<i>In vivo</i> transposon tagging in the nonheterocystous nitrogen‐fixing cyanobacterium <i>Leptolyngbya boryana</i>

Tomatsu, Chie; Uesaka, Kazuma; Yamakawa, Hisanori; Ihara, Kenji; Fujita, Yuichi

doi:10.1002/1873-3468.13079

Cited by 6 publications

(3 citation statements)

References 36 publications

(66 reference statements)

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“…In addition to the target gene disruption technique, we recently developed an in vivo transposon tagging system to isolate random mutants in L. boryana (Tomatsu et al, 2018). The technique may facilitate the identification of novel genes that are not in the nif gene cluster of L. boryana or in the genomes of heterotrophic bacteria such as A. vinelandii .…”

Section: Discussionmentioning

confidence: 99%

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana

et al. 2019

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Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

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Section: Discussionmentioning

confidence: 99%

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana

et al. 2019

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“…Transformation of L. boryana was performed using a conjugation method. The donor Escherichia coli S17-1 λ pir carrying the LBDG_21500-disrupting plasmid was prepared as described previously (Tomatsu et al 2018). L. boryana dg5 cells grown photosynthetically on a BG-11 agar plate were suspended in 0.3 mL of BG-11 medium at 0.5 OD 730 and mixed with the same volume of the donor E. coli .…”

Section: Methodsmentioning

confidence: 99%

Microevolution toward loss of photosynthesis: Mutations promoting dark-heterotrophic growth and suppressing photosynthetic growth in cyanobacteria

Hida,

Nishio,

Uesaka

et al. 2024

Preprint

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The prevalence of parasitic plants suggests frequent evolution of photosynthetic capacity loss in the natural environment. However, no studies have observed such evolutionary events as a loss of photosynthetic capacity. Herein, we report mutations that lead to a loss or decrease in photosynthetic growth capacity of dark-adapted variants of the cyanobacteriumLeptolyngbya boryana, which can grow heterotrophically even in the dark. We isolated 28 dark-adapted variants through long-term cultivation (7–49 months) under dark-heterotrophic conditions. All variants showed significantly faster dark-heterotrophic growth than the parental strains, accompanied by the loss of photosynthetic growth capacity in 15 variants. Genome resequencing of the variants revealed that 19 of the 28 variants carried various mutations in a common single gene (LBDG_21500) encoding a protein phosphatase 2C (PP2C) RsbU that is involved in the partner switching system (PSS). Phenotypic and transcriptomic analyses of aLBDG_21500-knockout mutant suggested that the PSS, including LBDG_21500, is involved in the global transcriptional regulation of various genes under both photoautotrophic and dark-heterotrophic conditions. We propose the renaming ofLBDG_21500tophsP(phototrophic–heterotrophic switching phosphatase). Our results imply that mutations in the global transcriptional regulatory system serve as the first evolutionary step leading to the loss of photosynthetic capacity.

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“…Lysozyme (final 10 mg mL -1 ) was added to the suspension and the sample was incubated at 37°C for 1 h. The suspension was centrifuged once (6,000 rpm for 1 min; RT15A3), and the precipitate was suspended in 100 µL of RNaseA (DNase free, final 20 µg mL -1 ) containing TE buffer. Then, 600 µL of DNA Suisui F (Rizo, Tsukuba) was added (Tomatsu et al 2018), the sample was mixed well by inversion, and then the sample was incubated at 70°C for 10 min for cell lysis. The cell lysate was transferred to a 2-mL microtube, 800 µL of TE-saturated phenol was added, and the sample was gently stirred with a reciprocal shaker (model, NR-1; TAITEC, Koshigaya) for 15 min.…”

Section: Preparation Of Genomic Dnamentioning

confidence: 99%

Restoration of the FunctionalnifGene Cluster by Seven Excision and Two Inversion Events during Heterocyst Development in the Nitrogen-Fixing CyanobacteriumCalothrixsp. NIES-4101

Uesaka,

Banba,

Chiba

et al. 2023

Preprint

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In the genome of the heterocystous cyanobacteriumCalothrixsp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB,nifH,nifD, andnifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intactnifBHDKgenes by the excision mechanism found in thenifDgene of theAnabaenasp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the completenifBHDKgenes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the completenifHDKandnifBgenes were found byde novoassembly of the sequencing reads. In addition, DNA fragments covering thenifBHDKoperon were successfully amplified by PCR. We propose that the process ofnifBHDKrestoration occurs as follows. First, thenifD-nifKgenes are restored by four excision events. Then the completenifHandnifBgenes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functionalnifgene cluster,nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of thenifBHDKgenes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

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

Cited by 6 publications

References 36 publications

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana

Microevolution toward loss of photosynthesis: Mutations promoting dark-heterotrophic growth and suppressing photosynthetic growth in cyanobacteria

Restoration of the FunctionalnifGene Cluster by Seven Excision and Two Inversion Events during Heterocyst Development in the Nitrogen-Fixing CyanobacteriumCalothrixsp. NIES-4101

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