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Biofilm formation is a widespread phenomenon in the microbial world. They may affect human and animal health and cause damage to various industries; at the same time, they can be useful in such fields as wastewater treatment or improving bioavailability of nutrients for plants. This fact actualizes the development of methods for studying biofilms. The present work is the first report on an optical sensor technique for the indication of bacterial biofilm formation with regard to biological variability, using plant growth-promoting rhizobacteria of the genus Azospirillum as a model. The correlation was found between the changes in electrophysical parameters recorded by the sensor system and the morphological features of bacteria from planktonic and/or biofilm cultures: the presence of motile organelles (flagella), polymorphism, and ultrastructure of cellular forms. It was established that the profiles of microbial cells recorded by the optical system were significantly different in planktonic and biofilm forms. When comparing the cells of different strains (the parental strain and its derivatives), or planktonic and biofilm bacteria, the variables recorded by the electro-optical sensor system were in agreement with the changes in bacterial micro- and ultrastructure recorded by other techniques. The results of analysis of the electrophysical profiles of A. baldaniorum Sp245 can be used as reference for revealing specific interactions between biofilm cells of this strain and various root surface components of a putative plant partner using an optical sensor system. [ABSTRACT FROM AUTHOR]

Inactivation of one of the genes (CDS AZOBR_p60123) of the ABC transporter ATP-binding protein Wzt in the p60123::aphAI mutant of Azospirillum baldaniorum Sp245 and the introduction of an additional copy of this gene into the parent or mutant strain affected the chain-length heterogeneity of O polysaccharides (OPSs) and lipopolysaccharides (LPSs), the OPS and LPS immunochemical characteristics, and the strain properties related to polysaccharide production (biofilm construction). Biofilms of the p60123::aphAI mutant Sp245.4-1-1 accumulated two times less biomass than did those of Sp245. Introduction of pRK415-p60123 into the cells of Sp245.4-1-1 or Sp245 resulted in a respective increase or decrease in the ability of the resulting derivative strains to accumulate biofilm biomass, as compared with the original strains. [ABSTRACT FROM AUTHOR]

Bacteria of the genusAgrobacteriumare capable of transferring a fragment of their Ti-plasmid T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through the membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of the virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, thevirE2gene was cloned into plasmid pQE31. VirE2 was isolated from the cells ofE. colistrain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein, with six histidine residues at the N-end, was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized theAgrobacterium tumefaciensvirulence protein VirE2, which is capable of binding to single-stranded T-DNA upon transfer to the plant cell. [ABSTRACT FROM AUTHOR]

Azospirillum alphaproteobacteria, which live in the rhizosphere of many crops, are used widely as biofertilizers. Two-component signal transduction systems (TCSs) mediate the bacterial perception of signals and the corresponding adjustment of behavior facilitating the adaptation of bacteria to their habitats. In this study, we obtained the A. baldaniorum Sp245 mutant for the AZOBR_150176 gene, which encodes the TCS of the hybrid histidine kinase/response sensory regulator (HSHK-RR). Inactivation of this gene affected bacterial morphology and motility. In mutant Sp245-HSHKΔRR-Km, the cells were still able to synthesize a functioning polar flagellum (Fla), were shorter than those of strain Sp245, and were impaired in aerotaxis, elaboration of inducible lateral flagella (Laf), and motility in semiliquid media. The mutant showed decreased transcription of the genes encoding the proteins of the secretion apparatus, which ensures the assembly of Laf, Laf flagellin, and the repressor protein of translation of the Laf flagellin's mRNA. The study examined the effects of polyethylene glycol 6000 (PEG 6000), an agent used to simulate osmotic stress and drought conditions. Under osmotic stress, the mutant was no longer able to use collective motility in semiliquid media but formed more biofilm biomass than did strain Sp245. Introduction into mutant cells of the AZOBR_150176 gene as part of an expression vector led to recovery of the lost traits, including those mediating bacterial motility under mechanical stress induced by increased medium density. The results suggest that the HSHK-RR under study modulates the response of A. baldaniorum Sp245 to mechanical and osmotic/water stress., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Angiosperm plants reproduce both sexually and asexually (by apomixis). In apomictic plants, the embryo and endosperm develop without fertilization. Modern maize seems to have a broken apomixis-triggering mechanism, which still works in Tripsacum and in Tripsacum -maize hybrids. For the first time, maize lines characterized by pronounced and inheritable high-frequency maternal parthenogenesis were generated 40 years ago, but there are no data on gene expression in parthenogenic maize proembryos. Here we examined for the first time gene expression in parthenogenic proembryos isolated from unpollinated embryo sacs (ESs) of a parthenogenic maize line (AT-4). The DNA-methylation genes ( dmt103 , dmt105 ) and the genes coding for the chromatin-modifying enzymes ( chr106 , hdt104 , hon101 ) were expressed much higher in parthenogenic proembryos than in unpollinated ESs. The expression of the fertilization-independent endosperm ( fie1 ) genes was found for the first time in parthenogenic proembryos and unpollinated ESs. In parthenogenic proembryos, the Zm_fie2 gene was expressed up to two times higher than it was expressed in unpollinated ESs.

VirE2 from Agrobacterium tumefaciens is a single-stranded (ss) DNA-binding protein involved in delivery of ssT-DNA (single-stranded transfer DNA) from the agrobacterial Ti plasmid into the eukaryotic cell nucleus. The crystallized part of VirE2 was studied by X-ray diffraction, and the noncrystallized parts of the C- (40 amino acid residues [aars]) and N- (111 aars) termini of the protein, which are presumably disordered, were evaluated by computational methods. We did a molecular dynamics simulation of VirE2 without VirE1 and observed no large changes in domain orientation. The interaction of VirE2 with ssDNA and formation of ssDNA-VirE2 complexes in silico were studied. We also used computer-aided methods to design model complexes consisting from two- and four-subunit VirE2 proteins. We examined the implication of disordered sites in formation of two- and four-subunit VirE2 complexes. Formation of VirE2 dimers and tetramers within ssDNA-VirE2 complexes was demonstrated by computational methods. Using the Platinum program, we found that hydrophilic amino acids were predominant on the surface of the four-subunit VirE2 complex.

