Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus
            <i>Cyanothece</i>

Huang, Min; Sherman, Louis A.

doi:10.1128/aem.01456-10

Cited by 27 publications

(22 citation statements)

References 37 publications

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“…The single-stranded DNA technique and electroporation (18) were used to transform Cyanothece sp. strain PCC 7822 with kanamycin as the selective agent (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Total chlorophyll a (Chl a) was extracted by methanol and quantified spectrophotometrically in a PerkinElmer lambda 40 spectrophotometer. The concentration was determined by using the equation [Chl] ϭ 14.97(A 678 Ϫ A 750 ) Ϫ 0.615(A 620 Ϫ A 750 ) by measuring light absorbance at 750, 678, and 620 nm (18). Protein concentrations were determined using the Bio-Rad protein assay according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…(ii) H 2 production. H 2 evolution was measured with 45 ml of cultures in 66-ml bottles, sparged with argon, and incubated for 24 h under continuous light (light intensity, 50 mol photons m Ϫ2 s Ϫ1 ) at 30°C for 24 h. Gas samples of 200 l were measured using a Hewlett Packard 5890 series II gas chromatograph (11,18).…”

Section: Methodsmentioning

confidence: 99%

“…The PCR-amplified hupL gene (primer pair hupLMF and hupLMR) ( Table 1) was cloned into a Topo-TA vector (Invitrogen Topo-TA cloning kit), and a neomycin/kanamycin cassette (Nm/ Km) from pRL448 (17) was inserted into hupL at an EcoRI site (phL-K) and then sequenced. Single-strand PCR products from asymmetrical PCR of hupL::Nm/Km were transformed into Cyanothece cells (18), and single colonies were selected from BG11 plates supplemented with 15 g/ml kanamycin. Asymmetric PCR with products carrying the hupL gene with a Km/Nm cassette insertion was conducted with primer hupLMF at 0.2 M final concentration and hupLMR at 1:100 of this concentration to produce single-stranded DNA (ssDNA).…”

Section: Methodsmentioning

confidence: 99%

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The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Zhang

Sherman²,

Sherman

2014

J Bacteriol

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Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H 2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H 2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (⌬hupL), and we determined how this would affect the amount of H 2 produced. The ⌬hupL mutant demonstrated virtually no nitrogenase activity or H 2 production when grown under N 2 -fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O 2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N 2 -fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H 2 produced by the nitrogenase.

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“…The single-stranded DNA technique and electroporation (18) were used to transform Cyanothece sp. strain PCC 7822 with kanamycin as the selective agent (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Zhang

Sherman²,

Sherman

2014

J Bacteriol

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“…Among nonheterocystous cyanobacteria, some Cyanothece strains have been studied by a combination of transcriptomic and proteomic analyses (20,21). However, the lack of an efficient transformation system has hindered the identification of the detailed mechanism underlying the regulation of nitrogen fixation (34). In the present study, we isolated 11 mutants with an efficient transformation system using the nonheterocystous strain L. boryana.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria

Tsujimoto¹,

Kamiya

Fujita³

2014

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

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Significance Nitrogen fixation is a process of conversion of atmospheric nitrogen to ammonia catalyzed by nitrogenase, which is quickly inactivated by oxygen. Cyanobacteria are a group of prokaryotes that perform oxygenic photosynthesis, and many cyanobacterial species have the ability to fix nitrogen. How nitrogen fixation is coordinated with oxygenic photosynthesis remains largely unknown. Here we report two transcriptional regulators, ChlR (chlorophyll regulator) and CnfR (cyanobacterial nitrogen fixation regulator), that activate the transcription of genes responsible for anaerobic chlorophyll biosynthesis and the nitrogen fixation genes, respectively, in response to low-oxygen conditions in Leptolyngbya boryana , a diazotrophic cyanobacterium lacking heterocysts.

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In vivo transposon tagging in the nonheterocystous nitrogen‐fixing cyanobacterium Leptolyngbya boryana

et al. 2018

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Nitrogenase is an oxygen-vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini-Tn5-derived vector, pKUT-Tn5-Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin-resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria.

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Genetic Transformation and Mutagenesis via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus Cyanothece

Cited by 27 publications

References 37 publications

The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria

In vivo transposon tagging in the nonheterocystous nitrogen‐fixing cyanobacterium Leptolyngbya boryana

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