Hansmeier Nicole, Albersmeier Andreas, Tauch Andreas, Damberg Thomas, Ros Robert, Anselmetti Dario, Pühler Alfred, Kalinowski Jörn
Institut für Genomforschung, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Universitätsstraße 25, D-33615 Bielefeld, Germany.
Microbiology (Reading). 2006 Apr;152(Pt 4):923-935. doi: 10.1099/mic.0.28673-0.
The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5.97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2(-) phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5' rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.
在粘粒克隆上鉴定了革兰氏阳性细菌谷氨酸棒杆菌ATCC 14067的表面（S）层基因区域，进行了测序，并与谷氨酸棒杆菌ATCC 13032的基因组序列进行了比较，后者的细胞表面没有有序的S层晶格。鉴定出谷氨酸棒杆菌ATCC 13032染色体中不存在的一个5.97 kb DNA区域。该区域包括cspB，即编码S层原体PS2的结构基因，以及另外六个编码序列。PCR实验表明，相应的DNA区域在能够形成S层的不同谷氨酸棒杆菌野生型菌株中是保守的。该DNA区域两侧有一个7 bp的直接重复序列，这表明非法重组可能是谷氨酸棒杆菌ATCC 13032中基因缺失的原因。克隆的cspB基因的转移恢复了谷氨酸棒杆菌ATCC 13032的PS2（-）表型，通过SDS-PAGE对PS2蛋白进行可视化以及通过原子力显微镜对活的谷氨酸棒杆菌细胞上有序的六边形S层晶格进行成像得到了证实。此外，通过5' cDNA末端快速扩增PCR对cspB基因的启动子进行了定位，并将相应的DNA片段用于DNA亲和纯化分析。纯化了一种与cspB基因启动子区域特异性结合的30 kDa蛋白质。对纯化蛋白质进行基质辅助激光解吸电离飞行时间质谱分析和肽质量指纹分析，鉴定出推定的转录调节因子Cg2831，它属于LuxR调节蛋白家族。谷氨酸棒杆菌中cg2831基因的破坏导致PS2合成几乎完全丧失。这些结果表明，Cg2831是谷氨酸棒杆菌中cspB基因表达的转录激活因子。
