Limits...
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 - BMC Genomics (2006)

Bottom Line: Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genomforschung, Universität Bielefeld, D-33594 Bielefeld, Germany. smormann@genetik.uni-bielefeld.de

ABSTRACT

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 3.1.3.25). 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 3.1.3.15) 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.

Show MeSH

Related in: MedlinePlus

Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour-joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobacterium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C.  jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1590026&req=5

Figure 4: Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour-joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobacterium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C. jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c).

Mentions: BLAST searches revealed four IMP paralogs in the C. glutamicum ATCC 13032 genome with significant similarity to cg0910, by name cg0911, cg0967 (cysQ), cg2090 (suhB), and cg2298 (impA). For a functional classification of the inositol monophosphatase family-like proteins, phylogenetic analysis in comparison to sequence-related proteins from other Actinobacteria and the model organism E. coli was conducted. For this, similarity searches with the deduced amino acid sequences of the C. glutamicum IMPs in non-redundant databases were carried out, comprising, beside E. coli K-12, the genomes of Actinobacteria including completed as well as draft status genomes. Proteins with reliable similarity scores were used to perform a multiple alignment. Based on this alignment a phylogenetic tree was created by the neighbour-joining method and, subsequently, the tree was evaluated by bootstrap analysis (Fig. 4).


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 - BMC Genomics (2006)

Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour-joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobacterium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C.  jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590026&req=5

Figure 4: Dendrogram showing the relationship of inositol monophosphatase family proteins (IMP) in Actinobacteria and E. coli K-12. A multiple alignment with amino acid sequences of proteins with high similarity to the C. glutamicum IMPs was generated with the use of the DIALIGN2 software. Based on this alignment an unrooted phylogenetic tree was constructed using the neighbour-joining algorithm integrated in the CLUSTALX package and visualized as a radial tree by the TreeTool software. The branches were combined to classes that delivered within the bootstrapping analyses in at least two thirds of the cases the same subtree. These classes, marked by different colours, were named according to the designations in the boxed leaves. The locus tags (leaves) were obtained from the GenBank genome entries. C. glutamicum proteins are printed in bold letters. Locus tag prefixes denote following organisms (c, complete genome sequence; da, draft assembly): Arth (Arthrobacter sp. FB24; da), BL (Bifidobacterium longum NCC2705; c), BLinB01 (Brevibacterium linens BL2; da), DIP (Corynebacterium diphtheriae NCTC13129; c), CE (C. efficiens YS-314; c), cg (C. glutamicum ATCC 13032; c), jk (C. jeikeium K411; c), Ecoli (Escherichia coli K-12; c), Francci3 (Frankia sp. CcI3; c), Franean1 (Frankia sp. EAN1pec; da), JNB (Janibacter sp. HTCC2649; da), Krad (Kineococcus radiotolerans SRS30216; da), Lxx (Leifsonia xyli subsp. xyli str. CTCB07; c), Micol (Micromonospora olivasterospora; da), MAP (Mycobacterium avium subsp. paratuberculosis K-10; c), ML (M. leprae TN; c), Rv (M. tuberculosis H37Rv; c), Mycsm (Mycobacterium smegmatis str. MC2 155; da), nfa (Nocardia farcinica IFM 10152; c), Noca (Nocardioides sp. JS614; da), PPA (Propionibacterium acnes KPA171202; c), RhoDS7 (Rhodococcus sp. DS7; da), Rxyl (Rubrobacter xylanophilus DSM 9941; da), SAV (Streptomyces avermitilis MA-4680; c), SCO (S. coelicolor A3(2); c) and Tfu (Thermobifida fusca YX; c).
Mentions: BLAST searches revealed four IMP paralogs in the C. glutamicum ATCC 13032 genome with significant similarity to cg0910, by name cg0911, cg0967 (cysQ), cg2090 (suhB), and cg2298 (impA). For a functional classification of the inositol monophosphatase family-like proteins, phylogenetic analysis in comparison to sequence-related proteins from other Actinobacteria and the model organism E. coli was conducted. For this, similarity searches with the deduced amino acid sequences of the C. glutamicum IMPs in non-redundant databases were carried out, comprising, beside E. coli K-12, the genomes of Actinobacteria including completed as well as draft status genomes. Proteins with reliable similarity scores were used to perform a multiple alignment. Based on this alignment a phylogenetic tree was created by the neighbour-joining method and, subsequently, the tree was evaluated by bootstrap analysis (Fig. 4).

Bottom Line: Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Genomforschung, Universität Bielefeld, D-33594 Bielefeld, Germany. smormann@genetik.uni-bielefeld.de

ABSTRACT

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 3.1.3.25). 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 3.1.3.15) 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.

Show MeSH
Related in: MedlinePlus