Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413

A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N(2)-fixing conditions, the mutant strain exhibited significantly increased rates in H(2) accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H(2) developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.

从鱼腥藻Anabaena variabilis ATCC 29413中克隆并测序了一段长10 kb的DNA区域，该区域包含吸氢酶（uptake hydrogenase）的结构基因hupSL。与鱼腥藻Anabaena sp.菌株PCC 7120的hupL基因不同——该基因在营养细胞中会被一段10.5 kb的DNA片段打断，A. variabilis的hupL基因在异形胞分化过程中未发生程序性重排。hupSL基因以2.7 kb的操纵子结构进行转录，且仅在固氮条件下被诱导表达，该结果经Northern印迹（Northern blot）实验与反转录PCR（reverse transcriptase PCR）证实。采用荧光标记寡核苷酸引物开展的引物延伸实验验证了上述结论，并确定mRNA转录起始位点位于ATG起始密码子上游103 bp处。研究人员在该启动子区域中检测到了可被富马酸硝酸盐还原酶调节因子（Fnr）识别的保守序列。鱼腥藻A. variabilis的hupSL操纵子经插入缺失突变后获得突变菌株AVM13。在固氮条件下，该突变菌株的氢气积累速率显著升高，产氢量为野生型菌株的3倍。上述结果表明，野生型菌株中的吸氢酶具备催化活性，该酶可对固氮酶生成的氢气进行再氧化。与野生型菌株相比，该突变菌株的固氮表型（Nif phenotype）表现为乙炔还原活性略有降低。