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

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).

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Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00220-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4425-4430 ◽

Cited By ~ 54

Author(s):

Ana Valladares ◽

Iris Maldener ◽

Alicia M. Muro-Pastor ◽

Enrique Flores ◽

Antonia Herrero

Keyword(s):

Nitrogenase Activity ◽

Nitrogen Deprivation ◽

Wild Type ◽

Cyanobacterium Anabaena ◽

In The Wild ◽

Membrane Reorganization ◽

Respiratory Oxidases ◽

And Performance ◽

Pcc 7120 ◽

Heterocyst Development

ABSTRACT Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N2 fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

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

Journal of Bacteriology ◽

10.1128/jb.00776-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5526-5533 ◽

Cited By ~ 49

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.

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c-di-GMP Homeostasis Is Critical for Heterocyst Development in Anabaena sp. PCC 7120

Frontiers in Microbiology ◽

10.3389/fmicb.2021.793336 ◽

2021 ◽

Vol 12 ◽

Author(s):

Min Huang ◽

Ju-Yuan Zhang ◽

Xiaoli Zeng ◽

Cheng-Cai Zhang

Keyword(s):

Nitrogen Starvation ◽

Filamentous Cyanobacterium ◽

Nitrogen Deprivation ◽

Heterocyst Differentiation ◽

Combined Nitrogen ◽

Heterocyst Frequency ◽

Genes Encoding ◽

Anabaena Sp ◽

Opposing Effects ◽

Pcc 7120

c-di-GMP is a ubiquitous bacterial signal regulating various physiological process. Anabaena PCC 7120 (Anabaena) is a filamentous cyanobacterium able to form regularly-spaced heterocysts for nitrogen fixation, in response to combined-nitrogen deprivation in 24h. Anabaena possesses 16 genes encoding proteins for c-di-GMP metabolism, and their functions are poorly characterized, except all2874 (cdgS) whose deletion causes a decrease in heterocyst frequency 48h after nitrogen starvation. We demonstrated here that c-di-GMP levels increased significantly in Anabaena after combined-nitrogen starvation. By inactivating each of the 16 genes, we found that the deletion of all1175 (cdgSH) led to an increase of heterocyst frequency 24h after nitrogen stepdown. A double mutant ΔcdgSHΔcdgS had an additive effect over the single mutants in regulating heterocyst frequency, indicating that the two genes acted at different time points for heterocyst spacing. Biochemical and genetic data further showed that the functions of CdgSH and CdgS in the setup or maintenance of heterocyst frequency depended on their opposing effects on the intracellular levels of c-di-GMP. Finally, we demonstrated that heterocyst differentiation was completely inhibited when c-di-GMP levels became too high or too low. Together, these results indicate that the homeostasis of c-di-GMP level is important for heterocyst differentiation in Anabaena.

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Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.2.c392 ◽

1995 ◽

Vol 269 (2) ◽

pp. C392-C402 ◽

Cited By ~ 41

Author(s):

D. Wang ◽

D. F. Balkovetz ◽

D. G. Warnock

Keyword(s):

Mutational Analysis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

Clear Cut ◽

Inhibition Concentration ◽

Nhe1 Activity ◽

Per Se ◽

Nhe Isoforms

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

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

Journal of Bacteriology ◽

10.1128/jb.183.22.6667-6675.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6667-6675 ◽

Cited By ~ 42

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.

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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 ◽

10.1099/mic.0.046128-0 ◽

2011 ◽

Vol 157 (3) ◽

pp. 617-626 ◽

Cited By ~ 13

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.

