T. Z. Esikova

The construction and monitoring of genetically tagged, plasmid-containing, naphthalene-degrading strains in soil

A genetically tagged, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142:: TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain’s fate in model soil systems to be monitored, whereas a minitransposon, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.

Ultrastructural organization and development cycle of soil ultramicrobacteria belonging to the class Alphaproteobacteria

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400–800 nm and ultrasmall cells of 200–300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB; these include the following: (a) piles of rod-like subunits, ca. 30 Å in diameter and 150–250 Å in length; (b) long bunches (up to 300–400 Å) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200–300 Å in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB cytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2–3) located directly on the mother cell, without formation of intermediate hyphae.

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.

Influence of soil pollution on the composition of a microbial community

The abundance dynamics and composition of indigenous soil microbial communities were studied in soils polluted with naphthalene, dioctyl phthalate, diesel fuel, and crude oil. DGGE analysis of the 16S rRNA genes amplified from the total soil DNA revealed that the bacterial community of uncontaminated soil was more diverse and included no dominant species. In the soil samples polluted with the crude oil, diesel fuel, or dioctyl phthalate, Pseudomonas became the dominant bacteria since the third day of the experiment. In the soil polluted with naphthalene, two genera of bacteria (Pseudomonas and Paenibacillus) were dominant in population on the third day of the experiment, while on the 21th day of the experiment Arthrobacter became dominant. During the experiment, the average number of indigenous bacterial degraders increased approximately by two orders of magnitude. While the key genes of naphthalene catabolism, nahAc and nahH, were not detected in the pristine soil, they were found in a significant amount on the third day after naphthalene addition. Three degrader strains harboring the plasmids of naphthalene biodegradation (IncP-9 group) were isolated on the third day from the soil polluted with naphthalene. Two of these plasmids, although isolated from various degraders, were shown to be identical.

[Novel ultramicrobacteria, strains NF4 and NF5, of the genus Chryseobacterium: Facultative epibionts of Bacillus subtilis ].

Two strains, NF4 and NF5, of a yellow-colored gram-negative bacterium were isolated from sediments of Lake Baikal and from old oil sludge of the Nizhnekamsk oil-processing plant. The cells of the strains are ultrasmall coccoids or short rods, measuring 0.2–0.4 × 0.2–0.5 μm; the average cell volume ranges from 0.004 to 0.04 μm3. A considerable proportion (30–60%) of cells have nanometer dimensions (180–300 nm in diameter and 0.004–0.02 μm3 in volume). The new isolates are thus among the smallest representatives of presently known free-living ultramicrobacteria. The two studied isolates are gram-negative nonmotile cells possessing a pronounced outer membrane. The cells do not have flagella and are not capable of gliding motility. They divide by constriction, budding, and multiple septation. The multiplicity of reproduction mechanisms results in a high degree of cell polymorphism. The isolates are chemoorganotrophic, aerobic, psychrotolerant, oxidase- and catalase-positive. Their characteristic trait is the absence of extracellular hydrolytic enzymes, such as proteases, lipases, pectinases, and cellulases. Menaquinone MK-6 is the main respiratory quinone; the flexirubin pigment was not detected. The G + C contents of the DNA of strains NF4 and NF5 are 40.8 and 40.5 mol %, respectively. The DNA-DNA hybridization level of strains NF4 and NF5 was close to 100%. Analysis of the 16S rRNA gene sequences and the fatty acid compositions showed that the isolates are most closely related to certain representatives of the genus Chryseobacterium (C. solincola, C. antarcticum, and C. jeonii). However, the differences in the 16S rRNA gene sequences, as well as in the phenotypic properties, such as formation of ultrasmall cells, the absence of extracellular hydrolases, oligotrophy, and the capacity for epibiosis on bacterial cells, suggest that the studied strains belong to a new species of the genus Chryseobacterium. The capacity for epibiosis, i.e., the ability to exist in a tightly adhered state on the surfaces of host Bacillus subtilis cells, is a peculiar trait of the studied isolates. It is assumed that adhesion of the cells of strains NF4 and NF5 (members of the phylum Bacteroidetes) occurs via by the same unique mechanism as the mechanism that we previously described for representatives of Alphaproteobacteria (Kaistia sp., NF1, and NF3), which use polysaccharide chains equipped with sticky granules as trapping and constricting cords.

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.

[Study of ectoparasitism of ultramicrobacteria of the genus Kaistia, strains NF1 and NF3 by electron and fluorescence microscopy].

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.

Transformation of low-molecular linear caprolactam oligomers by the caprolactam-degrading bacterium Pseudomonas putida BS394(pBS268)

A biosensor based on the most active caprolactam-degrading strain Pseudomonas putida BS394(pBS268) was used in the study of aerobic degradation of linear caprolactam oligomers by bacterial cells. The changes in the respiratory activity of the strain depend quantitatively on caprolactam dimer concentration, making it possible to develop biosensors for detection of caprolactam oligomers in aqueous media. Based on mass spectrometry data, the scheme of transformation of linear caprolactam oligomers by the degrader strain P. putida BS394(pBS268) was proposed for the first time. It was found that oxidative transamination to respective dicarbonic acids may be one of the mechanisms of transformation of linear caprolactam oligomers. According to the scheme proposed, the ability of the caprolactam-degrading strain to transform linear oligomers results from the broad substrate specificities of two enzymes of the caprolactam degradation pathway: 2-oxoglutarate-6-aminohexanoate transaminase and 6-oxohexanoate dehydrogenase. Transformation of linear oligomers is genetically controlled by the CAP biodegradation plasmid pBS268.

ScpA, a new salicylate hydroxylase gene localized in salicylate/caprolactam degradation plasmids

Both caprolactams and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders are controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids have been assigned to the P-7 incompatibility group. The new salicylate 1-hydroxylase gene (scpA) has been detected in SAL/CAP plasmids and partially sequenced. The scpA gene was equally related to the closest homolog genes nahG (NAH7), salA (P. reinekei MT1), and nahU (pND6-1); however, the identity rate did not exceed 72–74%. The synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited the highest specific activity toward 4-methylsalicylate and nonsubstituted salicylate substrates. Furthermore, conjugative pseudomonads’ plasmids of salicylate degradation without the classical nah2 operon, which harbors only salicylate 1-hydroxylase gene nahU have been described for the first time.

Key role of the dca genes in ε-caprolactam catabolism in Pseudomonas strains

CAP is a persistent pollutant with a toxic effect on living organisms (Gross, 1984). Although bacteria capable of utilizing CAP as a sole source of carbon and energy have been described (Baxi, 2013; Sanuth et al., 2013), bacterial catabolism of CAP is a poorly studied process, the initial stages of which the currently unknown genes and enzymes participate in. As a result of study of CAP catabolism in the strain Pseudomonas dacunhae, the following biochemical degradation pathway has been proposed: ε caprolactam (CAP) → 6 aminohexanoic acid (AHA) → → adipic acid (AD) → → → → Krebs cycle (Naumova, 1988). It was shown that the ability of pseudomonads to utilize CAP and its intermediates (AHA and AD) as the sole carbon and energy sources is controlled by CAP plas mids (Naumova, 1988; Esikova et al., 1990). The goal of the present study was to identify the regions of the CAP plasmid pBS270 that are responsible for the syn thesis of enzymes involved in the catabolism of CAP and its intermediates, and to investigate their molecu lar genetic organization.