scholarly journals Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

2001 ◽  
Vol 183 (22) ◽  
pp. 6667-6675 ◽  
Author(s):  
Ivan Y. Khudyakov ◽  
James W. Golden
Keyword(s):  
Sigma Factor ◽  
Double Mutant ◽  
Developmental Regulation ◽  
Single Gene ◽  
Sigma Factors ◽  
Nitrogen Deprivation ◽  
Insertional Inactivation ◽  
Group 2 ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD,sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD andsigE mutant strains to establish diazotrophic growth. AsigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigDor sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, thesigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes.luxAB reporter fusions with sigD,sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains,Anabaena sp. strain PCC 7120,Synechococcus sp. strain PCC 7002, andSynechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains,Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.

scholarly journals Inactivation of a Heterocyst-Specific Invertase Indicates a Principal Role of Sucrose Catabolism in Heterocysts of Anabaena sp.

2010 ◽  
Vol 192 (20) ◽  
pp. 5526-5533 ◽  
Author(s):  
Rocío López-Igual ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Fluorescent Protein ◽  
Northern Analysis ◽  
Filamentous Cyanobacterium ◽  
Nitrogen Deprivation ◽  
Vegetative Cells ◽  
Anabaena Sp ◽  
Green Fluorescent ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N2 fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO2. The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

scholarly journals Anabaena sp. strain PCC 7120 conR contains a LytR-CpsA-Psr domain, is developmentally regulated, and is essential for diazotrophic growth and heterocyst morphogenesis

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 617-626 ◽  
Author(s):  
Rodrigo A. Mella-Herrera ◽  
M. Ramona Neunuebel ◽  
James W. Golden
Keyword(s):  
Nitrogenase Activity ◽  
Growth Defect ◽  
Filamentous Cyanobacterium ◽  
Developmentally Regulated ◽  
Vegetative Cells ◽  
Septum Formation ◽  
Insertional Inactivation ◽  
Step Down ◽  
Pcc 7120 ◽  
Diazotrophic Growth

The conR (all0187) gene of the filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 is predicted to be part of a family of proteins that contain the LytR-CpsA-Psr domain associated with septum formation and cell wall maintenance. The conR gene was originally misannotated as a transcription regulator. Northern RNA blot analysis showed that conR expression was upregulated 8 h after nitrogen step-down. Fluorescence microscopy of a P conR –gfp reporter strain revealed increased GFP fluorescence in proheterocysts and heterocysts beginning 9 h after nitrogen step-down. Insertional inactivation of conR caused a septum-formation defect of vegetative cells grown in nitrate-containing medium. In nitrate-free medium, mutant filaments formed abnormally long heterocysts and were defective for diazotrophic growth. Septum formation between heterocysts and adjacent vegetative cells was abnormal, often with one or both poles of the heterocysts appearing partially open. In a conR mutant, expression of nifH was delayed after nitrogen step-down and nitrogenase activity was approximately 70 % of wild-type activity, indicating that heterocysts of the conR mutant strain are partially functional. We hypothesize that the diazotrophic growth defect is caused by an inability of the heterocysts to transport fixed nitrogen to the neighbouring vegetative cells.

scholarly journals Nitrogen-Regulated Group 2 Sigma Factor fromSynechocystis sp. Strain PCC 6803 Involved in Survival under Nitrogen Stress

2001 ◽  
Vol 183 (3) ◽  
pp. 1090-1095 ◽  
Author(s):  
Alicia Marı́a Muro-Pastor ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Sigma Factor ◽  
Nitrogen Starvation ◽  
Sigma Factors ◽  
Open Reading Frame ◽  
Nitrogen Stress ◽  
Nitrogen Control ◽  
Noticeable Effect ◽  
Reading Frame ◽  
Group 2 ◽  
Pcc 6803

ABSTRACT The expression of sll1689, an open reading frame from the cyanobacterium Synechocystis sp. strain PCC 6803 putatively encoding a member of the ς70 family of sigma factors, appears to be regulated by the nitrogen control transcription factor NtcA. Disruption of sll1689 had no noticeable effect on exponential growth, identifying its product as a member of the group 2, nonessential class of ς70-like sigma factors; however, this disruption decreased the viability of the cells after long periods of nitrogen starvation. We have named this generpoD2-V. The expression of glnN, encoding a type III glutamine synthetase, was impaired in strains bearing an inactivated copy of the rpoD2-Vgene.

