I. F. Puntus

Mycobacterium sp. mutant strain producing 9α-hydroxyandrostenedione from sitosterol

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9α-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9α-hydroxylase activity.

The efficiency of salicylate utilization by Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways.

Abstract Parameters of growth and substrate consumption by plasmid-harbouring Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways under batch cultivation on salicylate were estimated. The values of a specific growth rate, μ , and the yield coefficient, Y , for natural isolates with the functioning genes of the ortho-pathway and silent genes of meta cleavage of catechol oxidation were less than those for isogenous strains with the functioning genes of the meta-pathway. All strains under study with functioning genes of the meta-pathway were shown to possess the activities of the enzymes both of ortho- and meta pathway of catechol oxidation: catechol-1,2-dioxygenase and catechol-2,3-dioxygenase. The comparison of the functioning of the genes of the ortho- and meta-pathways was carried out in an isogenous host background. An approach to estimate the efficiency of the biodegradation process using the kinetics of microbial growth and substrate consumption under batch cultivation was proposed.

Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels

The effect of different moisture levels (from 20 to 70%) on the growth and survival of Pseudomonas putida strains G7 and BS3701 degrading naphthalene was studied in soil model systems. P.putida G7 contains plasmid NAH7 and P.putida BS3701 harbours plasmids pBS1141 and pBS1142. A mathematical model is proposed to describe the observed dynamics of the number of viable bacterial cells. Naphthalene and soil organic matter were considered as substrates available to bacteria. Data fitting allowed the estimation of model parameters characterizing microbial growth rate, utilization rate of substrates, specific maintenance rate and yield coefficient. Both the maximum bacterial concentration and the highest yield coefficient were observed at a soil moisture level of 40%. This optimal moisture level is close to but less than the water capacity (48%) of the soil used.

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10−5–10−4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.

Phenanthrene degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).

Biosynthesis of isocitric acid by the yeast Yarrowia lipolytica and its regulation

We studied the biosynthesis of isocitric acid from rapeseed (canola) oil by the yeast Yarrowia lipolytica and its regulation. We determined a fundamental possibility for directed biosynthesis of isocitric acid by Y. lipolytica yeast, with only minimal amounts of citric acid byproduct, when grown on a medium containing canola oil. Wild type strains of Y. lipolytica were mutagenized by UV irradiation and treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG). Subsequent selection on media with acetate and isocitrate resulted in isolation of a mutant Y. lipolytica UV/NG that synthesized isocitrate and citrate at a ratio of 2.7 : 1. In the parent strain, this ratio is 1 : 1. Inhibition of isocitrate lyase, a key enzyme in the metabolism of isocitric acid, by the addition of itaconic acid resulted in increased synthesis of isocitrate, with a ratio of isocitrate to citrate reaching 6 : 1. The cultivation of the Y. lipolytica UV/NG mutant in a pilot industrial fermenter in the presence of itaconic acid resulted in the production of 88.7 g/L of isocitric acid with a yield of 90%.

Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by Pseudomonas bacteria

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24°C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS1391 bearing plasmid pBS216 was found to be the most effective oil degrader.

Horizontal transfer of catabolic plasmids and naphthalene biodegradation in open soil.

The horizontal transfer of naphthalene biodegradation plasmids and the parallel process of its microbial degradation were studied for the first time. The tagged naphthalene-degrading strains bearing labeled biodegradation plasmids were used for the monitoring of horizontal plasmid transfer in open soil. The population kinetics of microorganisms, the survival rate and competitiveness of introduced strains, and the transfer of biodegradation plasmids to indigenous strains were investigated. The transfer of the labeled plasmid pNF142::TnMod-OTc to the introduced plasmid-free recipient P. putida KT2442 and to indigenous soil microorganisms of the genus Pseudomonas was shown both under selection pressure (in the presence of naphthalene) and in its absence. The 16S rRNA gene sequencing showed that the soil strains that had acquired plasmids were close to the species P. lini, P. frederiksbergensis, P. jessenii, P. graminis, P. putida, and P. alcaligenes. Thus, direct evidence of dissemination of the naphthalene biodegradation plasmids in microbial populations in open soil under selective and nonselective conditions has been obtained.

Effects of Naphthalene Degradative Plasmids on the Physiological Characteristics of Rhizosphere Bacteria of the Genus Pseudomonas

We studied the specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation in rhizosphere pseudomonades carrying the structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol-2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could “switch off” the genes of the catechol oxidation meta-pathway. It was shown that certain combinations of degradative plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and the stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).

The role of mineral phosphorus compounds in naphthalene biodegradation by Pseudomonas putida

The effect of phosphate concentration in the culture medium on the growth and naphthalene degradation by Pseudomonas putida BS 3701 was studied. The limiting concentration of phosphate was 0.4 mM and 0.1 mM under cultivation in media with naphthalene and glucose, respectively. The phosphate deficiency correlated with a decrease in the activities of naphthalene dioxygenase and salicylate hydroxylase and with salicylate accumulation in the culture medium. We suggest that this fact indicates the impaired regulation of gene expression of “upper” and “lower” pathways of naphthalene oxidation. Under naphthalene degradation, the cells accumulated three times more inorganic polyphosphates as compared with the consumption of glucose. The involvement of polyphosphates in the regulation of naphthalene metabolism has been considered.