Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway
<p>Abstract</p> <p>Background</p> <p><it>Corynebacterium glutamicum</it>, 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 r...
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BMC
2006-08-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/7/205 |
| _version_ | 1818751889062232064 |
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| author | Gaigalat Lars Rückert Christian Lömker Alexander Mormann Sascha Tauch Andreas Pühler Alfred Kalinowski Jörn |
| author_facet | Gaigalat Lars Rückert Christian Lömker Alexander Mormann Sascha Tauch Andreas Pühler Alfred Kalinowski Jörn |
| author_sort | Gaigalat Lars |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p><it>Corynebacterium glutamicum</it>, 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.</p> <p>Results</p> <p>A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, <it>C. glutamicum </it>RES167, with an IS<it>6100</it>-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 <it>cg0910</it>, which exhibited an auxotrophy for histidine. The protein sequence deduced from <it>cg0910 </it>showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of <it>cg0910 </it>delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that <it>cg0910 </it>encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in <it>C. glutamicum</it>. The <it>cg0910 </it>gene, renamed <it>hisN</it>, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes.</p> <p>Conclusion</p> <p>The absence of regional and sequence preferences of IS<it>6100</it>-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain <it>C</it>.<it>glutamicum </it>ATCC 13032. The identification of the <it>hisN </it>gene encoding histidinol-phosphate phosphatase in <it>C. glutamicum </it>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 IS<it>6100</it>.</p> |
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| spelling | doaj.art-5bde9ed3ee93440ab82dabe5abdc6aa12022-12-21T21:20:41ZengBMCBMC Genomics1471-21642006-08-017120510.1186/1471-2164-7-205Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathwayGaigalat LarsRückert ChristianLömker AlexanderMormann SaschaTauch AndreasPühler AlfredKalinowski Jörn<p>Abstract</p> <p>Background</p> <p><it>Corynebacterium glutamicum</it>, 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.</p> <p>Results</p> <p>A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, <it>C. glutamicum </it>RES167, with an IS<it>6100</it>-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 <it>cg0910</it>, which exhibited an auxotrophy for histidine. The protein sequence deduced from <it>cg0910 </it>showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of <it>cg0910 </it>delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that <it>cg0910 </it>encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in <it>C. glutamicum</it>. The <it>cg0910 </it>gene, renamed <it>hisN</it>, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes.</p> <p>Conclusion</p> <p>The absence of regional and sequence preferences of IS<it>6100</it>-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain <it>C</it>.<it>glutamicum </it>ATCC 13032. The identification of the <it>hisN </it>gene encoding histidinol-phosphate phosphatase in <it>C. glutamicum </it>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 IS<it>6100</it>.</p>http://www.biomedcentral.com/1471-2164/7/205 |
| spellingShingle | Gaigalat Lars Rückert Christian Lömker Alexander Mormann Sascha Tauch Andreas Pühler Alfred Kalinowski Jörn Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway BMC Genomics |
| title | Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| title_full | Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| title_fullStr | Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| title_full_unstemmed | Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| title_short | Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| title_sort | random mutagenesis in it corynebacterium glutamicum it atcc 13032 using an is it 6100 it based transposon vector identified the last unknown gene in the histidine biosynthesis pathway |
| url | http://www.biomedcentral.com/1471-2164/7/205 |
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