi M, Kurusu Y, Yukawa H., Mol. Gen. Genet. 245(4), 1994
PMID: 7808388
Transpositional activity of IS986 in Mycobacterium smegmatis.
Fomukong NG, Dale JW., Gene 130(1), 1993
PMID: 8393827
A new insertion sequence, IS14999, from Corynebacterium glutamicum.
Tsuge Y, Ninomiya K, Suzuki N, Inui M, Yukawa H., Microbiology (Reading, Engl.) 151(Pt 2), 2005
PMID: 15699199
Transposition of an antibiotic resistance element in mycobacteria.
Martin C, Timm J, Rauzier J, Gomez-Lus R, Davies J, Gicquel B., Nature 345(6277), 1990
PMID: 2163027
Insertion sequence IS6100 on plasmid pOAD2, which degrades nylon oligomers.
Kato K, Ohtsuki K, Mitsuda H, Yomo T, Negoro S, Urabe I., J. Bacteriol. 176(4), 1994
PMID: 8106333
Integrons found in different locations have identical 5' ends but variable 3' ends.
Hall RM, Brown HJ, Brookes DE, Stokes HW., J. Bacteriol. 176(20), 1994
PMID: 7929000
Integron-containing IncU R plasmids pRAS1 and pAr-32 from the fish pathogen Aeromonas salmonicida.
Sorum H, L'Abee-Lund TM, Solberg A, Wold A., Antimicrob. Agents Chemother. 47(4), 2003
PMID: 12654659
Bacterial insertion sequences
Galas DJ, Chandler M., 1989
Genes required for mycobacterial growth defined by high density mutagenesis.
Sassetti CM, Boyd DH, Rubin EJ., Mol. Microbiol. 48(1), 2003
PMID: 12657046
Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm.
Rigoutsos I, Floratos A., Bioinformatics 14(1), 1998
PMID: 9520502
A new method for the identification of biochemical mutants of microorganisms
Holliday R., 1956
Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.
Fernandes PJ, Powell JA, Archer JA., Microbiology (Reading, Engl.) 147(Pt 9), 2001
PMID: 11535792
Linkage map of Escherichia coli K-12, edition 6.
Bachmann BJ, Low KB., Microbiol. Rev. 44(1), 1980
PMID: 6997720
Patterns of polarity in the Escherichia coli car AB gene cluster.
Gigot D, Crabeel M, Feller A, Charlier D, Lissens W, Glansdorff N, Pierard A., J. Bacteriol. 143(2), 1980
PMID: 6162837
Expression of thiamin biosynthetic genes (thiCOGE) and production of symbiotic terminal oxidase cbb3 in Rhizobium etli.
Miranda-Rios J, Morera C, Taboada H, Davalos A, Encarnacion S, Mora J, Soberon M., J. Bacteriol. 179(22), 1997
PMID: 9371431
A sequence from a tryptophan-hyperproducing strain of Corynebacterium glutamicum encoding resistance to 5-methyltryptophan.
Heery DM, Fitzpatrick R, Dunican LK., Biochem. Biophys. Res. Commun. 201(3), 1994
PMID: 8024569
Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum.
McHardy AC, Tauch A, Ruckert C, Puhler A, Kalinowski J., J. Biotechnol. 104(1-3), 2003
PMID: 12948641
Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum.
Marienhagen J, Kennerknecht N, Sahm H, Eggeling L., J. Bacteriol. 187(22), 2005
PMID: 16267288
Histidine biosynthetic pathway and genes: structure, regulation, and evolution.
Alifano P, Fani R, Lio P, Lazcano A, Bazzicalupo M, Carlomagno MS, Bruni CB., Microbiol. Rev. 60(1), 1996
PMID: 8852895
The KEGG database.
Kanehisa M., Novartis Found. Symp. 247(), 2002
PMID: 12539951
cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.
Neuwald AF, Krishnan BR, Brikun I, Kulakauskas S, Suziedelis K, Tomcsanyi T, Leyh TS, Berg DE., J. Bacteriol. 174(2), 1992
PMID: 1729235
Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis.
Weaden J, Dyson P., Microbiology (Reading, Engl.) 144 ( Pt 7)(), 1998
PMID: 9695929
High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library.
Suzuki N, Okai N, Nonaka H, Tsuge Y, Inui M, Yukawa H., Appl. Environ. Microbiol. 72(5), 2006
PMID: 16672528
Polarity of Tn5 insertion mutations in Escherichia coli.
Berg DE, Weiss A, Crossland L., J. Bacteriol. 142(2), 1980
PMID: 6247321
Transpositional recombination in prokaryotes.
Grindley ND, Reed RR., Annu. Rev. Biochem. 54(), 1985
PMID: 2992361
Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction.
Ruckert C, Koch DJ, Rey DA, Albersmeier A, Mormann S, Puhler A, Kalinowski J., BMC Genomics 6(), 2005
PMID: 16159395
Inositol phosphate biochemistry.
