Nitrogenase Activity and Amounts of Nitrogenase Proteins in a <i>Frankia-Alnus incana</i> Symbiosis Subjected to Darkness

Lundquist, Per-Olof; Huss‐Danell, Kerstin

doi:10.1104/pp.95.3.808

Cited by 26 publications

(27 citation statements)

References 21 publications

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“…The separated polypeptides were transferred to a nitrocellulose membrane [23], and treated with antibodies raised against the phycobilisomes. The blot was developed using alkaline phosphatase conjugated swine anti-rabbit immunoglobulin G purchased from Dakopatts (Stockholm, Sweden) and used according to the instructions of the vendor.…”

Section: Isolation Of Proteins and Western Blottingmentioning

confidence: 99%

Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

1994

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Two open reading frames denoted as cpcE and cpcF were cloned and sequenced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are located downstream of the cpcB2A2 gene cluster in the phycobilisome rod operon and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by insertion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which display a substantial reduction in spectroscopically detectable phycocyanin. The levels of beta- and alpha-phycocyanin polypeptides were reduced in the R2EKM and R2FKM mutants although the phycocyanin and linker genes are transcribed at normal levels in the mutants as in the wild type indicating the requirement of the functional cpcE and cpcF genes for normal accumulation of phycocyanin. Two biliprotein fractions were isolated on sucrose density gradient from the R2EKM/R2FKM mutants. The faster sedimenting fraction consisted of intact phycobilisomes. The slower sedimenting biliprotein fraction was found to lack phycocyanin polypeptides, thus no free phycocyanin was detected in the mutants. Characterization of the phycocyanin from the mutants revealed that it was chromophorylated, had a lambda max similar to that from the wild type and could be assembled into the phycobilisome rods. Thus, although phycocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaining phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy transfer to the core.

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Section: Isolation Of Proteins and Western Blottingmentioning

confidence: 99%

Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

1994

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“…Protein from Frankia vesicle cluster preparations and Frankia cultures were extracted as described earlier [13]. After addition of 3 ml of anaerobic buffer containing 4% PVPP, 50 mM Tris.…”

Section: Protein Extractsmentioning

confidence: 99%

“…Rooted cuttings of grey alder (Ablus hwana(L.) (Moench) inoculated with the local source of Frankia [12] or with Frankia Cpll (see below), were grown in a climate chamber for 9-12 weeks as described earlier [13]. Frankia vesicle clusters were prepared from root nodules as described earlier [13,14] and in addition further washed five times in 0.1% Triton X-100, 50 mM Tris.…”

Section: Plant Material Frankia Resicle Clusters and Cldlllresmentioning

confidence: 99%

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Immunological studies of glutamine synthetase in Frankia-Alnus incana symbioses

Lundquist

1992

FEMS Microbiology Letters

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We have investigated the presence and form of glutamine synthetase (GS) in Frankia vesicle cluster preparations of two actively nitrogen‐fixing Frankia‐Alnus incana root‐nodule symbioses and in cultured Frankia sp. strain CpI1 (HFP070101). The symbioses contained Frankia CpI1 or the local source of Frankia. We used Western‐blot analysis with antisera raised against three types of GS. In symbiotic Frankia GS protein was not detected at a significant level when either antisera against Rhodospirillum rubrum GS or antisera against Rhizobium meliloti GSII were used. In cultured Frankia CpI1 GSI was detected both when grown with NH4+ or N2 as nitrogen source, and GSII was detected when grown on N2. Antiserum raised against the nodule‐specific GSn1 of Phaseolus vulgaris crossreacted with a 43‐kDa polypeptide corresponding to plant GS in root‐nodule extracts from Alnus, and with a 41‐kDa polypeptide corresponding to GSII in cultured Frankia CpI1 grown on N2. We conclude that both GSI and GSII are repressed in symbiotic Frankia and that NH4+ produced through nitrogen fixation is assimilated by the plant in Frankia‐Alnus incana symbioses. It thus appears that vesicle formation, synthesis of nitrogenase and synthesis of GS are separately regulated in symbiotic Frankia and that the plant has to supply symbiotic Frankia with organic nitrogen in some form in addition to the carbon.

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“…Proteins for western blols from the culture and from vesicle clusters were extracted as described earlier (Lundquist and Huss-Danell 1991). Western blols were ran with antisera made against FeSOD from Escherichia coli and Azotobacter vinelandii and against MnSOD from E. coli.…”

Section: Enzyme Analysismentioning

confidence: 99%

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Alskog¹,

Huss‐Danell²

1997

Physiol Plant

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Alskog, G. and Huss-Danell. K. 1997. Superoxide dismutase. catalase and nitrogenase activities of symbiolic Frankia [Alnus incana) in response to different oxygen Presence and activity of the enzymes superoxide dismula.se (SOD) and catalase were studied in Frankia in symbiosis with .\tnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and calalase. The activity of the enzymes was in Ihe same range as reported for cultured Frankia. Attempts to characterize SOD by western blots with antisera fi'om Escherichia colt and .\zotobacter vinelandii did not give clear-cut results with the antibodies used, .\lnus incana plants were grown with the root system in 5, 10. 21 or407r O; for up to 6 days. Nitrogenase activity, measured as ..\RA (acetylene reducing acli\ ity) dropped within 3 h when roots were exposed to low or high oxygen. .^1 409f O; ARA was almost completely lost while at 5 and 109; O; ARA decreased lo 69 and 74'/f of the initai value, respectively. Nitrogenase activity recovered al a!l oxygen tensions. Recovery rates resembled the continuous increa.se in AR.A in plants continuosly kept al 2I'7r O-. and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H^ evolution. There was a tendency for increased SOD and catatase activities in Frankia from rool systems exposed to 40'>r O: for 24 h but nol earlier or later than this. When data from all experimenlal times were pooled. SOD activity increased significantly with increased oxygen tension whereas catala.se activity decreased. Although ARA per plant varied wilh oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and calalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia.

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Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness

Cited by 26 publications

References 21 publications

Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Immunological studies of glutamine synthetase in Frankia-Alnus incana symbioses

Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

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