[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

[Functional analysis of the genome fragment involved in aerobic dichloromethane degradation by methylobacterium dichloromethanicum DM4].

The hypothetical genes of Methylobacterium dichloromethanicum DM4, METDI 2671 (bioD2), and METDI 2680 located within the chromosomal fragment (126 kb) associated with dichloromethane (DCM) degradation have been studied. The reverse transcription polymerase chain reaction method (RT-PCR) showed the presence of transcripts of both genes in cells grown on DCM and methanol. The mobilized suicidal vector pK18mob was used to obtain knockout mutants in these genes. The BIO mutant (with an insertion in the bioD2 gene) after cultivation on methanol was characterized by a lower growth rate on DCM compared to the wild-type DM4 strain, while the MT mutant (with an insertion in the METDI 2680 gene) did not differ from the initial strain in respect of these characteristics. The results demonstrate the involvement of the bioD2 gene in biotin biosynthesis coupled with DCM degradation.

[Adaptation of aerobic methylobacteria to dichloromethane degradation].

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCI. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.

[Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane].

Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.

[Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

[Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase]. / Fiziologo-biokhimicheskii analiz transformantov aérobnykh metilobakterii, ékspressiruiushchikh gen dikhlormetandegalogenazy dcm A.

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.