scholarly journals Sigma Factor Genes sigC, sigE, and sigG Are Upregulated in Heterocysts of the Cyanobacterium Anabaena sp. Strain PCC 7120

2007 ◽  
Vol 189 (22) ◽  
pp. 8392-8396 ◽  
Author(s):  
M. Ramona Aldea ◽  
Rodrigo A. Mella-Herrera ◽  
James W. Golden
Keyword(s):  
Sigma Factor ◽  
Fluorescent Protein ◽  
Developmental Regulation ◽  
Time Lapse ◽  
Green Fluorescent Protein Fluorescence ◽  
Protein Fluorescence ◽  
Cyanobacterium Anabaena ◽  
Reporter Strains ◽  
Green Fluorescent ◽  
Pcc 7120

ABSTRACT We used gfp transcriptional fusions to investigate the regulation of eight sigma factor genes during heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Reporter strains containing gfp fusions with the upstream regions of sigB2, sigD, sigI, and sigJ did not show developmental regulation. Time-lapse microscopy of sigC, sigE, and sigG reporter strains showed increased green fluorescent protein fluorescence in differentiating cells at 4 h, 16 h, and 9 h, respectively, after nitrogen step down.

scholarly journals Expression and Mutational Analysis of the glnB Genomic Region in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

2009 ◽  
Vol 191 (7) ◽  
pp. 2353-2361 ◽  
Author(s):  
Javier Paz-Yepes ◽  
Enrique Flores ◽  
Antonia Herrero
Keyword(s):  
Time Course ◽  
Mutational Analysis ◽  
Nitrogenase Activity ◽  
Genomic Region ◽  
Nitrogen Deprivation ◽  
Transport Activity ◽  
Wild Type ◽  
Combined Nitrogen ◽  
Pcc 7120 ◽  
Diazotrophic Growth

ABSTRACT In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the glnB gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional glnB gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [glnB all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent inducion of the hetC and nifH genes was unaltered, but induction of the urtA gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the glnB gene product. In the presence of a wild-type glnB gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the glnB gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for glnB can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).

scholarly journals NrrA directly regulates expression of the fraF gene and antisense RNAs for fraE in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (5) ◽  
pp. 844-850 ◽  
Author(s):  
Shigeki Ehira ◽  
Masayuki Ohmori
Keyword(s):  
Response Regulator ◽  
Repeat Sequence ◽  
Nitrogen Deprivation ◽  
Antisense Rnas ◽  
Regulated Expression ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Dna Strand ◽  
Pcc 7120 ◽  
Diazotrophic Growth

The heterocystous cyanobacterium Anabaena sp. strain PCC 7120 grows as linear multicellular filaments that can contain hundreds of cells. Heterocysts, which are specialized cells for nitrogen fixation, are regularly intercalated among photosynthetic vegetative cells, and these cells are metabolically dependent on each other. Thus, multicellularity is essential for diazotrophic growth of heterocystous cyanobacteria. In Anabaena sp. strain PCC 7120, the fraF gene, which is required to limit filament length, is induced by nitrogen deprivation. The fraF transcripts extend to the fraE gene, which lies on the opposite DNA strand and could possess dual functionality, mRNAs for fraF and antisense RNAs for fraE. In the present study, we found that NrrA, a nitrogen-regulated response regulator, directly regulated expression of fraF. Induction of fraF by nitrogen deprivation was abolished by the nrrA disruption. NrrA specifically bound to the promoter region of fraF, and recognized an inverted repeat sequence. Thus, it is concluded that NrrA controls expression of mRNAs for fraF and antisense RNAs for fraE in response to nitrogen deprivation.

scholarly journals Identification of genome-wide distribution of cyanobacterial group 2 sigma factor SigE accountable for its regulon

2021 ◽  
Author(s):  
Ryo Kariyazono ◽  
Takashi Osanai
Keyword(s):  
Sigma Factor ◽  
Promoter Sequence ◽  
Wide Distribution ◽  
Sigma Factors ◽  
In Vitro Assays ◽  
Expression Of Genes ◽  
Group 2 ◽  
Synechocystis Sp ◽  
Pcc 6803

