No abstract

A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid. When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication. Appearance of plasmid-free segregants is therefore effectively prevented under such conditions. Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition. The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation.Plasmids that replicate by using the replication origin (oriC) of the Escherichia coli chromosome are not stably maintained through cell division under nonselective conditions (1). We have previously found that when a particular segment of mini-F plasmid is inserted into such plasmids, the resulting oriC plasmids become stable (1, 2). The segment that contributes to this stability has been located within the 46.4-49.4 kilobase pairs (kb) coordinates on the F map (3), which is outside of the region essential for mini-F replication (44.0-46.35 kb), and the stabilization is achieved without a detectable increase in plasmid copy number (2). Apparently, this segment specifies the partition rather than the replication control of such plasmids. It has been shown that the segment includes three functional regions necessary for stable maintenance; two (sopA and sopB) act in trans and one (sopC) acts in cis (2) (Fig. 1). However, even oriC plasmids stabilized by the partition mechanism of mini-F are not fully stable. This observation prompted us to investigate additional DNA segments for stabilization properties. In this paper, we describe the characterization of a mini-F DNA seg-, ment that seems to play an important role in stable maintenance of plasmids in host bacteria. This segment (42.9-43.6 kb), designated ccd (coupled cell division), is located outside of the regions essential for autonomous replication and for partition of mini-F (Fig. 1). The ccd segment appears to act by coupling host cell division to proliferation of plasmids. We propose a hypothesis that explains the functions of the ccd segment. MATERIALS AND METHODSBacterial strains used were all derivatives of E. coli K-12. Strains KY7231 (F-trpB9578 tna-2. rpsL recAl) and KZ200 (F-ilv thr metE trp tyr thy rpsL recAl) were o...

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino‐terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha‐helical coiled‐coil domains and one globular domain, and a carboxyl‐terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild‐type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named muk4M, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mu)41 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin El protein, in addition to anucleate cel formation.

Using immunofluorescence microscopy, we have found that SeqA protein, a regulator of replication initiation, is localized as discrete fluorescent foci in E. coli wild-type cells. Surprisingly, SeqA foci were observed also in an oriC deletion mutant. Statistical analysis revealed that a SeqA focus is localized at midcell in newborn cells. The SeqA focus is duplicated and tethered at midcell until an FtsZ ring is formed. Subsequently, these foci migrate in opposite directions toward cell quarter sites and remain tethered there until the cell divides. The cell cycle-dependent bidirectional migration of SeqA-DNA complexes is quite different from the migration pattern of oriC Dna copies. MukB protein is required for correct localization of SeqA complexes by an unknown mechanism.