Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120.

Åsa Agervald,Karin Stensjö,Peter Lindblad,Marie Holmqvist

doi:10.1186/1471-2180-8-69

Abstract

BackgroundThe 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.ResultsRT-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.ConclusionThis 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.

Highlights

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
Transcription of the extended hyp-operon To determine if the hyp-genes are transcribed as a single operon and to identify putative 5'RACE transcriptional start points (TSPs), reverse transcriptase PCR (RT-PCR), Northern blot, and experiments were performed using total RNA isolated from N2-fixing cultures
Transcription of the extended hyp-operon This study demonstrates that the hyp-genes in Nostoc sp. strain PCC 7120 may be trancribed as a single operon which is in accordance with results from other cyanobacteria [22,25]

Summary

Introduction

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. Strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. Depending on the cyanobacterial strain, a single cell can harbour either an uptake hydrogenase or a bidirectional enzyme or both. N2-fixing strains contain at least an uptake hydrogenase which recycles the energy rich hydrogen molecule is produced as a by-product of the nitrogenase under N2-fixation. Hydrogenases, as well as nitrogenases, are very sensitive to oxygen which inactivates the activity of the proteins. To protect these enzymes they are physically located in the special cell type called heterocyst [5,6,10], or function under anaerobic conditions only [7,11]

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