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Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3.

ABSTRACT: Frankia is a representative genus of nitrogen-fixing (N₂-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N₂ fixation-specific structure (vesicle) and the regulation of N₂ fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 candidate N₂ fixation mutants. Twelve of these mutants were selected for further study, and their abilities to grow in NH₃-deficient (N-) liquid media and their rates of acetylene reduction activities were evaluated. Eleven mutant strains were confirmed to lack the ability to fix N₂. Five mutant strains formed significantly reduced numbers of vesicles, while some failed to form large mature vesicles. These vesicle mutants also exhibited an aberrant hyphal morphology, suggesting a relationship between vesicle differentiation and hyphal branching. Ten mutants showed significant reductions in the expression of nifE, nifH, and nifV genes under N- conditions. The genome sequencing of eight mutants identified 20 to 400 mutations. Although mutant strains N3H4 and N6F4 shared a large number of mutations (108), most were unique to each strain. Mutant strain N7C9 had 3 mutations in the nifD and nifH genes that may result in the inability to fix N₂. The other mutant strains did not have any mutations in any known N₂ fixation-related genes, indicating that they are novel N₂ fixation mutants.

SUBMITTER: Kucho KI

PROVIDER: S-EPMC5745019 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3.

Kucho Ken-Ichi KI Tamari Daiki D Matsuyama Shintaro S Nabekura Takeshi T Tisa Louis S LS

Microbes and environments 20171118 4

Frankia is a representative genus of nitrogen-fixing (N2-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N2 fixation-specific structure (vesicle) and the regulation of N2 fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 ...[more]

PMID: 29151446

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Project description:BackgroundFrankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb(2+), Al(+3), SeO2, Cu(2+), AsO4, and Zn(2+). With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison.ResultsUnlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia.ConclusionsEach strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils.

Dataset Information

Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3.

Publications

Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3.

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