I II 444 4 i

Rhodopseudomonas palustris orf orf nifZ-like fíxR nifA fdx nifB fdx y y orf nifzifíxU orf nifH nifD nifK nifE nifN

nifX i fdxBnifQ orf nifU nifS nifV nifW fixA fixB fixC fixX

1 kb vnfN vnfE vnfA

nifX mop vnfD vnfG vnfK like like

Il I

nifX i fdxBnifQ orf nifU nifS nifV nifW fixA fixB fixC fixX

I I i vnfN vnfE vnfA

Figure 1. Organization of nif and «¡^related genes m three diazotrophs. Note that genes shown on two lines representing the entire nif cluster of R. palustris are contiguous m the genome.

Figure 1. Organization of nif and «¡^related genes m three diazotrophs. Note that genes shown on two lines representing the entire nif cluster of R. palustris are contiguous m the genome.

7120 (Golden et al. 1985, 1988). In N. punctiforme the fdxN excision element is missing and the 24-kb nifD element in N. punctiforme is almost completely different from the 11-kb nifD element in Anabaena sp. PCC 7120. The two nifD elements share only the xisA gene that is required for site-specific recombination that removes the element during heterocyst differentiation and a small open reading frame of unknown function. Within the N. punctiforme excision element there is a bacterial retron-like gene. Anabaena sp. PCC 7120 also has a gene that appears to be a retron; however, it is not located in the excision element and it is not closely related phylogenetically to the gene in N. punctiforme. Upstream of nifH in N. punctiforme and in Nostoc commune (Potts et al. 1992) is a hemoglobin-like gene called cyanoglobin iglbN) whose function is not known. The nif genes of N. punctiforme are most closely related to those of Nostoc commune, as determined by phylogentic analysis (Thiel, unpublished). Upstream of the major «//'cluster in N. punctiforme are nifP, nifZ and nifT and downstream are genes for an uptake hydrogenase, including hupS and hupL which lack an excision element that is present in the hupL gene of Anabaena sp. PCC 7120 (Carrasco et al. 1995).

There is only one copy of most of the nif genes in N. punctiforme, supporting previous studies that indicated that this strain lacks alternative nitrogenases such as the V-nitrogenase in Anabaena variabilis ATCC 29413 (Thiel 1996). However, N. punctiforme has two additional copies of nifH and one additional copy of nifE and nifN. The organization of the multiple nif genes is shown in Figure 2 with multicopy nif genes of some other diazotrophic strains. Phylogenetic analysis indicates that the second copy of nifH in N. punctiforme (near the second copy of nifEN) clusters with the N. punctiforme and N. commune nifH genes that are part of the major «//cluster; however, it is less like those two genes than they are like each other (T. Thiel, unpublished). The third nifH in N. punctiforme is closely related to the nifH gene in A. variabilis that appears to encode the dinitrogenase reductase of the V-nitrogenase. None of the nifH genes in N. punctiforme is closely related to the second copy of nifH in Anabaena sp. PCC 7120

The genome of Rhodopseudomonas palustris was sequenced by the Joint Genome Institute at about the same time as the N. punctiforme genome. Analysis of the R. palustris genome indicates an unusually large number of nif-like genes. Like Rhodobacter capsulatus, R. palustris has genes that appear to encode a Mo-nitrogenase and an alternative Fe-nitrogenase. Unlike R. capsulatus, R. palustris also has genes that appear phylogenetically to be closely related to V-nitrogenase genes in Azotobacter vinelandii and A. variabilis. The organization of these three gene clusters is shown in Figure 1. In addition, R. palustris has additional copies of nifB, nifH, nifE, and nifN that are not associated with nifDK genes (Figure 2). Phylogenetic analysis indicates that these extra copies that are not associated with nifDK genes are only distantly related to homologs that have a known function. This is in contrast to the extra copies of nifH genes in cyanobacteria that cluster within the cyanobacterial nifH group. It will be of great interest to determine what role, if any, these multicopy nif gene families have in nitrogen fixation.

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