Cross-Activation of Two Nitrogenase Gene Clusters by CnfR1 or CnfR2 in the Cyanobacterium Anabaena variabilis.

In Anabaena variabilis, the genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the promoter or from the Co-inducible promoter, activated the expression of both and .

Comparative genomic insights into culturable symbiotic cyanobacteria from the water fern .

Species of the floating, freshwater fern form a well-characterized symbiotic association with the non-culturable cyanobacterium , which fixes nitrogen for the plant. However, several cyanobacterial strains have over the years been isolated and cultured from from all over the world. The genomes of 10 of these strains were sequenced and compared with each other, with other symbiotic cyanobacterial strains, and with similar strains that were not isolated from a symbiotic association.

Fluorescence Localization of Gene Expression Using a Reporter in the Heterocyst-forming Cyanobacterium .

One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene , encoding the enzyme β-galactosidase. We provide here a protocol for the localization of β-galactosidase activity in cyanobacterial cells.

Role of the nifB1 and nifB2 Promoters in Cell-Type-Specific Expression of Two Mo Nitrogenases in the Cyanobacterium Anabaena variabilis ATCC 29413.

Unlabelled: Anabaena variabilis ATCC 29413 has one Mo nitrogenase that is made under oxic growth conditions in specialized cells called heterocysts and a second Mo nitrogenase that is made only under anoxic conditions in vegetative cells. The two large nif gene clusters responsible for these two nitrogenases are under the control of the promoter of the first gene in the operon, nifB1 or nifB2 Despite differences in the expression patterns of nifB1 and nifB2, related to oxygen and cell type, the regions upstream of their transcription start sites (tss) show striking homology, including three highly conserved sequences (CS). CS1, CS2, and the region just upstream from the tss were required for optimal expression from the nifB1 promoter, but CS3 and the 5' untranslated region (UTR) were not.

Homologous regulators, CnfR1 and CnfR2, activate expression of two distinct nitrogenase gene clusters in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

The cyanobacterium Anabaena variabilis has two Mo-nitrogenases that function under different environmental conditions in different cell types. The heterocyst-specific nitrogenase encoded by the large nif1 gene cluster and the similar nif2 gene cluster that functions under anaerobic conditions in vegetative cells are under the control of the promoter for the first gene of each cluster, nifB1 or nifB2 respectively. Associated with each of these clusters is a putative regulatory gene called cnfR (patB) whose product has a C-terminal HTH domain and an N-terminal ferredoxin-like domain.

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

Unlabelled: In the cyanobacterium Anabaena variabilis ATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the large nif1 gene cluster, which is primarily under the control of the promoter for the first gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by RNA processing, which leads to increased nifH1 transcript stability.

Regulation of Three Nitrogenase Gene Clusters in the Cyanobacterium Anabaena variabilis ATCC 29413.

The filamentous cyanobacterium Anabaena variabilis ATCC 29413 fixes nitrogen under aerobic conditions in specialized cells called heterocysts that form in response to an environmental deficiency in combined nitrogen. Nitrogen fixation is mediated by the enzyme nitrogenase, which is very sensitive to oxygen. Heterocysts are microxic cells that allow nitrogenase to function in a filament comprised primarily of vegetative cells that produce oxygen by photosynthesis.

Complete genome sequence of Anabaena variabilis ATCC 29413.

Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc.

Regulation of nitrogenase gene expression by transcript stability in the cyanobacterium Anabaena variabilis.

The nitrogenase gene cluster in cyanobacteria has been thought to comprise multiple operons; however, in Anabaena variabilis, the promoter for the first gene in the cluster, nifB1, appeared to be the primary promoter for the entire nif cluster. The structural genes nifHDK1 were the most abundant transcripts; however, their abundance was not controlled by an independent nifH1 promoter, but rather, by RNA processing, which produced a very stable nifH1 transcript and a moderately stable nifD1 transcript. There was also no separate promoter for nifEN1.

Regulation of V-nitrogenase genes in Anabaena variabilis by RNA processing and by dual repressors.

Anabaena variabilis ATCC 29413 fixes nitrogen in specialized cells called heterocysts using either a Mo-nitrogenase or a V-nitrogenase. V-nitrogenase structural genes, vnfDGK, as well as vnfEN form an operon with ava4025, located upstream of vnfDG that is repressed by fixed nitrogen and by Mo. The ava4025-vnfDGKEN operon is under the control of a Mo-repressible promoter located nearly 600 bp upstream of ava4025.

RNA processing of nitrogenase transcripts in the cyanobacterium Anabaena variabilis.

Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1.

Regulation of fructose transport and its effect on fructose toxicity in Anabaena spp.

Anabaena variabilis grows heterotrophically using fructose, while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named frtRABC, into Anabaena sp.

Cross-functionality of nitrogenase components NifH1 and VnfH in Anabaena variabilis.

Anabaena variabilis fixes nitrogen under aerobic growth conditions in differentiated cells called heterocysts using either a Mo nitrogenase or a V nitrogenase. The nifH1 gene, which encodes the dinitrogenase reductase of the Mo nitrogenase that is expressed only in heterocysts, is cotranscribed with nifD1 and nifK1, which together encode the Mo dinitrogenase. These genes were expressed in the presence or absence of molybdate or vanadate.

High-affinity vanadate transport system in the cyanobacterium Anabaena variabilis ATCC 29413.

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M.