The single-stranded transfer DNA from the Ti plasmid of the soil bacteria Agrobacterium nonspecifically integrates into the plant chromosome and is inherited at subsequent cell divisions. How it is transferred across host membranes is unknown, but it is believed that VirE2 proteins form a membrane-spanning pore or channel in a lipid bilayer and possibly mediate the delivery of the single-stranded transfer DNA-VirD2-VirE2 complex to the plant cell chromosomes. The aim of this work was to perform a computer simulation of VirE2's pore-forming capacity and an evaluation of constructed VirE2 complexes. The oscillating motions of complexes consisting of two and four VirE2 subunits were estimated by the molecular dynamics and normal modes methods. We did not predict any large changes in domain orientation for two and four-subunit VirE2 complexes within simulation times of 1ns. A possible gating mechanism similar to that seen in the ion channels of the complex formed from two VirE2 proteins was proposed, whereas no conformational changes were predicted inside the pore in the complex formed from four VirE2 proteins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This article is devoted to the study of the double fertilization mechanism in plants, in particular of the maize gamete membrane fusion genes. We detected and analyzed for the first time gamete-fusion genes in the maize genome. Using the BLAST program, we searched for the hap2 gene (generative cell specific 1 (gcs1)) homologs from Arabidopsis in the maize genome. The ZM_BFb0162K03 maize transcript was found, which had 67% identity to the Athap2 gene and contained a conserved region similar to the Athap2 gene fragment. In mRNA samples from the haploid-inducing and control maize lines, an PCR was conducted by using primers specific to the ZM_BFb0162K03 sequence fragment. Sequences of the PCR products from a fragment (1467 bp) of the Zm_hap2 gene of the haploid-inducing and the control maize lines were identical and also were identical to the maize sequences from the GenBank (ZM_BFb0162K03). PCR products (656 bp region of Zm_hap2) for the ZM_BFb0162K03 (1925 bp) maize sequence were observed for the cDNA of pollen grains, ovary, leaves, and roots of the haploid-inducing and control maize lines. Using the Blastx program, we found significant homology of the maize translated proteins to the GEX2, TET11, and TET12 proteins, involved in Arabidopsis gamete-fusion contacts.

The transfer of single-stranded (ss) T-DNA from soil bacteria of the genus Agrobacterium with the help of the VirE2 protein, which possibly mediates the delivery of ss-T-DNA across the cell membrane, was demonstrated earlier, but how VirE2 participates in ssDNA transfer across artificial and natural membranes is not known. Using computational methods, we reconstructed model structures composed of two and four VirE2 proteins and showed by the MOLE program the formation of pores with channel diameters of 1.2-1.6 and 1.4-4.6 nm in a model structure formed from two and four VirE2 molecules, respectively. Using light scattering, we recorded the size distribution for recombinant VirE2-dependent complexes in aqueous solutions and found that VirE2 in a buffer solution is present as a complex made up of two or more proteins. We revealed single, long-lived jumps in voltage-dependent membrane conductance during coincubation of planar black membranes with the VirE2 protein. On the addition of VirE2 and FAM-labeled oligonucleotides to HeLa cells, the fluorescence intensity for the cells increased by 56% as compared to that for cells incubated only with oligonucleotides.

Abstract-A transfer DNA (T-DNA) carrying the marker gene nptII was detected in the genomes of diploid and haploid maize plants obtained after the treatment of pistil filaments with a suspension of Agrobacterium during artificial pollination. PCR analysis of total DNA isolated from 155 canamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 111 plants (32.7% of the total number of analyzed seeds). The example of matroclinal haploids was used to demonstrate that T-DNA may be transported to the egg cell by the growing pollen tube (PT). Twelve out of 16 analyzed haploid plants contained the T-DNA insertion. The possible mechanism of the transfer of the Agrobacterium T-DNA to the maize genome during pollination is discussed.

The recombinant virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into plant cells, was isolated, purified, and used for interactions with ssT-DNA. The in vitro interaction of VirE2 and ss-binding protein from Escherichia coli with single-stranded DNA (phage lambda) was determined by agarose gel electrophoresis by the formation of high-molecular-weight complexes after preliminary coincubation of purified protein preparations with ssDNA. We show that VirE2 binds to single-stranded DNA and protects it against nuclease S1 degradation much better than does E. coli SSB protein. We for first time observed the VirE2-ssT-DNA complex by using atomic force microscopy. The complex observed by atomic force microscopy after ssT-DNA and VirE2 protein mixing has a length of about 800 nm and a 5-8 nm width in sites with attached VirE2 protein.

Integration of T-DNA into the maize genome as a result of treatment of silks with Agrobacterium cells, containing activated vir genes, was demonstrated. In planta treatment of maize (Zea mays L) was performed during flowering in field. Cell suspension of Agrobacterium tumefaiciens strain GV3101(pTd33), carrying activated vir genes, was applied onto the previously isolated silks, which were afterwards pollinated with the pollen of the same cultivar. Integration of T-DNA into maize genome was confirmed by PCR (the nptII and gus reporter genes) and hystochemical staining of the seedling tissues, obtained from the transformed seeds. Amplification of the nptII gene showed the presence of about 60.3% of PCR-positive plants out of the total number of kanamycin-resistant seedlings examined, or 6.8% of the total of number of seedlings.

The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein.

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.