Evolution and Functional Analysis of orf1 Within nif Gene Cluster from Paenibacillus graminis RSA19

Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. Genomic sequence analysis has revealed that a compact nif (nitrogen fixation) gene cluster comprising 9–10 genes nifBHDKENX(orf1)hesAnifV is conserved in diazotrophic Paenibacillus species. The evolution and function of the orf1 gene within the nif gene cluster of Paenibacillus species is unknown. In this study, a careful comparison analysis of the compositions of the nif gene clusters from various diazotrophs revealed that orf1 located downstream of nifENX was identified in anaerobic Clostridium ultunense, the facultative anaerobic Paenibacillus species and aerobic diazotrophs (e.g., Azotobacter vinelandii and Azospirillum brasilense). The predicted amino acid sequences encoded by the orf1 gene, part of the nif gene cluster nifBHDKENXorf1hesAnifV in Paenibacillus graminis RSA19, showed 60–90% identity with those of the orf1 genes located downstream of nifENX from different diazotrophic Paenibacillus species, but shared no significant identity with those of the orf1 genes from different taxa of diazotrophic organisms. Transcriptional analysis showed that the orf1 gene was expressed under nitrogen fixation conditions from the promoter located upstream from nifB. Mutational analysis suggested that the orf1 gene functions in nitrogen fixation in the presence of a high concentration of O2.

Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.

Genetics of Nitrogen Regulation in Methanococcus maripaludis

We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation. We present evidence for a “nitrogen regulon” in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator. The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation. Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function. We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens. The phenotype resulting from a glnB null mutation in M. maripaludis provides direct evidence that glnB-like genes are involved in “ammonia switch-off,” the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia. Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria. These studies illustrate the utility of genetic methods in M. maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.