Majerus PW., Annu. Rev. Biochem. 61(), 1992
PMID: 1323235
The envelope of mycobacteria.
Brennan PJ, Nikaido H., Annu. Rev. Biochem. 64(), 1995
PMID: 7574484
Lipoarabinomannans: from structure to biosynthesis.
Nigou J, Gilleron M, Puzo G., Biochimie 85(1-2), 2003
PMID: 12765785
Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.
Nampoothiri KM, Hoischen C, Bathe B, Mockel B, Pfefferle W, Krumbach K, Sahm H, Eggeling L., Appl. Microbiol. Biotechnol. 58(1), 2002
PMID: 11831479
The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes.
Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crecy-Lagard V, Diaz N, Disz T, Edwards R, Fonstein M, Frank ED, Gerdes S, Glass EM, Goesmann A, Hanson A, Iwata-Reuyl D, Jensen R, Jamshidi N, Krause L, Kubal M, Larsen N, Linke B, McHardy AC, Meyer F, Neuweger H, Olsen G, Olson R, Osterman A, Portnoy V, Pusch GD, Rodionov DA, Ruckert C, Steiner J, Stevens R, Thiele I, Vassieva O, Ye Y, Zagnitko O, Vonstein V., Nucleic Acids Res. 33(17), 2005
PMID: 16214803

Sambrook J, Fritsch EF, Maniatis T., 1989
Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli.
Tauch A, Kirchner O, Wehmeier L, Kalinowski J, Puhler A., FEMS Microbiol. Lett. 123(3), 1994
PMID: 7988915
Efficient electrotransformation of corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1.
Tauch A, Kirchner O, Loffler B, Gotker S, Puhler A, Kalinowski J., Curr. Microbiol. 45(5), 2002
PMID: 12232668
Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library.
Tauch A, Homann I, Mormann S, Ruberg S, Billault A, Bathe B, Brand S, Brockmann-Gretza O, Ruckert C, Schischka N, Wrenger C, Hoheisel J, Mockel B, Huthmacher K, Pfefferle W, Puhler A, Kalinowski J., J. Biotechnol. 95(1), 2002
PMID: 11879709
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler DL., Nucleic Acids Res. 28(1), 2000
PMID: 10592170
The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000.
Bairoch A, Apweiler R., Nucleic Acids Res. 28(1), 2000
PMID: 10592178
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ., Nucleic Acids Res. 25(17), 1997
PMID: 9254694
The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG., Nucleic Acids Res. 25(24), 1997
PMID: 9396791
TreeTool - Interactive phylogenetic tree plotting program
Protoplast transformation of glutamate-producing bacteria with plasmid DNA.
Katsumata R, Ozaki A, Oka T, Furuya A., J. Bacteriol. 159(1), 1984
PMID: 6145700
The COG database: a tool for genome-scale analysis of protein functions and evolution.
Tatusov RL, Galperin MY, Natale DA, Koonin EV., Nucleic Acids Res. 28(1), 2000
PMID: 10592175
GenDB--an open source genome annotation system for prokaryote genomes.
Meyer F, Goesmann A, McHardy AC, Bartels D, Bekel T, Clausen J, Kalinowski J, Linke B, Rupp O, Giegerich R, Puhler A., Nucleic Acids Res. 31(8), 2003
PMID: 12682369
Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB.
Flachmann R, Kunz N, Seifert J, Gutlich M, Wientjes FJ, Laufer A, Gassen HG., Eur. J. Biochem. 175(2), 1988
PMID: 2841129
Linkage map of Escherichia coli strain K-12.
Taylor AL, Trotter CD., Bacteriol Rev 36(4), 1972
PMID: 4568762
A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase.
Keer J, Smeulders MJ, Williams HD., Microbiology (Reading, Engl.) 147(Pt 2), 2001
PMID: 11158364
Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.
Liu DX, Fan CS, Tao JH, Liang GX, Gao SE, Wang HJ, Li X, Song DX., World J. Gastroenterol. 10(24), 2004
PMID: 15534933
Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene.
Follettie MT, Sinskey AJ., J. Bacteriol. 167(2), 1986
PMID: 3525519
Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step.
Ankri S, Serebrijski I, Reyes O, Leblon G., J. Bacteriol. 178(15), 1996
PMID: 8755867
Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon.
Peoples OP, Liebl W, Bodis M, Maeng PJ, Follettie MT, Archer JA, Sinskey AJ., Mol. Microbiol. 2(1), 1988
PMID: 2835591
Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.
Pierard A, Glansdorff N, Gigot D, Crabeel M, Halleux P, Thiry L., J. Bacteriol. 127(1), 1976
PMID: 179975
Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.
Grant SG, Jessee J, Bloom FR, Hanahan D., Proc. Natl. Acad. Sci. U.S.A. 87(12), 1990
PMID: 2162051


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