Ubiquinone limits oxidative stress in Escherichia coli.

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- membranes compared to wild-type membranes, which possess superoxide-scavenging ubiquinol. Likewise, the rate of H2O2 production was twofold higher in the wild-type, but the overall production of H2O2 was again significantly higher in the Ubi- membranes. Inclusion of a water-soluble ubiquinone homologue (UQ-1) effectively decreased the amount of H2O2 produced in the Ubi- membranes in a concentration-dependent manner. Addition of UQ-2 to the membranes was even more effective in limiting accumulation of H2O2 than was UQ-1, suggesting a role for the side-chain in conferring liposolubility in the antioxidative defence mechanism. Intracellular H2O2 concentration was increased 1.8-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 CuSO4 or H2O2; 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.

Spatial and temporal organization of replicating Escherichia coli chromosomes.

The 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.

Recombination and chromosome segregation.

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.

A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered.

Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33.9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.

The aminoglycoside 6'-N-acetyltransferase type Ib encoded by Tn1331 is evenly distributed within the cell's cytoplasm.

The multiresistance transposon Tn1331, which mediates resistance to several aminoglycosides and beta-lactams, includes the aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1) genes. The nucleotide sequence of aac(6')-Ib includes a region identical to that of the bla(TEM-1) gene. This region encompasses the promoter and the initiation codon followed by 15 nucleotides. Since there were three possible translation initiation sites, the amino acid sequence at the N terminus of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was determined and was found to be SIQHF. This result indicated that aac(6')-Ib includes a translational fusion: the first five amino acids of the leader peptide of the TEM beta-lactamase are fused to the rest of the AAC(6')-Ib protein. This gene fusion could have formed during the genesis of Tn1331 as a consequence of the generation of a 520-nucleotide duplication (M. E. Tolmasky, Plasmid 24:218-226, 1990). An identical gene isolated from a Serratia marcescens strain has been previously described (G. Tran van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987). Extraction of the periplasmic proteins of E. coli harboring aac(6')-Ib by spheroplast formation showed that most of the AAC(6')-Ib protein is present in the cytoplasm. A genetic fusion to phoA confirmed these results. AAC(6')-Ib was shown to be evenly distributed inside the cell's cytoplasm by fluorescent microscopy with an AAC(6')-Ib-cyan fluorescent protein fusion.