Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.

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 ...

Circadian (daily) rhythms are a fundamental and ubiquitous property of eukaryotic organisms. However, cyanobacteria are the only prokaryotic group for which bona fide circadian properties have been persuasively documented, even though homologs of the cyanobacterial kaiABC central clock genes are distributed widely among Eubacteria and Archaea. We report the purple non-sulfur bacterium Rhodopseudomonas palustris (that harbors homologs of kaiB and kaiC) only poorly sustains rhythmicity in constant conditions–a defining characteristic of circadian rhythms. Moreover, the biochemical characteristics of the Rhodopseudomonas homolog of the KaiC protein in vivo and in vitro are different from those of cyanobacterial KaiC. Nevertheless, R. palustris cells exhibit adaptive kaiC-dependent growth enhancement in 24-h cyclic environments, but not under non-natural constant conditions. Therefore, our data indicate that Rhodopseudomonas does not have a classical circadian rhythm, but a novel timekeeping mechanism that does not sustain itself in constant conditions. These results question the adaptive value of self-sustained oscillatory capability for daily timekeepers and establish new criteria for circadian-like systems that are based on adaptive properties (i.e., fitness enhancement in rhythmic environments), rather than upon observations of persisting rhythms in constant conditions. We propose that the Rhodopseudomonas system is a "proto" circadian timekeeper, as in an ancestral system that is based on KaiC and KaiB proteins and includes some, but not necessarily all, of the canonical properties of circadian clocks. These data indicate reasonable intermediate steps by which bona fide circadian systems evolved in simple organisms.

The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction-based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction-amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.

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.