Mormann Sascha, Lömker Alexander, Rückert Christian, Gaigalat Lars, Tauch Andreas, Pühler Alfred, Kalinowski Jörn
Institut für Genomforschung, Universität Bielefeld, D-33594 Bielefeld, Germany.
BMC Genomics. 2006 Aug 10;7:205. doi: 10.1186/1471-2164-7-205.
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.
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.
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.
谷氨酸棒杆菌是放线菌纲的革兰氏阳性菌，是一种与工业相关的氨基酸生产菌。已经开发了几种针对该生物体的靶向基因操作和合理菌株改良方法。一个适用于完全测序的模式菌株ATCC 13032的高效转座子诱变系统将显著推进该细菌的功能基因组分析。
通过将菌株ATCC 13032的限制缺陷衍生物谷氨酸棒杆菌RES167与含IS6100的非复制性质粒进行电转化，构建了一个包含10,080个独立克隆的综合转座子突变文库。转座子突变体在转座子载体和染色体之间具有稳定的共整合体。通过对染色体插入片段的测序共确定了172个转座子整合位点，表明每个整合发生在不同的位点。统计靶位点分析表明明显不存在靶位点偏好。从文库中获得营养缺陷型突变体的频率为2.9%。通过营养缺陷型平板分析法进一步鉴定了近三分之二的营养缺陷型，包括具有单一、双重和替代营养需求的突变体。在大多数情况下，观察到的营养需求与参与氨基酸、核苷酸或维生素生物合成的突变基因的注释相关。一个显著的例外是通过转座插入基因cg0910诱变的一个克隆，其表现出对组氨酸的营养缺陷型。从cg0910推导的蛋白质序列与肌醇-1（或4）-单磷酸酶（EC 3.1.3.25）显示出高度的序列相似性。随后对cg0910进行基因缺失产生了相同的组氨酸营养缺陷型表型。突变体的基因互补以及组氨醇的补充表明，cg0910在谷氨酸棒杆菌中编码迄今未知的必需L-组氨醇磷酸磷酸酶（EC 3.1.3.15）。cg0910基因，重命名为hisN，及其编码的酶在几乎所有放线菌中都有假定的直系同源物，包括分枝杆菌和链霉菌。
IS6100转座缺乏区域和序列偏好，表明所建立的系统适用于对已测序的模式菌株谷氨酸棒杆菌ATCC 13032进行高效的全基因组随机诱变。在谷氨酸棒杆菌中鉴定出编码组氨醇磷酸磷酸酶的hisN基因填补了放线菌中组氨酸合成的最后一个空白。由于IS6100具有广泛的宿主谱，该系统在其他细菌中也可能是一种有价值的遗传工具。
