scholarly journals The NtcA-Regulated amtB Gene Is Necessary for Full Methylammonium Uptake Activity in the Cyanobacterium Synechococcus elongatus

2007 ◽  
Vol 189 (21) ◽  
pp. 7791-7798 ◽  
Author(s):  
Javier Paz-Yepes ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Synechococcus Elongatus ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Noticeable Effect ◽  
Unicellular Cyanobacterium ◽  
Net Uptake ◽  
In The Wild ◽  
Uptake Activity ◽  
Step Down ◽  
Pcc 7942

ABSTRACT The Amt proteins constitute a ubiquitous family of transmembrane ammonia channels that permit the net uptake of ammonium by cells. In many organisms, there is more than one amt gene, and these genes are subjected to nitrogen control. The mature Amt protein is a homo- or heterooligomer of three Amt subunits. We previously characterized an amt1 gene in the unicellular cyanobacterium Synechococcus elongatus strain PCC 7942. In this work, we describe the presence in this organism of a second amt gene, amtB, which encodes a protein more similar to the bacterial AmtB proteins than to any other characterized cyanobacterial Amt protein. The expression of amtB took place in response to nitrogen step-down, required the NtcA transcription factor, and occurred parallel to the expression of amt1. However, the transcript levels of amtB measured after 2 h of nitrogen deprivation were about 100-fold lower than those of amt1. An S. elongatus amtB insertional mutant exhibited an activity for uptake of [14C]methylammonium that was about 55% of that observed in the wild type, but inactivation of amtB had no noticeable effect on the uptake of ammonium when it was supplied at a concentration of 100 μM or more. Because an S. elongatus amt1 mutant is essentially devoid of [14C]methylammonium uptake activity, the mature Amt transporter is functional in the absence of AmtB subunits but not in the absence of Amt1 subunits. However, the S. elongatus amtB mutant could not concentrate [14C]methylammonium within the cells to the same extent as the wild type. Therefore, AmtB is necessary for full methylammonium uptake activity in S. elongatus.

scholarly journals Alanine Dehydrogenase Activity Is Required for Adequate Progression of Phycobilisome Degradation during Nitrogen Starvation in Synechococcus elongatus PCC 7942

2006 ◽  
Vol 188 (14) ◽  
pp. 5258-5265 ◽  
Author(s):  
Roxane Lahmi ◽  
Eleonora Sendersky ◽  
Alexander Perelman ◽  
Martin Hagemann ◽  
Karl Forchhammer ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Cellular Response ◽  
Nitrogen Starvation ◽  
Synechococcus Elongatus ◽  
Wild Type ◽  
Sulfur Starvation ◽  
Alanine Dehydrogenase ◽  
In The Wild ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942

ABSTRACT Degradation of the cyanobacterial light-harvesting antenna, the phycobilisome, is a general acclimation response that is observed under various stress conditions. In this study we identified a novel mutant of Synechococcus elongatus PCC 7942 that exhibits impaired phycobilisome degradation specifically during nitrogen starvation, unlike previously described mutants, which exhibit aberrant degradation under nitrogen, sulfur, and phosphorus starvation conditions. The phenotype of the new mutant, AldΩ, results from inactivation of ald (encoding alanine dehydrogenase). AldΩ is deficient in transcription induction of a number of genes during nitrogen starvation. These genes include the “general nutrient stress-related” genes, nblA and nblC, the products of which are essential for phycobilisome degradation. Furthermore, transcripts of several specific nitrogen-responsive genes accumulate at lower levels in AldΩ than in the wild-type strain. In contrast, ald inactivation did not decrease the accumulation of transcripts during sulfur starvation. Transcription of ald is induced upon nitrogen starvation, which is consistent with the ability of wild-type cells to maintain a low cellular content of alanine under these conditions. Unlike wild-type cells, AldΩ accumulates alanine upon nitrogen starvation. Our analyses suggest that alanine dehydrogenase activity is necessary for an adequate cellular response to nitrogen starvation. Decomposition of alanine may be required to provide a sufficient amount of ammonia. Furthermore, the accumulated alanine, or a related metabolite, may interfere with the cues that modulate acclimation during nitrogen starvation. Taken together, our results provide novel information regarding cellular responses to nitrogen starvation and suggest that mechanisms related to nitrogen-specific responses are involved in modulation of a general acclimation process.

scholarly journals Signal Transduction Protein PII Is Required for NtcA-Regulated Gene Expression during Nitrogen Deprivation in the Cyanobacterium Synechococcus elongatus Strain PCC 7942

2003 ◽  
Vol 185 (8) ◽  
pp. 2582-2591 ◽  
Author(s):  
M. Fadi Aldehni ◽  
Jörg Sauer ◽  
Christian Spielhaupter ◽  
Roland Schmid ◽  
Karl Forchhammer
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Synechococcus Elongatus ◽  
Dependent Manner ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Combined Nitrogen ◽  
Signal Transduction Protein ◽  
Regulated Gene Expression ◽  
Pcc 7942

