Characterization of the hupSL promoter activity in Nostoc punctiformeATCC 29133

Marie Holmqvist,Pia Lindberg,Karin Stensjö,Paulo Oliveira,Peter Lindblad

doi:10.1186/1471-2180-9-54

Marie Holmqvist, Pia Lindberg + Show 3 more

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https://doi.org/10.1186/1471-2180-9-54

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Abstract

BackgroundIn cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H2.Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the hupSL promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the hupSL promoter, truncated versions of the hupSL promoter were fused to either gfp or luxAB, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively.ResultsInterestingly, all hupsSL promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity.ConclusionObtained data suggest that the hupSL promoter in N. punctiforme is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (-57 to tsp) is enough for a high and heterocyst specific expression of hupSL. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.

Highlights

In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H2.Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase
Little is known about the regulation and maturation of the uptake hydrogenases in cyanobacteria and the knowledge is mainly based on studies made in Escherichia coli
Binding of NtcA to the hupSL promoter To elucidate if NtcA binds to the identified NtcA binding site (TGT-N9-ACA), centred at 258.5 bp upstream the tsp (Fig. 1), in the hupSL promoter, Electrophoretic Mobility Shift Assays (EMSA), using the hupSL promoter from N.punctiforme and NtcA protein from Nostoc PCC 7120, were performed (Fig. 2)

Summary

Introduction

In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H2.Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. In cyanobacteria there are three enzymes directly involved in hydrogen metabolism; nitrogenase, uptake hydrogenase and bidirectional hydrogenase [1,2,3]. The active sites in the large subunits of hydrogenases are very complex and require a set of accessory proteins for their correct assembly and folding, which in E. coli are encoded by hypA-F [7,8]. Homologues of these genes are present in cyanobacteria [2,9]. Recently a set of genes within the extended hyp-operon was suggested to be involved in the maturation of the small subunit of the cyanobacterial uptake hydrogenase [10]

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