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NrrA Directly Regulates Expression of hetR during Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.01314-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8520-8525 ◽

Cited By ~ 62

Author(s):

Shigeki Ehira ◽

Masayuki Ohmori

Keyword(s):

Transcription Initiation ◽

Response Regulator ◽

Nitrogen Deprivation ◽

Heterocyst Differentiation ◽

Combined Nitrogen ◽

Regulatory Cascade ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

ABSTRACT Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 requires NtcA, the global nitrogen regulator in cyanobacteria, and HetR, the master regulator of heterocyst differentiation. Expression of hetR is upregulated by nitrogen deprivation, and its upregulation depends on NtcA. However, it has not yet been revealed how NtcA regulates the expression of hetR. In the experiments presented here, it was confirmed that NrrA (All4312), a nitrogen-responsive response regulator, was required for the upregulation of hetR. The use of the nitrogen-responsive transcription initiation sites (TISs) for the hetR gene depended upon NrrA. NrrA bound specifically to the region upstream of TISs located at positions −728 and −696 in vitro. Overexpression of nrrA resulted in enhanced hetR expression and heterocyst formation. A molecular regulatory cascade is proposed whereby NtcA upregulates the expression of nrrA upon limitation of combined nitrogen in the medium and then NrrA upregulates the expression of hetR, leading to heterocyst differentiation.

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hetL Overexpression Stimulates Heterocyst Formation in Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.184.24.6873-6881.2002 ◽

2002 ◽

Vol 184 (24) ◽

pp. 6873-6881 ◽

Cited By ~ 41

Author(s):

Duan Liu ◽

James W. Golden

Keyword(s):

Null Mutant ◽

Wild Type ◽

Heterologous Promoter ◽

Repeat Proteins ◽

Overexpression Strain ◽

In The Wild ◽

Anabaena Sp ◽

Pcc 7120 ◽

Diazotrophic Growth ◽

Heterocyst Development

ABSTRACT The cyanobacterium Anabaena sp. strain PCC 7120 forms single heterocysts about every 10 to 15 vegetative cells along filaments. PatS is thought to be a peptide intercellular signal made by developing heterocysts that prevents neighboring cells from differentiating. Overexpression of the patS gene suppresses heterocyst formation. The hetL gene (all3740) was isolated in a genetic screen to identify genes involved in PatS signaling. Extracopy hetL allowed heterocyst formation in a patS overexpression strain. hetL overexpression from a heterologous promoter in wild-type Anabaena PCC 7120 induced multiple-contiguous heterocysts (Mch) in nitrate-containing medium. The predicted HetL protein is composed almost entirely of pentapeptide repeats with a consensus of A(D/N)L*X, where * is a polar amino acid. Thirty Anabaena PCC 7120 genes contain this repeat motif. A synthetic pentapeptide corresponding to the last 5 amino acids of PatS, which suppresses heterocyst formation in the wild type, did not suppress heterocyst formation in a hetL overexpression strain, indicating that HetL overexpression is affecting heterocyst regulation downstream of PatS production. The transcription regulator NtcA is required for the initiation of heterocyst formation. hetL overexpression allowed the initiation of heterocyst development in an ntcA-null mutant, but differentiation was incomplete. hetR and hetC mutations that block heterocyst development are epistatic to hetL overexpression. A hetL-null mutant showed normal heterocyst development and diazotrophic growth, which could indicate that it is not normally involved in regulating development, that it normally plays a nonessential accessory role, or perhaps that its loss is compensated by cross talk or redundancy with other pentapeptide repeat proteins.

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Control of the Streptomyces Subtilisin Inhibitor Gene by AdpA in the A-Factor Regulatory Cascade in Streptomyces griseus

Journal of Bacteriology ◽

10.1128/jb.00662-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6207-6216 ◽

Cited By ~ 27

Author(s):

Setsu Hirano ◽

Jun-ya Kato ◽

Yasuo Ohnishi ◽

Sueharu Horinouchi

Keyword(s):

