L. E. Mikheeva

Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans

Among cyanobacteria, the heterocystous, N2‐fixing Anabaena variabilis and the unicellular Anacystis nidulans have recently been shown to possess an NAD+‐dependent, bidirectional hydrogenase. A 5.0 kb DNA segment of the A. nidulans genome is now identified to harbor the structural genes hoxUYH coding for three subunits of the bidirectional hydrogenase. The gene arrangement in A. nidulans and in A. variabilis is remarkably dissimilar. In A. nidulans, but not in A. variabilis, the four accessory genes hoxW, hypA, hypB and hypF could be identified downstream of hoxH. An insertional homozygous mutant in hoxH from A. nidulans was completely inactive in performing Na2S2O4‐dependent H2 evolution but could utilize the gas with almost 50% of the activity of the wild type. These findings with the first defined hydrogenase mutant in any photosynthetic, O2‐evolving microorganism indicate that the unicellular cyanobacterium A. nidulans possesses both an uptake and a bidirectional hydrogenase. The physiological role(s) of the two hydrogenases in unicellular non‐N2‐fixing cyanobacteria is not yet understood.

MUTANTS OF THE CYANOBACTERIUM ANABAENA VARIABILIS ALTERED IN HYDROGENASE ACTIVITIES

Abstract Two mutants of the cyanobacterium Anabaena variabilis impaired in the utilization or formation of molecular hydrogen have been obtained by nitroso-guanidine mutagenesis. Cultures of both mutants did not show alterations in the growth characteristics or in the heterocysts frequency but evolved molecular hydrogen from nitrogenase with enhanced rates. Activity measurements in extracts showed that one mutant (PK84) did not perform Na2S2O4- dependent H2-formation and was, therefore, unable to express an active bidirectional hydrogenase. Both mutants (PK84, PK 17R) were characterized by lower activity of phenazine-methosulphate-dependent H2-uptake when extracts were assayed from younger cultures. In older cells, particularly when grown with nitrate in the medium , this H2-uptake activity was, how ever, enhanced. Both mutants are likely affected in regulatory hydrogenase genes. The mutant PK84 offers perspectives for potential applications in solar energy conversion programs.

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H2 producers.

The Role of Mobile Genetic Elements in the Evolution of Cyanobacteria

Possible pathways of cyanobacterial evolution are discussed on the basis of in silico analysis of the fully sequenced genomes of 45 species/strains of cyanobacteria. Information on the quantity and functions of different mobile elements (IS, MITE elements, and group II introns) has been reviewed. A positive correlation between entire genome sizes and the number of genes that encode transposases has been revealed. It is suggested that transpositions play a significant role in genomic rearrangements and take part in gene regulation and adaptation processes that determine the directions of microevolutionary processes in cyanobacterial populations.

On the nature of lengthy nucleotide repeated sequences in genomes of prokaryotic organisms

The whole genomic analysis in silico of 64 free-living prokaryotic species has been performed to determine the number, length, distribution, and localization of directed and inverted intragenomic repeated sequences (LRS). Three main types of lengthy (≥500 bp) repeated sequences were revealed: (a) associated with ribosomal RNA genes; (b) with copies of protein coding genes; (c) with IS-elements and genes encoding the hypothetical transposases. Lengthy repeated sequences related to transposases comprise 50 to 95% of the total number of LRS depending on the species. Intragenomic LRS associated with transposases and IS-elements can reflect the recombination potential of different prokaryotic species determining the capability for adaptive gene rearrangements as well as the cell capacity for integration of foreign genes acquired through horizontal transfer paths.

Effects of Medium-Wave Ultraviolet Radiation on Levels and Spectrum of Polyamines in Leaves and Roots of Wild-Growing Plants

The work continues serial studies on short-term effects of medium-wave ultraviolet radiation (UV-B) at 12.5 kJ/m2 on plants. Special attention is paid to the rapid response of the antioxidant system. Free and conjugated forms of putrescine polyamines (putrescine, spermine, and spermidine), as well as those of cadaverine, are recognized to be constituents of the antioxidant system. These compounds were analyzed in plants 24 h after UV-B irradiation. Thellungiella salsuginea (Pallas) O.E.Schulz, Salvia officinalis L, Plantago major L., and Geum urbanum L. grown in aquatic culture under phytotron conditions were examined. The results support the hypothesis that putrescine plays the chief role in the plant defense response against medium-wave ultraviolet irradiation. Three of four plants manifested an increase in the content of this polyamine in leaves. It is the change that determines the enhanced total level of free polyamines. We failed to reveal a general tendency in dynamics of levels of conjugated forms of spermine, spermidine, and cadaverine; only conjugates of putrescine demonstrated a distinct increase. This study allows a conclusion that contributions of particular polyamines to the protective response primarily depend on the species to which the investigated plant belongs. It is likely that conjugated polyamines can be reserved as a pool necessary for rapid recovery of free polyamine levels.

New low-copy plasmid in cyanobacterium Anabaena variabilis

Complete genome sequencing was performed for Anabaena variabilis ATCC 29413 from the collection of the Chair of Genetics, Department of Biology, Moscow State University, Russia. In addition to known plasmids A, B, and C, a new circular low-copy plasmid was detected and named D. It was also sequenced completely and found to have 27051 bp. The plasmid contained the parA and parB genes of the partition system, two genes that encode replication proteins, a gene for site-specific recombinase, a type-I restriction-modification system, and several genes with unknown functions. Analysis by PCR revealed the presence of plasmid D in two epiphytic strains from Vietnam, i.e., Anabaena sp. 182 and Anabaena sp. 281, as well as in Anabaena sp. V5 and A. azollae (Newton’s isolate).