Proteogenomic Study of Nitrogen-Fixing Cyanobacterium Anabaena Cylindrica

Cyanobacteria are gram-negative photosynthetic bacteria. In some filamentous cyanobacteria such as Anabaena cylindrica, every 10th to 20th vegetative cells can differentiate into nitrogen fixing heterocysts. Heterocyst can induce the neighboring cells developing into spore-like akinetes. The specialized cell functions and resourceful networks have contributed to the prosperity of cyanobacteria for over 2 billion years, but the genetic mechanisms for multi-cellular differentiation are barely known, especially for akinete formation. The heterocysts, akinetes and vegetative cells of Anabaena cylindrica were isolated for proteomic study. This study identified a total of 1395 proteins, including 664 proteins from akinetes, 751 proteins from heterocysts, and 1236 proteins from vegetative cells. There were 45 proteins (33 novel proteins) found exclusive in akinetes, 57 heterocyst-specific proteins (33 novel proteins), including nif gene products, and 485 proteins exclusively in vegetative cells. HAVe model was proposed that akinetes, unlike the typical spores of bacteria, perform unique biochemical functions that collaborate with both heterocysts and vegetative cells. Regardless of nitrate availability, some vegetative cells of Anabaena cylindrica are programed to differentiate semi-regularly spaced, single heterocysts along filaments. Since heterocysts are non-dividing cells, with the sole known function for solar-powered N2-fixation, is it necessary for heterocyst to retain entire genome (7.1Mb) from its progenitor vegetative cell? By sequencing the genome of isolated heterocyst, I discovered that at least six DNA elements (0.12 Mbp) are deleted from the heterocyst genome during heterocyst development. The six-element deletions restore five genes (nifH1, nifD, hupL, primase P4, acyl_5725 (hypothetical protein) that were interrupted in the genome of vegetative cells. More deletions are expected to be identified in the completed genome of heterocyst, a uniquely solar-powered, oxic N2-fixing cell. To my best knowledge, this is the first report that (1) different genomes may occur in distinct cell types in a single bacterium; and (2) genome editing is coupled to cellular differentiation and/or cellular function in a multicellular cyanobacterium. In response to environmental changes, Anabaena cylindrica differentiate three cell types, vegetative cells for photosynthesis, heterocysts for nitrogen fixation, and akinetes for stress survival. Cell-surface polysaccharides play important roles in bacterial ecophysiology. In this study, specific cell-surface sugars were discovered in heterocysts, akinetes and vegetative cells of A. cylindrica using 20 fluorescein labeled lectins. Both Nacetylglucosamineor- binding lectins WGA and succinylated WGA bound specifically to the vegetative cells. Akinetes bound to three mannose-binding lectins (LCA, PSA, and ConA), and one of the galactose-binding lectins (GSL-I). ConA also bound to heterocyst, and the binding was diminished in the heterocysts of an all4388 mutant, in which the putative polysaccharide export protein gene all4388 was disrupted. Above proteomics, genomics and genetic research greatly added to our understanding of the cell development in A. cylindrica and clarify the patterns of gene expression in heterocysts, akinetes and vegetative cells, which pave the way for further study of Anabaena cylindrica

Isolation of Potential Photosynthetic N\u3csub\u3e2\u3c/sub\u3e-Fixing Microbes from Topsoil of Native Grasslands in South Dakota

Nitrogen fertilizer is one of the most limiting factors and costly inputs in agriculture production. Current fossil fuel-dependent ammonia production is both energy intensive and environmentally damaging. An economically practical and environmentally friendly solution for the production of ammonia is urgently needed. Solar-powered N2-fixing cyanobacteria provide a unique opportunity and promise for applications in agriculture compared to all other N2-fixing bacteria that cannot use solar energy. Isolation of nitrogen-fixing microbes from the topsoil of native grasslands may have the potential to use them in crop fields as living ammonia factories. This may be a mechanism to free farmers from heavy reliance on fossil fuels-dependent chemical nitrogen fertilizers and to improve soil health for sustainable agriculture. To screen for solar-powered N2-fixing cyanobacteria in topsoil of native grasslands in South Dakota, we collected 144 topsoil samples from several native grasslands. Six photosynthetic microbial strains were isolated that are capable of growing well autotrophically in a nitrogen-free medium, suggesting that these six microbial strains have the ability to fix N2. They were assigned the names: Xu15, Xu81, Xu86, Xu111, Xu141, and WW3. Based on cell morphology and its 18S rRNA gene sequence that we obtained, strain Xu15 was reassigned as Chloroidium saccharophilum Xu15, a common terrestrial coccoid green alga. An acetylene reduction assay detected substantial ethylene production, suggesting nitrogenase activity occurrences in cultures Xu81 and Xu15. The other four are in the process of purification for testing their nitrogenase activity. Xu81, Xu111 and Xu141 are probably unicellular microalga, while WW3 and Xu86 are likely filamentous cyanobacteria. Future research will focus on developing these validated N2-fixing microbes as in situ living ammonia factories in crop fields