Binding Site ◽

Type Strain ◽

Time Course ◽

Wild Type Strain ◽

Mutational Analysis ◽

Streptomyces Griseus ◽

Wild Type ◽

Regulatory Cascade ◽

Number Of Genes ◽

Subtilisin Inhibitor

ABSTRACT AdpA in the A-factor regulatory cascade in Streptomyces griseus activates a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. The Streptomyces subtilisin inhibitor (SSI) gene, sgiA, in S. griseus was transcribed in response to AdpA, showing that sgiA is a member of the AdpA regulon. AdpA bound a single site upstream of the sgiA promoter at approximately position −70 with respect to its transcriptional start point. Mutational analysis of the AdpA-binding site showed that the AdpA-binding site was essential for transcriptional activation. Mutants in which sgiA was disrupted had higher trypsin, chymotrypsin, metalloendopeptidase, and total protease activities than the wild-type strain, which showed that SgiA modulated the activities of these extracellularly produced proteases. Because a number of genes encoding chymotrypsins, trypsins, and metalloendopeptidases, most of which are SSI-sensitive proteases, are also under the control of AdpA, the A-factor regulatory cascade was thought to play a crucial role in modulating the extracellular protease activities by triggering simultaneous production of the proteases and their inhibitor at a specific timing during growth. Mutants in which sgiA was disrupted grew normally and formed aerial hyphae and spores with the same time course as the wild-type strain. However, exogenous addition of purified SgiA to substrate mycelium grown on agar medium resulted in a delay in aerial mycelium formation, indicating that SgiA is involved in aerial hypha formation in conjunction with proteases.

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The Inorganic Nutrient Regime and the mre Genes Regulate Cell and Filament Size and Morphology in the Phototrophic Multicellular Bacterium Anabaena

mSphere ◽

10.1128/msphere.00747-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Cristina Velázquez-Suárez ◽

Ignacio Luque ◽

Antonia Herrero

Keyword(s):

Cell Size ◽

Inorganic Nitrogen ◽

Oxygenic Photosynthesis ◽

Filament Length ◽

Wild Type ◽

Active Growth ◽

Lower Growth ◽

Combined Nitrogen ◽

Size And Morphology ◽

Diazotrophic Growth

ABSTRACT The model cyanobacterium Anabaena sp. PCC 7120 exhibits a phototrophic metabolism relying on oxygenic photosynthesis and a complex morphology. The organismic unit is a filament of communicated cells that may include cells specialized in different nutritional tasks, thus representing a paradigm of multicellular bacteria. In Anabaena, the inorganic carbon and nitrogen regime influenced not only growth, but also cell size, cell shape, and filament length, which also varied through the growth cycle. When using combined nitrogen, especially with abundant carbon, cells enlarged and elongated during active growth. When fixing N2, which imposed lower growth rates, shorter and smaller cells were maintained. In Anabaena, gene homologs to mreB, mreC, and mreD form an operon that was expressed at higher levels during the phase of fastest growth. In an ntcA mutant, mre transcript levels were higher than in the wild type and, consistently, cells were longer. Negative regulation by NtcA can explain that Anabaena cells were longer in the presence of combined nitrogen than in diazotrophic cultures, in which the levels of NtcA are higher. mreB, mreC, and mreD mutants could grow with combined nitrogen, but only the latter mutant could grow diazotrophically. Cells were always larger and shorter than wild-type cells, and their orientation in the filament was inverted. Consistent with increased peptidoglycan width and incorporation in the intercellular septa, filaments were longer in the mutants, suggesting a role for MreB, MreC, and MreD in the construction of septal peptidoglycan that could affect intercellular communication required for diazotrophic growth. IMPORTANCE Most studies on the determination of bacterial cell morphology have been conducted in heterotrophic organisms. Here, we present a study of how the availability of inorganic nitrogen and carbon sources influence cell size and morphology in the context of a phototrophic metabolism, as found in the multicellular cyanobacterium Anabaena. In Anabaena, the expression of the MreB, MreC, and MreD proteins, which influence cell size and length, are regulated by NtcA, a transcription factor that globally coordinates cellular responses to the C-to-N balance of the cells. Moreover, MreB, MreC, and MreD also influence septal peptidoglycan construction, thus affecting filament length and, possibly, intercellular molecular exchange that is required for diazotrophic growth. Thus, here we identified new roles for Mre proteins in relation to the phototrophic and multicellular character of a cyanobacterium, Anabaena.

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