Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis

Abstract

A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4' into medium.Mutant strains capable of producing nitrogenase in the presence of NH4' were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, N03 , or low concentrations of NH4' (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4' produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4' when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4' produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.In free-living, nitrogen-fixing organisms, nitrogenase synthesis and activity are regulated by the presence of NH4' in the medium (3,10,31,45). That NH4+-mediated regulation of nitrogenase synthesis is not exerted by NH4+ itself but is actually a consequence of metabolic products of NH4+ assimilation has been revealed by several lines of evidence.(i) Mutant strains incapable of NH4' assimilation were derepressed for nitrogenase synthesis in the presence of NH4+ (28,31,43,44). (ii) Addition of amino acids to the growth medium repressed nitrogenase synthesis even in nitrogenase-derepressed mutant strains (30). (iii) Inhibition of glutamine synthetase, a primary enzyme responsible for NH4+ assimilation, by a substrate (glutamate) analog, Lmethionine-D,L-sulfoximine (MSX) as well as by other inhibitors led to derepression of nitrogenase synthesis in the presence of NH4+ in all organisms tested (8,13,14,21,31,39). The above described derepression of nitrogenase synthesis is achieved by alteration of cellular physiology leading to a decrease in the rate of glutamate production, since addition of amino acids to the medium reversed the effect (22,29). This set of conditions also derepressed other NH4+-as well as glutamate-producing enzyme systems (NO3 assimilation; histidine and proline utilization) in the cell (16,29).Mutant strains that are blocked at the level of NH4+ assimilation were first described in Klebsiella pneumoniae (32), and such mutant strains not only derepress nitrogenase synthesis but also excrete the NH4+ produce...

Physiological conditions for nitrogen fixation in a unicellular marine cyanobacterium, Synechococcus sp. strain SF1

“…169,1987 on May 12, 2018 by guest http://jb.asm.org/ Downloaded from nitrogenase activity in the presence of DCMU (Table 4), it failed to stimulate growth ( Table 1). The observed enhancement of nitrogenase activity by either pyruvate or glucose is in agreement with reports on the utilization of organic compounds to support nitrogenase activity in other nitrogenfixing cyanobacteria, including heterocystous species capable of photoheterotrophic growth (15,20,24). Cyclic photophosphorylation obviously supplies the ATP needed, an interpretation supported by the high nitrogenase activity of the samples containing DCMU.…”

“…The ammonia concentration was monitored with Nessler reagent (20 (16,22), this isolate, designated strain SF1, was identified as a Synechococcus sp.…”

“…Nitrogenase and hydrogenase activities were determined as described previously (19,20). The general protocol for measuring hydrogenase activity by the tritium exchange assay is described below.…”

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis

“…An MSXresistant mutant strain of A. variabilis that requires glutamine, but only when grown on N2, NO3, or low concentrations of ammonia, has recently been reported (26). It has been shown that glutamine is taken up by filaments of A. variabilis but growth of the cells on glutamine as the sole nitrogen source occurred only at high concentrations of the amino acid; this growth, which occurred under an Ar-02-CO2 atmosphere, was attributed to ammonia produced from glutamine (5).…”

Cyanobacterial Toxins of the Laurentian Great Lakes, their Toxicological Effects, and Numerical Limits in Drinking Water

“…For example, lack of N promotes N fixation by diazotrophic cyanobacteria, but this is more associated with the lack of ammonia than other N sources [96]. As such, N fixation occurs in the presence of nitrate [85,97,98]. Despite the association with eutrophic environments, on a temporal scale, cyanoHABs generally occur during periods when the standing stock of N and P are at their lowest [78,99].…”