SummaryThe positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.

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Crossing over by homologous recombination between monomeric circular chromosomes generates dimeric circular chromosomes that cannot be segregated to daughter cells during cell division. In Escherichia coli, homologous recombination is biased so that most homologous recombination events generate noncrossover monomeric circular chromosomes. This bias is lost in ruv mutants. A novel protein, RarA, which is highly conserved in eubacteria and eukaryotes and is related to the RuvB and the DnaX proteins, ␥ and , may influence the formation of crossover recombinants. Those dimeric chromosomes that do form are converted to monomers by Xer site-specific recombination at the recombination site dif, located in the replication terminus region of the E. coli chromosome. The septum-located FtsK protein, which coordinates cell division with chromosome segregation, is required for a complete Xer recombination reaction at dif. Only correctly positioned dif sites present in a chromosomal dimer are able to access septum-located FtsK. FtsK acts by facilitating a conformational change in the Xer recombination Holliday junction intermediate formed by XerC recombinase. This change provides a substrate for XerD, which then completes the recombination reaction.homologous recombination ͉ Ruv͞Xer recombination ͉ dimer resolution ͉ FtsK B arbara McClintock, during her work on ring chromosomes in maize, inferred in 1932 that whereas crossing over between rod-shaped (linear) chromosomes does not alter their topology, crossing over between ring (circular) chromosomes generates larger ring chromosomes (circular dimers) that cannot be segregated normally at cell division (1). This topological complication arising from crossing over between circular chromosomes was largely ignored until the 1980s, when it was demonstrated that site-specific recombination systems act to convert dimeric plasmid molecules, formed by homologous recombination, to monomers, and thereby facilitate stable plasmid inheritance (2, 3). Subsequently, it was shown that one of these site-specific recombination systems, XerCD site-specific recombination, also functions in the conversion of dimeric Escherichia coli chromosomes to monomers (4-6). Xer recombination uses two related recombinases, XerC and XerD, belonging to the tyrosine recombinase family, each recombinase catalyzing the exchange of one pair of strands in a reaction that proceeds through a Holliday junction (HJ) intermediate (7)(8)(9). XerCD act at the recombination site dif, located in the replication terminus region of the E. coli chromosome and at related sites in multicopy plasmids, for example, psi in plasmid pSC101 and cer in ColE1 (3, 10).Here we outline the processes that limit dimer formation by homologous crossing over in E. coli. We also discuss the mechanism that restricts Xer recombination at chromosomal dif to converting dimers to monomers by making a part of the recombination machine only accessible to dif sites when they are present in chromosomal dimers at the time of cell division.Homologous recom...

Ubiquinone is an essential redox component of the aerobic respiratory chains of bacteria and mitochondria. It is well established that mammalian ubiquinone can function in its reduced form (ubiquinol) as a lipid-soluble antioxidant preventing lipid peroxidation. The objective of this study was to test the hypothesis that prokaryotic ubiquinone is involved in the defence against oxidative stress in the cytoplasmic membrane. The rate of superoxide production by rapidly respiring wild-type Escherichia coli membranes was twofold higher than in the slowly respiring membranes from a ubiCA knockout mutant. However, large amounts of superoxide accumulated in the Ubi N membranes compared to wild-type membranes, which possess superoxidescavenging ubiquinol. Likewise, the rate of H 2 O 2 production was twofold higher in the wild-type, but the overall production of H 2 O 2 was again significantly higher in the Ubi N membranes. Inclusion of a water-soluble ubiquinone homologue (UQ-1) effectively decreased the amount of H 2 O 2 produced in the Ubi N membranes in a concentration-dependent manner. Addition of UQ-2 to the membranes was even more effective in limiting accumulation of H 2 O 2 than was UQ-1, suggesting a role for the side-chain in conferring liposolubility in the antioxidative defence mechanism. Intracellular H 2 O 2 concentration was increased 18-fold in the ubiCA mutant, and expression of the katG gene, encoding the catalase hydroperoxidase I, as well as catalase enzyme activity, were increased twofold in this mutant. The ubiCA mutant was hypersensitive to oxidative stress mediated by CuSO 4 or H 2 O 2 ; sensitivity to the latter could be abolished by addition of cysteine. This phenotype was also exhibited by a ubiG mutant, defective in the last step of UQ biosynthesis and therefore expected to accumulate several UQ biosynthetic intermediates. These observations support the participation of reduced ubiquinone as an antioxidant in E. coli. The ubiCA mutant exhibited a pleiotropic phenotype, being resistant to heat, linolenic acid and phleomycin. Resistance to the two latter compounds is probably due to reduced uptake. Like mutants unable to synthesize the quinol oxidase, cytochrome bd, the ubiCA mutant was also sensitive to dithiothreitol, an effect that is attributed to inability of the respiratory chain to maintain an appropriate redox balance in the periplasm.

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.