E. S. Shumkova

Phenol degradation by Rhodococcus opacus strain 1G

During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two catechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to catechol 1,2-dioxygenases of the classical ortho-pathway. Chlorocatechols and chlorophenols served as competitive inhibitors of catechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.

Degradation of chlorinated biphenyls and products of their bioconversion by Rhodococcus sp. B7a strain

Strain Rhodococcus sp. B7a isolated from artificially polluted soil destructs mono- and di-substituted ortho- and/or para-chlorinated biphenyls with utilization of chlorinated benzoic acids and shows high degradation activity as regards trichlorinated biphenyls. It is shown that p-hydroxybenzoic and protocatehoic acids are the products of p-chlorobenzoic acid catabolism.

Degradation of 4-chlorobiphenyl and 4-chlorobenzoic acid by the strain Rhodococcus ruber P25

The strain Rhodococcus ruber P25 utilizes 4-chlorobiphenyl (4CB) and 4-chlorobenzoic acid (4CBA) as sole carbon and energy sources. 4CB degradation by washed cells of strain P25 was accompanied by transient formation of 4CBA, followed by its utilization and release of equimolar amounts of chloride ions into the medium. The strain R. ruber P25 possessed active enzyme systems providing 4CBA degradation via the stages of formation of intermediates, para-hydroxybenzoate (PHBA) and protocatechuic acid (PCA), to compounds of the basic metabolism. The involvement of protocatechuate 4,5-dioxygenase in 4CBA degradation by rhodococci was revealed. It was established that the initial stage of 4CBA degradation (dehalogenation) in the strain R. ruber P25 was controlled by the fcbA and fcbB genes encoding 4-CBA-CoA ligase and 4-CBA-CoA dehalogenase, respectively. The genes encoding 4CBA dehalogenase components have not been previously detected and characterized in bacteria of the genus Rhodococcus.

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores.

Detection of n-alkane biodegradation genes alkB and ladA in thermophilic hydrocarbon-oxidizing bacteria of the genera Aeribacillus and Geobacillus

Ability to degrade crude oil n-alkanes was revealed in new strains of thermophilic bacilli isolated from petroleum reservoirs and a hot spring: Geobacillus toebii В-1024, Geobacillus sp. 1017, and Aeribacillus pallidus 8m3. The strains utilized С10–С30n-alkanes (В-1024), С10, C11, and С13–С19,22n-alkanes (1017), and C11–C29n-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucleotide primers revealed the alkB gene encoding rubredoxin-dependent alkane monooxygenase. In strains В-1024 and 1017, fragments of the genes homologous to the ladA gene determining flavin-dependent alkane monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those of the genes ladAαB23, ladAβB23, and ladBB23, which were revealed previously in Geobacillus thermoleovorans strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain n-alkanes has been shown. Thus, simultaneous presence of the alkB and ladA genes coding alkane monooxygenases responsible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the first time.

Molecular biological characterization of biphenyl-degrading bacteria and identification of the biphenyl 2,3-Dioxygenase α-subunit genes

Bacterial isolates from soils contaminated with (chlorinated) aromatic compounds, which degraded biphenyl/chlorinated biphenyls (CB) and belonged to the genera Rhodococcus and Pseudomonas, were studied. Analysis of the 16S rRNA gene sequences was used to determine the phylogenetic position of the isolates. The Rhodococcus cells were found to contain plasmids of high molecular mass (220–680 kbp). PCR screening for the presence of the bphA1 gene, a marker indicating the possibility for induction of 2,3-dioxygenase (biphenyl/toluene dioxygenase subfamily), revealed the presence of the bphA1 genes with 99–100% similarity to the homologous genes of bacteria of the relevant species in all pseudomonad and most Rhodococcus isolates. A unique bphA1 gene, which had not been previously reported for the genus, was identified in Rhodococcus sp. G10. The absence of specific amplification of the bphA1 genes in some biphenyl-degrading bacteria (Rhodococcus sp. B7b, B106a, G12a, P2kr, P2(51), and P2m), as well as in an active biphenyl degrader Rhodococcus ruber P25, indicated the absence of the genes encoding the proteins of the biphenyl/toluene dioxygenase subfamily and participation of the enzymes other than this protein family in biphenyl/CB degradation.

Identification of hydrocarbon-oxidizing Dietzia bacteria from petroleum reservoirs based on phenotypic properties and analysis of the 16S rRNA and gyrB genes

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol %, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002T, respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classification of strain 32d as a D. maris strain and strain 263 as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Dioxygenases of chlorobiphenyl-degrading species Rhodococcus wratislaviensis G10 and chlorophenol-degrading species Rhodococcus opacus 1CP induced in benzoate-grown cells and genes potentially involved in these processes

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2 g/liter benzoate during 30 h and 10 g/liter during 200 h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding αand β-subunits of protocatechuate 3,4-dioxygenase and to two genes of theR. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.

Whole-cell bacterial biosensors for the detection of aromatic hydrocarbons and their chlorinated derivatives (Review)

The review summarizes the data on new directions in biosensor technologies based on whole bacterial cells. Biosensors for the monitoring of mono(poly)aromatic hydrocarbons and their chlorinated derivatives, which are constructed with genetically modified bacterial cells bearing a reporter gene fusion, are considered. The operating principle of these biosensors is based on the expression of reporter genes (luc, lux, gfp, rfp) under the control of a promoter and a regulator that specifically respond to a detected compound.

Draft Genome Sequence of Rhodococcus ruber Strain P25, an Active Polychlorinated Biphenyl Degrader

ABSTRACT We report the 5,728,255-bp draft genome sequence of Rhodococcus ruber P25, isolated from a soil polluted with halogenated aromatic compounds in the city of Perm, Russia. The strain degrades polychlorinated biphenyls and a broad range of aromatic compounds. It possesses genes that mediate the degradation of biphenyls/polychlorinated biphenyls, naphthalene, and monoaromatic compounds.