Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152

Reade, John P.H.; Dougherty, Lisa J; Rogers, Lyndon J.; Gallon, John

doi:10.1099/13500872-145-7-1749

Cited by 24 publications

(29 citation statements)

References 31 publications

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“…This is interesting since Gloeothece sp. ATCC 27152 has been shown to fix nitrogen mainly in the dark (Reade et al, 1999), and consequently displays a higher hydrogen-uptake activity during the dark cycle (confirmed in this study using a hydrogen electrode, data not shown). The appearance of the transcript(s) prior to a detectable hydrogen uptake activity may be due to the fact that the uptake hydrogenase requires a complex maturation process.…”

Section: Hupslw Transcription Under Different Growth Conditionssupporting

confidence: 66%

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

et al. 2004

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The structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N 2 fixation, were identified and sequenced. 39-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW ) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at "41?5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW. INTRODUCTIONSeveral species of bacteria and cyanobacteria are capable of N 2 fixation. During the N 2 fixation process H 2 is formed as a by-product. This nitrogenase-dependent H 2 production is often compromised by the presence of an uptake hydrogenase (encoded by hupSL) that rapidly consumes the H 2 generated. In addition, a bi-directional enzyme (encoded by hoxEFUYH) may be present which, depending on the growth conditions, may display the capacity of both producing and consuming H 2 (Lambert & Smith, 1981;Houchins, 1984;Schmitz et al., 2002;Tamagnini et al., 2002). All cyanobacteria examined so far contain an uptake, a bi-directional or both the hydrogenases (Schmitz et al., 1995(Schmitz et al., , 2002Boison et al., 1996;Tamagnini et al., 2000Tamagnini et al., , 2002Sheremetieva et al., 2002; Schütz et al., 2004). Moreover, the uptake-type enzyme has been found in all nitrogen fixing cyanobacterial strains studied to date (Carrasco & Golden, 1995;Oxelfelt et al., 1998;Happe et al., 2000;Tamagnini et al., 2000Tamagnini et al., , 2002 Schütz et al., 2004).The first data on cyanobacterial hupSL transcription appeared in 1995 (Carrasco & Golden, 1995). RT-PCR experiments on Anabaena/Nostoc sp. strain PCC 7120 demonstrated that hupL transcription coincides with the formation of heterocysts. Subsequent studies, in other filamentous strains, have confirmed the induction of an hupL transcript under nitrogen-fixing conditions only (Axelsson et al., 1999;Happe et al., 2000; Hansel et al., 2001). One exception is Anabaena variabilis ATCC 29413, where a low level of hupL expression has been detected in vegetative cells grown with the addition of ammonia (Boison et al., 2000). In all the cyanobacterial strains 3Present address: Department of Physiological Botany, Evolutionary Biology Centre,...

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Section: Hupslw Transcription Under Different Growth Conditionssupporting

confidence: 66%

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

et al. 2004

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“…The total Fe content of the nitrogenase complex depends on the ratio of Fe to MoFe protein. This ratio has not, to our knowledge, been measured in Trichodesmium, but a ratio of 3 was observed in vivo in the single-celled nitrogen-fixing cyanobacterium Gloeothece ATCC 27152 (Reade et al 1999). In vitro protein titrations indicate that maximal specific activity for the enzyme occurs at a ratio near 5 for many species (Eady and Smith 1979;Johnson et al 2000).…”

