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.

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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 degra dation by rhodococci was revealed. It was established that the initial stage of 4CBA degradation (dehaloge nation) 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.

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The taxonomic position of hydrocarbon oxidizing bacterial strains 263 and 32d isolated from for mation 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. cerci diphylli YIM65002 T , 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. natrono limnaea 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. rRNA and gyrB genes, primers for amplification and sequencing of the gyrB genes in bacteria of the genus Dietzia