A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium.

Abstract

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may ri nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Filamentous cyanobacteria of the genus Anabaena serve as a simple prokaryotic model for developmental control of gene expression. When deprived of a source of fixed nitrogen, about every 10th photosynthetic vegetative cell in the cyanobacterial filament differentiates into a morphologically and physiologically distinct cell called a heterocyst (1, 2). The primary function of heterocysts is nitrogen fixation, the reduction of atmospheric dinitrogen to ammonia mediated by the enzyme nitrogenase. Nitrogenase is very oxygen labile; hence, nitrogen fixation is restricted to anaerobic environments. Heterocysts provide the requisite anaerobic environment because their cell envelope limits oxygen entry and they lack oxygen-evolving photosystem II, which is characteristic of vegetative cells (2). Within a filament heterocysts differentiate in a semiregular pattern, thus providing spatial separation of nitrogen fixation from oxygenic photosynthesis in what is functionally a onedimensional multicellular organism (3).Among nitrogen-fixing cyanobacteria that do not differentiate heterocysts there does not appear to be a single mechanism for protection of nitrogenase from oxygen and different strains show a range in oxygen tolerance (4,5). In many nonheterocystous cyanobacteria, photosynthesis is temporally separated from nitrogen fixation, which occurs only at night (6-8). For other nonheterocystous cyanobacteria that fix nitrogen aerobically in the light without apparently differentiated cells, little is known of the mechanisms for protecting nitrogenase from oxygen (9, 10); however, nitrogenase activity in laboratory-grown cultures is significantly enhanced by lower oxygen tensions (4). Thus, low oxygen tensions are probably necessary for optimal nitrogenase activity.The heterocystous cyanobacterium, Anabaena sp. strain PCC 7120 (hereafter, Anabaena PCC 7120), has a large cluster of nif genes (including nifBSUHDKEN) that encode a Modependent nitrogenase system (11). The nifB-fdxN-nifS-nifU operon is interrupted by a 55-kb insertion infdxN and the nifD gene has an 11-kb insertion, both of which are excised during heterocyst differentiation (12-14). The 11-kb element is prevalent in heterocystous cyanobacteria (15) but is missing in all nonheterocystous cyanobacteria examined to date (4). The nif genes of Anabaena variabilis ATCC 29413 homologous to those of Anabaena PCC 7120 have been cloned and partially mapped (16); they contain the 11-kb excision element, but not the 55-kb excision element (17). In addition to that nifHDK cluster, a different putative nifHD segment, transcribed within hours after the onset of nitrogen starvation under anaerobic conditions, was cloned from ...

Horizontal transfer of the nitrogen fixation gene cluster in the cyanobacterium Microcoleus chthonoplastes

“…This organism possesses three nifclusters, one of which is interrupted by an insertion sequence including an integrase (Brusca et al, 1989). Under anaerobic conditions, this insertion sequence is excised, leaving an intact nif operon that functions in vegetative cells (Thiel et al, 1995). The third nif-cluster in A. variabilis encodes an alternative, vanadium-dependent nitrogenase that functions under aerobic conditions in the heterocyst when molybdate is unavailable.…”

The NtcA-activated amt1 gene encodes a permease required for uptake of low concentrations of ammonium in the cyanobacterium Synechococcus sp. PCC 7942 The GenBank accession number for the nucleotide sequence of the amt1 gene described in this paper is AJ311900.

“…The 1n3 kb Cm R gene cassette C.C2, from pRL479 (Elhai & Wolk, 1988), excised with HindIII and filled with the Klenow enzyme, was inserted into a unique NcoI site that is present in the pCSF50 insert, after filling it with the Klenow enzyme, to generate plasmids pCSF72 and pCSF73 (both orientations). The 5n1 kb lacZ neomycin\kanamycin-resistant (Nm\Km) R gene cassette (Thiel et al, 1995), excised with SmaI from pPE20, was also inserted into the Klenow-enzymefilled NcoI site of pCSF50, and a construct, pCSP3a, in which lacZ was in the same orientation as amt1, was chosen for further work.…”

“…Rate of expression of β-galactosidase activity [U (µg Chl) Low carbon 0n90p0n25 0n90p0n28 0n84p0n09 High carbon 1n11p0n35 3n01p0n49 2n56p1n48 Low carbon Low carbon 1n62p0n91 2n68p0n71 2n31p0n35 High carbon 1n64p0n42 3n34p0n70 4n52p0n95 consisting of a promoterless lacZ gene followed by a (Nm\Km) R encoding gene (Thiel et al, 1995) was inserted into the NcoI site of Synechococcus amt1. A Synechococcus clone homozygous for mutant chromosomes in which the lacZ-(Nm\Km) R insert was located such that the amt1 promoter drives lacZ was isolated and named CSP2.…”

Role of Diazotrophic Bacteria in Biological Nitrogen Fixation and Plant Growth Improvement

“…Azotobacter have been observed to have alternative nitrogenases such as vnfH and anfH (Bishop et al, 1986). Cyanobacteria have possible multiple copies of nitrogenase, including a vnfH as well as a second distinct copy of nifH (Thiel et al, 1995). Archaea often have two nifH homologues, in which two copies are in Cluster IV and Cluster III, and in other cases, the homologues are in Cluster II and Cluster III.…”