Alternative Naphthalene Metabolic Pathway Includes Formation of ortho-Phthalic Acid and Cinnamic Acid Derivatives in the Rhodococcus opacus Strain 3D.

Naphthalene, as a component of crude oil, is a common environmental pollutant. Biochemical and genetic aspects of naphthalene catabolism have been examined in most detail in the bacteria of Pseudomonas genus. In pseudomonads, the key intermediate in naphthalene degradation is salicylate. In this study, we investigated the ability of Rhodococcus opacus strain 3D to utilize naphthalene as a sole carbon and energy source. The characteristic feature of this strain is the inability to grow in the mineral medium supplemented with salicylate (typical intermediate of naphthalene degradation in Gram-negative bacteria). The absence of salicylate hydroxylase activity and salicylate accumulation in the course of R. opacus 3D cultivation in the mineral medium supplemented with naphthalene indicated existence of an alternative pathway of naphthalene oxidation. At the same time, R. opacus 3D was able to use monoaromatic compounds (salts of gentisic, ortho-phthalic, and 2-hydroxycinnamic acids and coumarin) as growth substrates. Based on the analysis of enzymatic activities, identification of the reaction intermediates, genetic determinants, and growth substrates, we concluded that R. opacus 3D carries out naphthalene degradation through an alternative pathway via formation of ortho-phthalic acid, which is untypical for pseudomonads. Using mass spectrometry, we showed for the first time that salicylic acid associate formed in trace amounts in the process of naphthalene degradation is not further metabolized and accumulated in the growth medium in a form of a dimer.

[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 < or =0.3 microm and volume of < or =0.014 microm3) 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 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. 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.

[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 A in diameter and 150-250 angstroms in length: (b) long bunches (up to 300-400 angstroms) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200-300 angstroms 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 eytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2-3) located directly on the mother cell, without formation of intennediate hyphae.