Section: Discussionmentioning

confidence: 96%

Diel variation of molybdenum and iron in marine diazotrophic cyanobacteria

Tuit

Waterbury

Ravizza

2004

Limnology & Oceanography

137

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Measurements of Mo : C and Fe : C ratios in cultured cells of two N 2 -fixing cyanobacteria, Crocosphaera watsonii strain WH8501 and Trichodesmium erythraeum strain IMS101, agree with estimated metal : carbon ratios based on growth rate and the metal use efficiency of the nitrogenase enzyme. Crocosphaera, a single-celled nocturnal N 2 fixer, showed two-to eightfold increases in Mo and Fe cellular concentrations in response to nitrogen fixation activity. Mo required for N 2 assimilation can account for almost the entire Mo pool measured in the cells, implying that Crocosphaera synthesizes its entire nitrogenase pool de novo each night. In contrast, cultures of Trichodesmium, a filamentous, diurnal N 2 -fixing cyanobacterium, did not show diel variations in Mo or Fe carbon ratios or in cellular metal concentrations. Trichodesmium appears to maintain an internal pool of Mo. In Trichodesmium cultures, Mo concentrations were up to 30% higher than needed to support measured N 2 fixation. Trichodesmium colonies collected from the field had Mo : C ratios 10-fold larger than those measured in culture, far in excess of what is needed to fix N 2 at rates normally measured in the field, despite equivalent Fe : C ratios (66 Ϯ 39 [field samples] and 87 Ϯ 64 [cultures] mol mol Ϫ1 ). The average Fe : C ratio measured in N 2 -fixing Crocosphaera (16 Ϯ 11 mol mol Ϫ1 ) was equivalent to theoretical estimates of Fe demand based on nitrogenase requirements (13 Ϯ 5 mol mol Ϫ1 ). These results demonstrate the extremely efficient use of Fe by these organisms and provide support for the use of theoretical estimates of Fe : C ratios to calculate biological Fe demand for N 2 fixation.Nitrogen fixation, the conversion of N 2 gas into bioavailable forms, is an important source of nitrogen for primary production in the oligotrophic ocean (Capone et al. 1997;Karl et al. 1997). Nitrogen fixation is catalyzed by the nitrogenase complex, a metal-rich enzyme containing 2 mol molybdenum (Mo) and 38-50 mol iron (Fe) mol Ϫ1 complex. Recent studies have focused on Fe as a potentially limiting nutrient for both photoautotrophic growth and N 2 fixation in the marine environment (Sunda and Huntsman 1995;Berman-Frank et al. 2001a;Kustka et al. 2002). Although there is an ongoing debate whether the high levels of sulfate (SO ) in seawater can competitively inhibit molybdate 2Ϫ 4 1 To whom correspondence should be addressed. Present address: Department of Geosciences, Princeton University, Princeton, New Jersey 08544 (ctuit@princeton.edu).2 Present address: Department of Geology & Geophysics, SOEST, University of Hawaii, Manoa, 1680 East-West Road, Honolulu, Hawaii 96822. AcknowledgmentsWe are grateful to Eric Webb for his input throughout this study and for his analysis of the Trichodesmium genome for the putative Mo transporter. We thank James Howard for discussions about nitrogenase and acetylene reduction methods. We also thank Rob Sherrell, Dan McCorkle, and two anonymous reviewers for their comments on the early versions of this manuscript. M...

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“…Global transcriptional studies have also observed large scale changes in the transcriptomes of the unicellular diazotrophs Crocosphaera watsonii (21), Cyanothece (22), Gloeothece (23), and a hot-spring Synechococcus (24) during the diel cycle, consistent with a temporal separation of photosynthesis and nitrogen fixation. Yet the possibility that diel cycling of the transcriptome or proteome might affect the iron requirement has not been previously discussed, likely because transcriptional studies provide information about gene expression rather than actual enzyme inventories, and immunological protein studies thus far, while having detected diel oscillations of the NifH subunit in Crocosphaera and Gloeothece (23,25), are limited to a few select proteins with no global or absolute quantitative proteome studies of diazotrophs published as of yet. As a result, it has remained uncertain whether the observed diel transcriptome cycling was needed for the maintenance of a relatively consistent proteome or was in fact causing large changes in the global proteome composition during each diel cycle.…”

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confidence: 99%

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii

Saito¹,

Bertrand²,

Dutkiewicz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

204

228

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The marine nitrogen fixing microorganisms (diazotrophs) are a major source of nitrogen to open ocean ecosystems and are predicted to be limited by iron in most marine environments. Here we use global and targeted proteomic analyses on a key unicellular marine diazotroph Crocosphaera watsonii to reveal large scale diel changes in its proteome, including substantial variations in concentrations of iron metalloproteins involved in nitrogen fixation and photosynthesis, as well as nocturnal flavodoxin production. The daily synthesis and degradation of enzymes in coordination with their utilization results in a lowered cellular metalloenzyme inventory that requires ∼40% less iron than if these enzymes were maintained throughout the diel cycle. This strategy is energetically expensive, but appears to serve as an important adaptation for confronting the iron scarcity of the open oceans. A global numerical model of ocean circulation, biogeochemistry and ecosystems suggests that Crocosphaera's ability to reduce its iron-metalloenzyme inventory provides two advantages: It allows Crocosphaera to inhabit regions lower in iron and allows the same iron supply to support higher Crocosphaera biomass and nitrogen fixation than if they did not have this reduced iron requirement.cyanobacteria | marine iron cycle | nitrogen cycle

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Synthesis and proteolytic degradation of nitrogenase in cultures of the unicellular cyanobacterium Gloeothece strain ATCC 27152

Cited by 24 publications

References 31 publications

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

Diel variation of molybdenum and iron in marine diazotrophic cyanobacteria

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii

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