Agervald Asa, Stensjö Karin, Holmqvist Marie, Lindblad Peter
Department of Photochemistry and Molecular Science, The Angström Laboratories, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden.
BMC Microbiol. 2008 Apr 28;8:69. doi: 10.1186/1471-2180-8-69.
The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120.
RT-PCRs demonstrated that the six hyp-genes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hyp-operon, and two downstream ORFs from the hyp-genes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifU-like protein. Orthologues of the four ORFs asr0689-alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2-fixing cyanobacteria, but are absent in non N2-fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hyp-operon, appearing between 11 and 79 times in the genome.
This study demonstrated that five ORFs upstream of the hyp-gene cluster are co-transcribed with the hyp-genes, and identified three TSPs in the extended hyp-gene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.
氢化酶成熟为活性酶是一个复杂的过程，例如，一个正确组装的活性位点至少需要七种蛋白质的参与，这些蛋白质由hypABCDEF编码，还有一种氢化酶特异性蛋白酶，由hupW或hoxW编码。固氮蓝藻念珠藻属PCC 7120菌株可能同时含有一种吸收型氢化酶和一种双向氢化酶。本研究探讨了念珠藻属PCC 7120菌株中hyp基因的存在及表达情况。
逆转录聚合酶链反应（RT-PCR）表明，六个hyp基因与一个开放阅读框（ORF）可能作为一个单一操纵子进行转录。分别在hypF上游280 bp和hypC上游445 bp处鉴定到转录起始点（TSP），这表明存在多种转录本。此外，位于编码吸收型氢化酶小亚基和大亚基的hupSL与hyp操纵子之间的五个上游ORF，以及hyp基因下游的两个ORF被证明是同一转录单元的一部分。在该操纵子中五个ORF中的第一个asr0689上游45 bp处鉴定到第三个TSP。除alr0692被鉴定为类NifU蛋白外，这些ORF注释为编码未知蛋白质。在其他几种固氮蓝藻中发现了asr0689 - alr0692这四个ORF的直系同源物，它们在基因组中的排列高度保守，位于hupSL和hyp基因之间，但在仅含有双向氢化酶的非固氮蓝藻中不存在。在扩展的hyp操纵子的六个基因间区域发现了短保守序列，在基因组中出现11至79次。
本研究表明，hyp基因簇上游的五个ORF与hyp基因共同转录，并在念珠藻属PCC 7120菌株扩展的hyp基因簇中鉴定到三个TSP。这可能表明其与功能性吸收型氢化酶的组装有关，推测是在该酶小亚基的组装过程中发挥作用。
