Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants

Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments

Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes

Two Thimet Oligopeptidase-Like Pz Peptidases Produced by a Collagen- Degrading Thermophile, Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site