Phospholipid changes in seqA and dam mutants of Escherichia coli

SeqA and Dam proteins were known to be responsible for regulating the initiation of replication and to affect the expression of many genes and metabolisms. We have examined here the fatty acids composition and phospholipids membrane in dam and/or seqA mutants. The dam mutant showed an accumulation of the acidic phospholipids cardiolipin, whereas, the seqA mutant showed a higher proportion of phosphatidylglycerol compared with the wild-type strain. The seqA dam double mutant showed an intermediate proportion of acidic phospholipids compared with the wild-type strain. Based on these observations, we discuss the role of Dam and SeqA proteins in the regulation of phospholipids synthesis.     

Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage

SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two‐ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one‐ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus under‐replication, indicating inhibition of replication forks. Pre‐/post‐labelling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear‐ending into each other. We suggest that, in the absence of SeqA, the sister‐chromatid cohesion ‘safety spacer’ is destabilized and completely disappears if the replication fork is inhibited, leading to the segregation fork running into the inhibited replication fork and snapping the latter at single‐stranded DNA regions.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage λ forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage λ development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage λ to the dnaA seqA mutant cells is decreased at 0°?C , but not at 30°?C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for β-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants

Abstract The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.     

Differentiation between mutants of Escherichia coli K defective in oxidative phosphorylation.

Hybrid membrane particles from two mutants of Escherichia coli K12, Bv4 and K11, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant K11 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K11 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant Bv14 or to parental particles depleted of ATPase. Mutant Bv4 was found to be devoid of coupoing factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant K11 is impaired in respiration-driven amino acid transport, in contrast to mutant Bv4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largest subunit (alpha component) or against the intact catalytic subunits (alpha + beta components) inhibit both ATP-Pi exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (beta or gamma components) also inhibit these two reactions, but were found to be less effective. Mutant N144, which lacks ATPase activity, shows no precipitin lines with anti-alpha, anti-beta, anti-gamma, or anti (alpha + beta) preparations. In contrast, mutants Bv4 and K11, exhibit cross-reactivity with all of the antisera.     

Oxidative phosphorylation in intact chl-r mutants of Escherichia coli K 12.

The efficiency of oxidative phosphorylation was estimated in intact resting cells of Escherichia coli K 12, strain PA 601 (chl-s) and its chl-r mutants, all of them grown anaerobically in the presence of nitrate. The oxidation of endogenous NADH in intact chl-s cells was accompanied by the formation of ATP whatever the terminal electron acceptor, oxygen or nitrate, so that it was possible to conclude that the energy conservation sites are operating with either of the two acceptors in cells grown anaerobically in the presence of nitrate. For chl-r mutants oxidation of endogenous NADH correlated with ATP-production was found only with oxygen as electron acceptor. It is concluded that the energy-conservation sites are preserved in these mutants, the nitrate respiratory chain of which is altered. This assumption is corroborated by the effects of uncouplers of oxidative phosphorylation on ATP-synthesis.     

Aggregation deficient Escherichia coli K-12 female mutants with altered lipopolysaccharide.

Two F- mutants deficient in conjugation with F-donors have been characterized. They map at about 83 minut position, show resistance to T3 and T7 bacteriophages, and form mating aggregates in the liquid medium with lowered efficiency. Mutants have no detectable alterations in their outer membrane protein composition.     

Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression.     

Bacteriophage-resistant mutants of Escherichia coli K12. Location of receptors within the lipopolysaccharide.

A series of mutants of Escherichia coli K12 resistant to lipopolysaccharide (LPS)-specific bacteriophages were isolated, and examined with regard to their general properties, phage typing, chemical analysis of their LPS, and genetic analysis. Fourteen classes of mutants were distinguished on the basis of phage typing and sensitivity to bile salts. Three of the mutant classes are sensitive to phages to which the parent is resistant. Mutants which are sensitive to bile salts generally lack heptose in their LPS, but two mutant classes are exceptions to this rule. Analyses of the sugars in the purified LPS of all mutant classes indicated that mutants were obtained which are blocked at most stages in core polysaccharide synthesis. On the basis of the chemical analysis, in conjunction with phage typing data and other known properties of the mutants, it is deduced which residue(s) is involved as a receptor for each of the phages used and which residues hinder these receptors. Some of the mutant classes do not seem to be changed in their LPS structure. Many of the mutations map in or near the rfa locus, but some are far removed from this region.     

Flagellar assembly mutants in Escherichia coli

Genetic and biochemical analysis of mutants defective in the synthesis of flagella in Escherichia coli revealed an unusual class of mutants. These mutants were found to produce short, curly, flagella-like filaments with low amplitude ( approximately 0.06 mum). The filaments were connected to characteristic flagellar basal caps and extended for 1 to 2 mum from the bacterial surface. The mutations in these strains were all members of one complementation group, group E, which is located between his and uvrC. The structural, serological, and chemical properties of the filament derived from the mutants closely resemble those of the flagellar hook structure. On the basis of these properties, it is suggested that these filaments are "polyhooks", i.e., repeated end-to-end polymers of the hook portion of the flagellum. Polyhooks are presumed to be the result of a defective cistron which normally functions to control the length of the hook region of the flagellum.     

Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12.

Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.