Article Figures & Data
Figures
- Fig. 1.
Southern blot analysis of the polA gene ofM. dichloromethanicum DM4. (A) Chromosomal DNA (5 μg) from wild-type M. dichloromethanicum DM4 (lanes 1), mutantM. dichloromethanicum DM4-1445 (lanes 2), and mutant DM4-1445 conjugated with complementing plasmid pME8112 and digested with PstI or with HindIII/EcoRI (lanes 3) was hybridized with a polA-specific DIG-labeled probe after agarose gel electrophoresis. Lanes M, molecular size markers. (B) Corresponding schematic map of cloned DNA fragments with the DM4 polA gene, indicating the point of insertion of the mini-Tn5 element (12) in mutant DM4-1445 (top), and vector pVK100 (30) used for mutant complementation with the wild-type (wt) polA gene. Restriction sites relevant for Southern analysis and the location of the DIG-labeled probe (striped box) are shown. Segments corresponding to 5′-3′ exonuclease, 3′-5′ exonuclease, and DNA polymerase domains of the polA gene product are indicated as light grey, dark grey and black boxes, respectively.
- Fig. 2.
Effect of DCM on growth of wild-type M. dichloromethanicum DM4 and of the polA mutant. Strains DM4 wild-type (squares), mutant DM4-1445 (circles), and complemented mutant DM4-1445(pME8112) (triangles) were grown at 30°C in minimal medium with 40 mM MeOH as the unique carbon source (open symbols). The growth behavior of cultures additionally treated with 10 mM DCM at an OD600 of 0.05 (arrow) is depicted with filled symbols. Data from one representative experiment are shown.
- Fig. 3.
Induction of DCM dehalogenase after addition of DCM. DCM dehalogenase induction during growth is given as a function of culture turbidity. (A) Specific activity in crude extracts harvested at various stages of growth; (B) concentration of chloride ions released in the growth medium (symbols as defined in the legend to Fig. 2). Error bars, standard errors of triplicate measurements.
- Fig. 4.
DNA polymerase I activity of wild-type M. dichloromethanicum DM4 and of the polA mutant. Shown is an autoradiogram of cell extract protein (100 μg) of wild-type strain DM4 (lane 1), mutant DM4-1445 (lane 2), and complemented mutant DM4-1445(pME8112) (lane 3), separated by SDS-PAGE in a gel containing nicked DNA, after incubation with dideoxynucleotides and α-32P-labeled dCTP (see Materials and Methods). Lane M, prestained marker from the scanned gel; lanes 4 and 5, E. coli DNA polymerase I and Klenow fragment, respectively (0.02 U [∼0.1 ng] each).
- Fig. 5.
Effect of DCM on the expression of thepolA′-lacZ fusion in the DM4-1445 polA mutant background. The β-galactosidase specific activity (sp. act.) arising from the polA′-lacZ transcriptional fusion was determined in cell extracts of M. dichloromethanicum DM4-1445 (circles) and DM4-1445(pME8112) (triangles) grown with MeOH as the carbon source with (filled symbols) and without (open symbols) prior treatment with 10 mM DCM at an OD600 of 0.05 and expressed as a function of the optical density of the cultures at the time of cell harvest. Error bars, standard errors of triplicate measurements.
- Fig. 6.
Effects of UV light and DCM on the viability of wild-type M. dichloromethanicum DM4 and of thepolA mutant. Methanol-grown cultures of M. dichloromethanicum DM4 wild-type (squares), DM4-1445 (circles), and DM4-1445(pME8112) (triangles) were exposed to UV light at 254 nm (5 J · m−2 · s−1) (A) or treated with 10 mM DCM at an OD600 of 0.05 (B) for various times before plating on MM with 40 mM MeOH. Bacterial viability is expressed relative to that of untreated cultures.
