Volume 53, Nº 2 (2017)

Recent studies of the immune system of leguminous plants infected with nodular bacteria (rhizobia) are summarized. The possibility of blocking the invasion of rhizobia into plant organs not affected by the primary infection is discussed. The concept of local and systemic resistance of the leguminous plant to rhizobial infection is introduced. The Nod factors of rhizobia are considered, as well as the plant receptors that interact with these factors upon the formation of symbiosis of the plant and bacteria. The role of bacterial surface exopolysaccharides in the suppression of the protective system of the plants is discussed. The innate immunity of leguminous plant cells is assumed to affect the formation and functioning of the symbiosis of the plant and the bacteria.

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl₂ and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k_cat = 17 s^–1; k_cat/K_m = 855 mM^–1 s^–1) has been 100–300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni²⁺-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni²⁺ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.

A knockout mutant of Methylobacterium dichloromethanicum DM4 with an inactivated gene of a putative transcription regulator METDI5511 (ΔMETDI5511) has been obtained. The expression of this gene increases many times when the strain is grown on dichloromethane compared to methanol. The mutant had a low growth rate on dichloromethane as compared with the original strain and was found to be more sensitive to influences of various types of stress (oxidative, osmotic stress, heat, and drying). The cells were stained with Fluorescent Brightener 28 (Calcofluor white), and the intensity of their fluorescence showed that the ΔMETDI5511 mutant had significantly increased numbers of surface polysaccharides with β-1,3 and β-1,4-glycoside bonds. The results indicate that the METDI5511 gene is involved in the regulation of surface polysaccharides that play an important role in adaptation of cells to growth on dichloromethane.

Three indigenous pseudomonads, Pseudomonas putida DLL-E4, Pseudomonas reactans and Pseudomonas fluorescens, were isolated from chlorophenol-contaminated soil samples collected from a sawmill located in Durban (South Africa). The obtained isolates were tested for their ability to degrade chlorophenolic compounds: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in batch cultures. The isolates were found to effectively degrade up to 99.5, 98.4 and 94.0% with a degradation rate in the range of 0.67–0.99 (2,4-D), 0.57–0.93 (2,4-DCP) and 0.30–0.39 (2,4,6-TCP) mgL^–1 day^–1 for 2,4-D; 2,4-DCP and 2,4,6-TCP, respectively. The degradation kinetics model revealed that these organisms could tolerate up to 600 mg/L of 2,4-DCP. Catechol 2,3-dioxygenase activity detected in the crude cell lysates of P. putida DLL-E4 and P. reactans was 21.9- and 37.6-fold higher than catechol 1,2-dioxygenase activity assayed, suggesting a meta-pathway for chlorophenol degradation by these organisms. This is also supported by the generally high expression of C23O gene (involved in meta-pathway) relative to tfdC gene (involved in ortho-pathway) expression. Results of this study will be helpful in the exploitation of these organisms and/or their enzymes in bioremediation strategies for chlorophenol-polluted environment.

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.

A change in the contents of endogenous salicylic and jasmonic acids in the roots of the host plant at the preinfectious stage of interaction with symbiotic (Rhizobium leguminosarum) and pathogenic (Agrobacterium rizogenes) bacteria belonging for to the family Rhizobiaceae was studied. It was found that the jasmonic acid content increased 1.5–2 times 5 min after inoculation with these bacterial species. It was shown that dynamics of the change in the JA and SA contents depends on the type of infection. Thus, the JA content decreased in the case of pathogenesis, while the SA content increased. At the same time, an increased JA content was observed during symbiosis. The observed regularities could indicate the presence of different strategies of hormonal regulation for interaction with symbiotic and pathogenic bacteria belonging to the family Rhizobiaceae in peas plants.

Bacterial communities in anode microbial fuel cells (MFC) obtained from anaerobic digester sludge in a municipal wastewater treatment plant (Nanjing, China) were investigated. Glucose, propyl alcohol and methanol were used as sole carbon source in two-chamber MFC. The results showed that a reproducible cycle of power production can be formed in MFC fed with 3 substrates and glucose-fed MFC had the highest peak power density of 1499 ± 33 mW/m³, followed by methanol- (1264 ± 47 mW/m³) and propyl alcohol-fed MFC (1192 ± 36 mW/m³). Firmicutes, Bacteroidetes, Verrucomicrobia, Proteobacteria, Synergistetes and Armatimonadetes were dominant phyla in 3 MFC. Firmicutes was the most dominant phylum in glucose-fed MFC samples and Bacteroidetes prevailed in methanol- and propyl alcohol-fed MFC. These data indicate that propyl alcohol and methanol along with glucose can be used as substrates of MFC.

The maximal rates and effective constants of 2,6-dichlorphenolindophenol and oxygen reduction by bacterim Gluconobacter oxydans in bacterial fuel cells under different conditions were evaluated. In an open-circuit mode, the rate of 2,6-dichlorphenolindophenol reduction coupled with ethanol oxidation under oxygen and nirogen atmospheres were 1.0 and 1.1 μM s^–1 g^–1, respectively. In closed-circuit mode, these values were 0.4 and 0.44 μM s^–1 g^–1, respectively. The initial rate of mediator reduction with the use of membrane fractions of bacteria in oxygen and nitrogen atmospheres in open-circuit mode were 6.3 and 6.9 μM s^–1 g^–1, whereas these values in closed-circuit mode comprised 2.2 and 2.4 μM s^–1 g^–1, respectively. The oxygen reduction rates in the presence and absence of 2,6-dichlorphenolindophenol were 0.31 and 0.32 μM s^–1 g^–1, respectively. The data obtained in this work demonstrated independent electron transfer from bacterial redox centers to the mediator and the absence of competition between the redox mediator and oxygen. The results can make it possible to reduce costs of microbial fuel cells based on activity of acetic acid bacteria G. oxydans.