Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme

Abstract

The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

“…Cyanobacterial genomic DNA was extracted using a phenol/chloroform method (Tamagnini et al, 1997). Complete segregation of the mutants was confirmed by agarose gel electrophoresis of PCR amplification products using standard protocols (Sambrook and Russell, 2001) and Southern blot (oligonucleotides in Supporting Information Table S1).…”

The cyanobacterium Anabaena sp. PCC 7120 has two distinct β‐carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

“…Genomic DNA was isolated from mutant cells by the glass bead method [12]. The two adjoining parts of the target genes ( Fig.…”

Transcriptional regulation of Nostoc uptake hydrogenase

“…PCC 73102 and N. muscorum), and a continuous irradiance of 40 Wmol m 3P s 3I (see [5]). To induce heterocyst formation/nitrogen-¢xing conditions, the Nostoc cultures were pelleted by centrifugation, washed once in BG 11 H medium (medium lacking combined nitrogen) before being resuspended in the same medium.…”

“…Genomic DNA was isolated from nitrogen-¢xing cultures of A. PCC 7120, A. variabilis ATCC 29413, N. PCC 73102, and N. muscorum Agardh CCAP 1453/12 as described [5]. Southern blot hybridizations (using cloned xisC as the probe, see below) and DNA sequencing (PCR-generated DNA fragments of N. muscorum) were performed as detailed before [3,5].…”

“…The uptake hydrogenase is a dimeric enzyme consisting of a large subunit (encoded by hupL) and a small subunit (encoded by hupS) [1^3]. The cyanobacterial bidirectional hydrogenase is a tetrameric enzyme where the diaphorase moiety is encoded by hoxFU and the hydrogenase part by hoxYH [1,4,5]. The cyanobacterial uptake hydrogenase has been shown to be predominantly active in heterocysts, the site for N P -¢xation [6].…”