Brenda S. Pratte

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413.

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.

Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo‐nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo‐dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High‐affinity transport of molybdate in A. variabilis was mediated by an ABC‐type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate‐binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo‐nitrogenase. Growth using the alternative V‐nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 µM) supported normal growth of the modBC mutant using the Nif1 Mo‐nitrogenase, indicating that there was a low‐affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low‐affinity molybdate transport system in this strain.

Transport of molybdate in the cyanobacteriumAnabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such asAnahaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase.A. variabilis efficiently transported99Mo (molybdate) at concentrations less than 10−9 M. Competition experiments with other oxyanions suggested that the molybdate-transport system ofA. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of99Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system inA. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strainAnabaena sp. PCC 7120 transported99Mo in a very similar way toA. variabilis.

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. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frtA was very highly expressed, with or without fructose. In the frtR mutant, fructose uptake was immediate, in contrast to that in the wild-type strain, which required about 40 min for induction of transport. In the frtR mutant, high-level expression of the fructose transporter resulted in cells that were extremely sensitive to fructose. Even in the presence of the inducer, fructose, expression of frtA was low in the wild-type strain compared to that in the frtR mutant, indicating that FrtR repressed the transporter genes even in the presence of fructose. FrtR bound to the upstream region of frtA, but binding was not visibly altered by fructose, further supporting the hypothesis that fructose has only a modest effect in relieving repression of frtA by FrtR. A. variabilis grew better with increasing concentrations of fructose up to 50 mM, showing increased cell size and heterocyst frequency. Anabaena sp. strain PCC 7120 did not show any of these changes when it was grown with fructose. Thus, although Anabaena sp. strain PCC 7120 could take up fructose and use it in the dark, fructose did not improve growth in the light.

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. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.

Hydrogen production in nitrogenase mutants in Anabaena variabilis

Nitrogenase produces hydrogen as a normal byproduct of the reduction of dinitrogen to ammonia. The Nif2 nitrogenase in Anabaena variabilis is an alternative Mo-nitrogenase and is expressed in vegetative cells grown with fructose under strictly anaerobic conditions. We report here that the V75I substitution in the alpha-subunit of Nif2 showed greatly impaired acetylene reduction and reduced levels of (15)N(2) fixation but had similar hydrogen production rates as the wild-type enzyme under argon. Another mutant containing a substitution in the alpha-subunit, V76I, would result in a decrease in the size of the putative gas channel of nitrogenase and, thus, was hypothesized to affect substrate selectivity of nitrogenase. However, this substitution had no effect on the enzyme selectivity, suggesting that access by gases to the active site through this putative gas channel is not limited by the increased size of the amino acid side chain in the alpha-subunit, V76I substitution.

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. The vnfH gene, which encodes the dinitrogenase reductase of the V nitrogenase, was located about 23 kb from vnfDGK, which encodes the V dinitrogenase; however, like vnfDGK, vnfH was expressed only in the absence of molybdate, with or without vanadate. Like nifH1, the vnfH gene was expressed exclusively in heterocysts under either aerobic or anaerobic growth conditions and thus is under the control of developmental factors. The vnfH mutant was able to grow diazotrophically using the V nitrogenase, because NifH1, which was also made in cells starved for molybdate, could substitute for VnfH. Under oxic conditions, the nifH1 mutant grew in the absence of molybdate but not in its presence, using VnfH, while the nifH1 vnfH double mutant did not grow diazotrophically with or without molybdate or vanadate. A nifH1 mutant that expressed nifDK and vnfH but not vnfDGK was able to grow and fix nitrogen normally, indicating that VnfH could substitute for NifH in the Mo nitrogenase and that these dinitrogenase reductases are not involved in determining the metal specificity of the Mo nitrogenase or the V nitrogenase.

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. The strain is a moderate thermophile, growing well at approximately 40° C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.

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. A. variabilis is unique among well-characterized cyanobacteria in that it has three nitrogenase gene clusters that encode different nitrogenases, which function under different environmental conditions. The nif1 genes encode a Mo-nitrogenase that functions only in heterocysts, even in filaments grown anaerobically. The nif2 genes encode a different Mo-nitrogenase that functions in vegetative cells, but only in filaments grown under anoxic conditions. An alternative V-nitrogenase is encoded by vnf genes that are expressed only in heterocysts in an environment that is deficient in Mo. Thus, these three nitrogenases are expressed differentially in response to environmental conditions. The entire nif1 gene cluster, comprising at least 15 genes, is primarily under the control of the promoter for the first gene, nifB1. Transcriptional control of many of the downstream nif1 genes occurs by a combination of weak promoters within the coding regions of some downstream genes and by RNA processing, which is associated with increased transcript stability. The vnf genes show a similar pattern of transcriptional and post-transcriptional control of expression suggesting that the complex pattern of regulation of the nif1 cluster is conserved in other cyanobacterial nitrogenase gene clusters.

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. In addition to the nifB1 promoter, there were weak promoters inside the nifU1 gene and inside the nifE1 gene, and both promoters were heterocyst specific. In an xisA mutant, which effectively separated promoters upstream of an 11-kb excision element in nifD1 from the downstream genes, the internal nifE1 promoter was functional. Transcription of the nif1 genes downstream of the 11-kb element, including the most distant genes, hesAB1 and fdxH1, was reduced in the xisA mutant, indicating that the nifB1 promoter contributed to their expression. However, with the exception of nifK1 and nifE1, which had no expression, the downstream genes showed low to moderate levels of transcription in the xisA mutant. The hesA1 gene also had a promoter, but the fdxH gene had a processing site just upstream of the gene. The processing of transcripts at sites upstream of nifH1 and fdxH1 correlated with increased stability of these transcripts, resulting in greater amounts than transcripts that were not close to processing sites.