Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli

Abstract

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element TnlO into recD resulted in increased concatemerization and loss of pSC101 and ColEl-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coi chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.The maintenance of plasmids as extrachromosomal elements in bacterial cells is dependent on a number of complex processes. Of primary importance is the requirement for plasmid replication, which commonly involves at least some host-encoded replication functions (see Scott [39] for a review). Replication of the pSC101 plasmid (7) requires the Escherichia coli dnaA gene product (21). Mutations in other genes involved in chromosomal replication, including dnaB, dnaC, and dnaG (14,22), also affect replication of this plasmid. Stable maintenance of pSC101 in growing cell populations also requires a plasmid locus (named par for partitioning) that is involved in the distribution of plasmid molecules to daughter cells at the time of cell division (29,45). Earlier work has shown that the pSC101 par region does not encode a protein but instead includes partition-related segments (30) that appear to be necessary for plasmids in the intracellular pool to be counted and partitioned as individual molecules (45). To better understand the mechanism by which pSC101 segregates during cell division, we undertook to identify and characterize E. coli genes encoding products involved in the plasmid maintenance (Pma) phenotype.We report here the isolation and characterization of mutations that affect the stable maintenance of pSC101, as well as that of certain other plasmids. We present evidence that these mutations, which can alter plasmid stability by increasing the frequency of multimer formation and are located in a previously unidentified E. coli gene, affect the activity of exonuclease V (ExoV). MATERIALS AND METHODSStrains, media, and general methodology. Relevant bacterial genotypes, plasmids, and phages are listed in Table 1. The media used for these studies have been previously described (31). Minimal medium was M9 or M63 supplemented with 0.2% glucose (or another carbon source), 1 ,ug of thiamine per ml, and any required L-amino acids (Sigma Chemical Co.) at 40 ,ug/ml. Solid media contained 1.5% agar (Difco Laboratories) or 0.7% agar for soft agar overlays. Lactose indicator plates contained 40 ,ug of X-Gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside; Boehringer Mannheim) per ml in minimal medium containing...

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

“…Plasmid stability and copy number were determined as described by Meacock and Cohen (1980) and Biek and Cohen (1986), respectively, or by gene dosage of kanamycin resistance (Nordströ m et al, 1980).…”

“…Clones containing chromosomally integrated interrupted genes were confirmed by Southern hybridization. Subsequently, P1 transduction was performed to move the mutation to appropriate strains (Biek and Cohen, 1986).…”

“…Plasmid partitioning is also affected by host-encoded genes that influence the ability of replicating plasmid DNA molecules to form concatamers (i.e. recD ;Biek and Cohen, 1986) or undergo negative supercoiling (i.e. topA, gyrA and gyrB ;Biek and Cohen, 1989a;Miller et al, 1990).…”

A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex.

“…Escherichia coli K-12 strain MG1655 and LB growth medium have been described (15). When selecting for inheritance of plasmid pZC209 or its derivatives after transformation or when scoring for the presence of plasmid by replica plating, the concentrations of streptomycin sulfate and spectinomycin dihydrochloride were 20 pg/ml and 40 pg/ml, respectively; selection under other conditions was in medium containing antibiotics at one-half these concentrations, which allowed better growth of the cells.…”

“…When selecting for inheritance of plasmid pZC209 or its derivatives after transformation or when scoring for the presence of plasmid by replica plating, the concentrations of streptomycin sulfate and spectinomycin dihydrochloride were 20 pg/ml and 40 pg/ml, respectively; selection under other conditions was in medium containing antibiotics at one-half these concentrations, which allowed better growth of the cells. Plasmid stability studies were performed as described (15). Loss rates per cell per generation were calculated from curves fitted to the data.…”