Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.     

Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the East European Plain

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.     

Molecular analysis of some genes from plasmid p19 of the Bacillus subtilis 19 soil strain involved in conjugation

Two fragments of conjugative plasmid p19 (95 kb) from the Bacillus subtilis 19 soil strain were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1–ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the B. subtilis 72 soil strain and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.     

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain

The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110. To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed. The frequency of transfer of the large plasmid between cells of B. subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B. subtilis 168 was a recipient. However, when the restriction-deficient strain B. subtilis 168 was a recipient, the transfer efficiency was almost completely recovered. The effectiveness of pUB110 mobilization was virtually not altered in all these cases. pC194 was not mobilized by p19. The kinetics of p19 conjugative transfer is also presented.     

Molecular analysis of some genes from plasmid p19 of the soil strain Bacillus subtilis 19 involved in conjugation

Two fragments of conjugative plasmid p19 (95 kb) from the soil strain Bacillus subtilis 19 were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1-ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the soil strain B. subtilis 72 and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.     

Inter- and intraspecies conjugal transfer of different plasmids in bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Functioning of malate dehydrogenase system in mesophyll and bundle sheath cells of maize leaves under salt stress conditions

The formation of adaptive response to salt stress in mesophyll and bundle sheath cells of maize (Zea mays L.) leaves was studied at the level of operation of enzyme systems that participate in oxidation of malate. Functioning of four malate dehydrogenases (MDH), the components of this system, was studied and found to maintain malate and pyruvate pools, which are required for operation of the Hatch-Slack cycle and actively used for neutralization of salt treatment. The increase in activity of NAD-MDH was related to salt-induced synthesis of the additional isoform of MDH in mesophyll cells. Such changes in the isozyme pattern were not found in bundle sheath cells.