Abstract
Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters.
 Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state.
 Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation.
 Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.
Highlights
C Исследована возможность модификации регуляции азотфиксации у клубеньковых бактерий Mesorhizobium, Ensifer и Rhizobium путем привнесения в их геном дополнительной копии гена nifA под регуляцией бактериальных индуцируемых промоторов
The possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters
A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created
Summary
For citation: Baymiev AnK, Gumenko RS, Vladimirova AA, et al Artificial activation of nif gene expression in nodule bacteria ex planta. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. Поскольку в этом процессе могут участвовать не только представители одного вида, но и бактерии разных таксонов, то это приводит к комбинации nif-генов разных бактерий, что является элементом комбинативного эволюционного процесса [2,3,4]. Это позволит использовать ген nifA клубеньковых бактерий в качестве удобного маркера при изучении функциональности гетерологичных комбинаций nif-генов, поскольку данный ген также: 1) является неотъемлемой частью азотфиксирующего аппарата всех ризобий; 2) не имеет жесткой сцепленности с генами коровых белков нитрогеназы. По появлению нитрогеназной активности у клубеньковых бактерий ex planta при искусственной активации гена nifA можно будет судить о функциональности данного гена в гетерологичной системе nif-генов. Придание им способности фиксировать азот ex planta может значительно улучшить их ростостимулирующие свойства
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