DNA Polymerase I Is Essential for Growth of Methylobacterium dichloromethanicum DM4 with Dichloromethane

Abstract

Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase-glutathione S-transferase. A mutant of the dichloromethane-degrading strain M. dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the polA mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into the polA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The polA -lacZ transcriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of M. dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.Dichloromethane (DCM) is an organic solvent produced industrially in large amounts for a wide range of technical applications (Halogen Solvents Industry Alliance [http://www .hsia.org/white_papers/methchlor.htm]). Its low boiling point and high solubility in water make it a frequently encountered environmental contaminant (36,46). The toxicity of DCM to mammals continues to be investigated intensively (9,14,21,40,45), but its causes are not yet fully characterized at the molecular level. Many specialized aerobic methylotrophic bacteria have been isolated from soil and groundwater environments contaminated with DCM for their ability to grow with DCM as the sole source of carbon and energy (49). Such bacteria rely on a single enzyme, DCM dehalogenase, for this purpose. DCM dehalogenase, which can make up to 20% of the soluble protein during bacterial growth with DCM, was purified and shown to catalyze the glutathione-dependent transformation of DCM to formaldehyde, used in both biomass and energy production, and to two molecules of hydrochloric acid (31). The corresponding gene dcmA was cloned (33) from Methylobacterium dichloromethanicum DM4 (15) (formerly Methylobacterium sp. strain DM4), Methylophilus sp. strain DM11 (3), and, more recently, from several other DCM-degrading strains (49, 50). Sequence analysis indicates that DCM dehalogenases belong to the glutathione S-transferase (GST) enzyme family (27,47). DCM dehalogenases were the first bacterial GSTs to be characterized, but it is becoming clear that the genom...

Microbial dehalogenation

“…Recently, it has been shown that a functional DNA polymerase I, which is involved in DNA repair, is necessary, in addition to a functional catabolic pathway, for the utilisation of dichloromethane [21]. The requirement for additional detoxification mechanisms may be a more general phenomenon, which would explain the failure of various experiments aimed at the genetic construction of organisms with new catabolic pathways.…”

Dichloromethane metabolism and C1 utilization genes in Methylobacterium strains The GenBank accession numbers for the sequences determined in this work are AJ421476 and AJ421477.

“…In particular, the genotoxic effects associated with DCM-dehalogenasedependent conversion of DCM (Kayser & Vuilleumier, 2001) indicate that an efficient DNA repair system is required. Indeed, DNA polymerase I, an enzyme well known to participate in several DNA repair mechanisms, was shown to be essential for growth with DCM in strain DM4 (Kayser et al, 2000).…”

“…This is a source of concern because of the genotoxic effects associated with DCM in bacteria as well as in mammals (Kayser et al, 2000 ;Landi, 2000 ;Kayser & Vuilleumier, 2001 ;Vuilleumier, 2002). Methylobacterium dichloromethanicum DM4 is able to use DCM as the sole source of carbon and energy (Ga$ lli & Leisinger, 1988 ;Doronina et al, 2000 ;Vuilleumier, 2002).…”

Growth inhibition of Escherichia coli by dichloromethane in cells expressing dichloromethane dehalogenase/glutathione S-transferase

“…These mutants grow well with methanol, which is oxidized during growth to formaldehyde, a central metabolic intermediate in methylotrophic bacteria. In one of these mutants, the transposon insertion is located in the gene encoding DNA polymerase I, a key enzyme in DNA repair (Kayser et al, 2000). Thus, it seems possible that the unexpected severity of the growth inhibition observed during exposure to DCM of E. coli cells expressing DCM dehalogenase is due in part to genotoxic GSH adducts.…”