ABSTRACT The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the PII signaling protein play central roles in global nitrogen control in cyanobacteria. A dependence on PII for NtcA-regulated transcription, however, has not been observed. In the present investigation, we examined alterations in gene expression following nitrogen deprivation in Synechococcus elongatus strain PCC 7942 and specifically the roles of NtcA and PII. Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo [35S]methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis. Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of S. elongatus failed to respond in PII- and NtcA-deficient mutants. One of the proteins whose synthesis was down-regulated in a PII- and NtcA-dependent manner was RbcS, the small subunit of RubisCO. Quantification of its mRNA revealed that the abundance of the rbcLS transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in PII and NtcA mutant cells. To investigate further the relationship between PII and NtcA, fusions of the promotorless luxAB reporter genes to the NtcA-regulated glnB gene were constructed and these constructs were used to transform wild-type cells and PII − and NtcA− mutants. Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a PII-independent manner. By contrast, NtcA-dependent activation of glnB expression following combined-nitrogen deprivation was impaired in the absence of PII. Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the PII signal transduction protein.

scholarly journals The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

2001 ◽  
Vol 183 (8) ◽  
pp. 2654-2661 ◽  
Author(s):  
Francis C. Y. Wong ◽  
John C. Meeks
Keyword(s):  
Fluorescent Protein ◽  
Filamentous Cyanobacterium ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Combined Nitrogen ◽  
In The Wild ◽  
Step Down ◽  
Green Fluorescent ◽  
Heterocyst Development

ABSTRACT A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF intrans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription ofhetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetFmutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

scholarly journals Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942

2009 ◽  
Vol 191 (15) ◽  
pp. 4863-4869 ◽  
Author(s):  
Javier Espinosa ◽  
Miguel Angel Castells ◽  
Karim Boumediene Laichoubi ◽  
Asunción Contreras
Keyword(s):  
Molecular Basis ◽  
Synechococcus Elongatus ◽  
Wild Type ◽  
Growth Defects ◽  
Genetic Inactivation ◽  
Domains Of Life ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942 ◽  
Severe Growth ◽  
Null Mutants

ABSTRACT The PII proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of PII proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which PII proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where PII forms complexes with the NtcA coactivator PipX, attempts to engineer PII-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that PII is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.

scholarly journals Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942

Microbiology ◽  
2016 ◽  
Vol 162 (12) ◽  
pp. 2029-2041 ◽  
Author(s):  
You Chen ◽  
Arnaud Taton ◽  
Michaela Go ◽  
Ross E. London ◽  
Lindsey M. Pieper ◽  
...  
Keyword(s):  
Synechococcus Elongatus ◽  
Unicellular Cyanobacterium ◽  
Shuttle Vectors ◽  
Synechococcus Elongatus Pcc 7942 ◽  
Pcc 7942

scholarly journals pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

2001 ◽  
Vol 183 (2) ◽  
pp. 628-636 ◽  
Author(s):  
Sara Lázaro ◽  
Francisca Fernández-Piñas ◽  
Eduardo Fernández-Valiente ◽  
Amaya Blanco-Rivero ◽  
Francisco Leganés
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Molecular Size ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reading Frame ◽  
Cyanobacterium Anabaena ◽  
In The Wild ◽  
Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

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

2007 ◽  
Vol 189 (12) ◽  
pp. 4425-4430 ◽  
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.

scholarly journals Role of RelGsu in Stress Response and Fe(III) Reduction in Geobacter sulfurreducens

2006 ◽  
Vol 188 (24) ◽  
pp. 8469-8478 ◽  
Author(s):  
Laurie N. DiDonato ◽  
Sara A. Sullivan ◽  
Barbara A. Methé ◽  
Kelly P. Nevin ◽  
Reg England ◽  
...  
Keyword(s):  
Stress Responses ◽  
Physiological Role ◽  
Growth Stress ◽  
Geobacter Sulfurreducens ◽  
Nitrogen Deprivation ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Subsurface Environments ◽  
In The Wild

ABSTRACT Geobacter species are key members of the microbial community in many subsurface environments in which dissimilatory metal reduction is an important process. The genome of Geobacter sulfurreducens contains a gene designated relGsu , which encodes a RelA homolog predicted to catalyze both the synthesis and the degradation of guanosine 3′,5′-bispyrophosphate (ppGpp), a regulatory molecule that signals slow growth in response to nutrient limitation in bacteria. To evaluate the physiological role of Rel Gsu in G. sulfurreducens, a relGsu mutant was constructed and characterized, and ppGpp levels were monitored under various conditions in both the wild-type and relGsu mutant strains. In the wild-type strain, ppGpp and ppGp were produced in response to acetate and nitrogen deprivation, whereas exposure to oxygen resulted in an accumulation of ppGpp alone. Neither ppGpp nor ppGp could be detected in the relGsu mutant. The relGsu mutant consistently grew to a higher cell density than the wild type in acetate-fumarate medium and was less tolerant of oxidative stress than the wild type. The capacity for Fe(III) reduction was substantially diminished in the mutant. Microarray and quantitative reverse transcription-PCR analyses indicated that during stationary-phase growth, protein synthesis genes were up-regulated in the relGsu mutant and genes involved in stress responses and electron transport, including several implicated in Fe(III) reduction, were down-regulated in the mutant. The results are consistent with a role for Rel Gsu in regulating growth, stress responses, and Fe(III) reduction in G. sulfurreducens under conditions likely to be prevalent in subsurface environments.

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