Sigma factors are the subunits of bacterial RNA polymerase that govern the expression of genes by recognizing the promoter sequence. Cyanobacteria, which are oxygenic phototrophic eubacteria, have multiple alternative sigma factors that respond to various environmental stresses. The subgroup highly homologous to the primary sigma factor (SigA) is called the group 2 sigma factor. The model cyanobacterium, Synechocystis sp. PCC 6803, has four group 2 sigma factors (SigB-E) conserved within the phylum Cyanobacteria. Among the group 2 sigma factors in Synechocystis sp. PCC 6803, SigE is unique because it alters metabolism by inducing the expression of genes related to sugar catabolism and nitrogen metabolism. However, the features of promoter sequence of the SigE regulon remains elusive. Here, we identified the direct targets of SigA and SigE by chromatin immunoprecipitation sequencing (ChIP-seq). We then showed that the binding sites of SigE and SigA overlapped substantially, but SigE exclusively localized to SigE-dependent promoters. We also found consensus sequences from SigE-dependent promoters and confirmed their importance. ChIP-seq analysis showed both the redundancy and specificity of SigE compared with SigA, integrating information obtained from a previously adopted genetic approach and in vitro assays. The features of SigE elucidated in our study indicate its similarity with group 2 sigma factors of other bacteria, even though they are evolutionally irrelevant. Our approach is also applicable to other organisms and organelles, such as plant plastids, which have multiple group 2 sigma factors.

scholarly journals Roles for Sigma Factors in Global Circadian Regulation of the Cyanobacterial Genome

2002 ◽  
Vol 184 (13) ◽  
pp. 3530-3538 ◽  
Author(s):  
Usha Nair ◽  
Jayna L. Ditty ◽  
Hongtao Min ◽  
Susan S. Golden
Keyword(s):  
Sigma Factor ◽  
Sigma Factors ◽  
Circadian Regulation ◽  
Cyanobacterial Genome ◽  
Circadian Period ◽  
Unicellular Cyanobacterium ◽  
Circadian Expression ◽  
A Cell ◽  
Group 2 ◽  
Pcc 7942

ABSTRACT The circadian clock of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 imposes a global rhythm of transcription on promoters throughout the genome. Inactivation of any of the four known group 2 sigma factor genes (rpoD2, rpoD3, rpoD4, and sigC), singly or pairwise, altered circadian expression from the psbAI promoter, changing amplitude, phase angle, waveform, or period. However, only the rpoD2 mutation and the rpoD3 rpoD4 and rpoD2 rpoD3 double mutations affected expression from the kaiB promoter. A striking differential effect was a 2-h lengthening of the circadian period of expression from the promoter of psbAI, but not of those of kaiB or purF, when sigC was inactivated. The data show that separate timing circuits with different periods can coexist in a cell. Overexpression of rpoD2, rpoD3, rpoD4, or sigC also changed the period or abolished the rhythmicity of PpsbAI expression, consistent with a model in which sigma factors work as a consortium to convey circadian information to downstream genes.

scholarly journals Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

2012 ◽  
Vol 78 (16) ◽  
pp. 5638-5645 ◽  
Author(s):  
Yoon-Suk Kang ◽  
Brian Bothner ◽  
Christopher Rensing ◽  
Timothy R. McDermott
Keyword(s):  
Binding Site ◽  
Sigma Factor ◽  
Model Organism ◽  
Coding Region ◽  
Rt Pcr ◽  
Obvious Effect ◽  
Content Type ◽  
Definitive Evidence ◽  
Relative Contribution ◽  
Insertional Inactivation

ABSTRACTIn this study with the model organismAgrobacterium tumefaciens, we used a combination oflacZgene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of AsIIIoxidation. A deletion mutation that removed the RpoN binding site from theaioBApromoter and anaacC3(gentamicin resistance) cassette insertional inactivation of therpoNcoding region eliminatedaioBAexpression and AsIIIoxidation, althoughrpoNexpression was not related to cell exposure to AsIII. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters foraioB,phoB1,pstS1,dctA,glnA,glnB, andflgBthat were examined by using qualitative RT-PCR andlacZreporter fusions to assess the relative contribution of RpoN to their transcription. The expressions ofaioBanddctAin the wild-type strain were considerably enhanced in cells exposed to AsIII, and both genes were silent in therpoN::aacC3mutant regardless of AsIII. The expression level ofglnAwas not influenced by AsIIIbut was reduced (but not silent) in therpoN::aacC3mutant and further reduced in the mutant under N starvation conditions. TherpoN::aacC3mutation had no obvious effect on the expression ofglnB,pstS1,phoB1, orflgB. These experiments provide definitive evidence to document the requirement of RpoN for AsIIIoxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by AsIIIor by this sigma factor.

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