DNA polymerase I is not required for replication of linear chromosomes in streptomyces

Abstract

Both polA (encoding DNA polymerase I; Pol I) and a paralog were deleted from Streptomyces strains. Despite the UV sensitivity and slow growth caused by the DeltapolA mutation, the double mutant was viable. Thus, in contrast to a previous postulate, Pol I and its paralog are not essential for replication of Streptomyces chromosomes.

FIG. 1.

Creation and characterization of ΔpolA mutants. (A) Design. The polA and relevant surrounding genes in the wild type (top) are shown as open arrows. In the mutant (bottom), polA was replaced by a cassette (filled box) containing aac3(IV) (white arrow). Two hybridization probes, A and B, are indicated by open boxes. Ps, PstI site. The sizes (in kilobases) of the hybridization PstI fragments are indicated. (B) Hybridization analysis of the ΔpolA mutants. PstI-digested total DNA of each culture was electrophoresed in an agarose gel and hybridized to digoxigenin-labeled probes A (left panel) and B (right panel), as described by Kieser et al. (12). (C) UV sensitivity of the mutants (9). Filled circles, strain 3456; open circles, strain HT1-3; filled triangles, strain 3454; open triangles, strain HT4-4. The error bars indicate 1 standard deviation calculated by Poisson distribution based on the number of colonies scored. (D) Growth retardation of the mutants. The cultures were plated on LB agar (12) and incubated at 30° for 4 days.

FIG. 2.

Creation and characterization of ΔSCO3434 ΔpolA double mutants. (A) Design. SCO3434 and relevant surrounding genes in the wild type (top) are shown as open arrows. In the mutant (bottom), SCO3434 was replaced by a cassette (filled box) containing aadA or aac3(IV) (white arrow). The digoxigenin-labeled hybridization probe C is indicated by an open box. Ba, BamHI site. The sizes of the hybridization BamHI fragments are indicated by numbers. (B) Hybridization analysis of the ΔSCO3434 mutants. BamHI-digested total DNA of each culture was electrophoresed in an agarose gel and hybridized to probe C. HL-5m, ΔSCO3434 mutant of M145; HL-13n, ΔSCO3434 mutant of 3456; HD21-16, ΔSCO3434 mutant of HT1-3. (C) UV sensitivity of the mutants (9). Filled circles, strain 3456; open circles, strain HL-13n; filled triangles, strain HT1-3; open triangles, strain HD21-16. The error bars indicate 1 standard deviation calculated by Poisson distribution based on the number of colonies scored. The results for M145 and HL-5m were identical to those for 3456 and HL-13n, respectively.