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.     

Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.     

Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.

The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.     

Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN⁻) from thiocyanate (SCN⁻), using H₂O₂ as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN⁻.     

Proteins induced by high osmotic pressure in Escherichia coli

Abstract The protein composition of Escherichia coli grown under conditions of high and low osmotic pressure and following shifts from one condition to the other was examined by two-dimensional gel electrophoresis. Upon shift to high osmotic pressure there is a rapid induction of a group of proteins whose synthesis persists during prolonged growth at elevated osmotic pressure. An osmotic down-shift results in the repression of these proteins. Neither shift induced the heat shock regulon.     

Stress response of Escherichia coli to elevated hydrostatic pressure.

The response of exponentially growing cultures of Escherichia coli to abrupt shifts in hydrostatic pressure was studied. A pressure upshift to 546 atm (55,304 kPa) of hydrostatic pressure profoundly perturbed cell division, nucleoid structure, and the total rate of protein synthesis. The number of polypeptides synthesized at increased pressure was greatly reduced, and many proteins exhibited elevated rates of synthesis relative to total protein synthesis. We designated the latter proteins pressure-induced proteins (PIPs). The PIP response was transient, with the largest induction occurring approximately 60 to 90 min postshift. Fifty-five PIPs were identified. Many of these proteins are also induced by heat shock or cold shock. The PIP demonstrating the greatest pressure induction was a basic protein of 15.6 kDa. High pressure inhibits growth but does not inhibit the synthesis of stringently controlled proteins. Cold shock is the only additional signal which has been found to elicit this type of response. These data indicate that elevated pressure induces a unique stress response in E. coli, the further characterization of which could be useful in delineating its inhibitory nature.     

An SOS response induced by high pressure in Escherichia coli

Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore used in food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary. Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the first time that pressure induces a bona fide SOS response in E. coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA. Moreover, it was shown that pressure is capable of triggering lambda prophage induction in E. coli lysogens. The remnant lambdoid e14 element, however, could not be induced by pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response. Furthermore, the pressure-induced SOS response seems not to be initiated by DNA damage, since DeltarecA and lexA1 (Ind-) mutants, which are intrinsically hypersensitive to DNA damage, were not sensitized or were only very slightly sensitized for pressure-mediated killing and since pressure treatment was not found to be mutagenic. In light of these findings, the current knowledge of pressure-mediated effects on bacteria is discussed.     

Functional analysis of minichromosome replication: bidirectional and unidirectional replication from the Escherichia coli replication origin, oriC.

Replicating molecules of minichromosomes pCM959 and pOC24 were analyzed by electron microscopy. Replication of pCM959 proceeded bidirectionally from the replication origin, oriC, in about 60% of the molecules; the rest of the molecules replicated unidirectionally in either direction. pOC24, in which deoxyribonucleic acid to the right (clockwise) of the oriC segment is deleted, seemed to replicate predominantly unidirectionally counterclockwise from oriC.     

Induction of oxidative stress by high hydrostatic pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Electromechanical coupling model of gating the large mechanosensitive ion channel (MscL) of Escherichia coli by mechanical force.

We have developed a theoretical electromechanical coupling (EMC) model of gating of the large-conductance mechanosensitive ion channel (MscL). The model presents the first attempt to explain the pressure-dependent transitions between the closed and open channel conformations on a molecular level by assuming 1) a homohexameric structural model of the channel, 2) electrostatic interactions between various domains of the homohexamer, 3) structural flexibility of the N-terminal portion of the monomer, and 4) mechanically and electrostatically induced displacement of the N-terminal domain relative to other structural domains of the protein. In the EMC model, 12 membrane-spanning alpha-helices (six each of the M1 and M2 transmembrane domains of the MscL monomer), are envisaged to line the channel pore with a diameter of 40 A, whereas the N- and C-termini are oriented toward each other inside the pore when the channel is closed. The model proposes that stretching the membrane bilayer by mechanical force causes the monomers to be pulled away from and slightly tilted toward each other. This relative movement of alpha-helices could serve as a trigger to initiate a "swing-like" motion of the N-terminus around the glycine residue G14 that may act as a pivot. The analysis of the attractive and repulsive coulomb forces between all domains of the channel homohexamer suggested that an inclination angle of approximately 3.0 degrees - 4.1 degrees between the oppositely oriented channel monomers should suffice for the N-terminus to turn away from other domains causing the channel to open. According to the EMC model the minimal free energy change, deltaG, that could initiate the opening of the channel was 2 kT. Also, the model predicted that the negative pressure required for channel open probability, Po = 0.5, should be between 50 and 80 mmHg. These values were in a good agreement with the experimentally estimated pressures of 60-70 mmHg obtained with the MscL reconstituted in liposomes. Furthermore, consistent with a notion that the N-terminus may present a mechanosensitive structural element providing a mechanism to open the MscL by mechanical force, the model provides a simple explanation for the variations in pressure sensitivity observed with several MscL mutants having either deletions or substitutions in N- or C-terminus, or site-directed mutations in the S2-S3 loop.     

Superhelical Escherichia coli DNA: relaxation by coumermycin.

A class of compounds of the form: NH₂NHCO(CH₂)_nC(OR)₂CHR′X has been designed to allow selective blocking of specific genetic sequences of DNA and RNA (Summerton, unpublished data). This paper describes the synthesis and use of 6-bromo-5,5-dimethoxyhexanohydrazide for such site-specific inactivation. In model reactions it is shown that this compound can be attached to the C-4 position of cytidine and that, after activation, the cytidine-bound agent crosslinks to the N-7 position of guanosine. This reaction sequence has been applied to the crosslinking of bacteriophage T7 RNA to its complementary DNA in a highly specific fashion. The RNA is derivatized with the hydrazide reagent, activated, and incubated under annealing conditions with the complementary DNA, resulting in crosslinks between the two strands that are stable to denaturing conditions, dependent on the presence of the crosslinking agent, and specific for the complementary DNA sequence. These studies show that the title compound is a promising sequence-specific blocking agent for nucleic acids. The capability of introducing site-specific blocks in DNA and RNA in this way may have a wide variety of applications in the study of genetic processes. In particular, the combination of this compound with appropriate restriction fragments may enable systematic mapping and characterization of viral genomes.     

Reversible high hydrostatic pressure inactivation of phosphofructokinase from Escherichia coli.

Tetrameric Escherichia coli phosphofructokinase dissociates reversibly on incubation under hydrostatic pressures of 80 MPa and above, yielding inactive dimers and monomers. The transition is dependent upon enzyme concentration and presence of ligands. The substrate, D-fructose 6-phosphate, which bridges the intersubunit interface at the active site, produces a massive stabilization to pressure, whereas ATP, which binds to only one subunit, induces only a mild stabilization. Both the positive allosteric regulator, GDP, and the negative allosteric regulator, phosphoenolpyruvate, whose binding sites lie at the other subunit interface, produce an intermediate effect. Of these ligands, only ATP increases the rate of reactivation after depressurization.     

Recycling of murein by Escherichia coli.

The tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid [A2pm]), tetrapeptide (L-Ala-D-Glu-A2pm-D-Ala), and dipeptide (A2pm-D-Ala) which are shed by Escherichia coli from the murein sacculus were found to be reused by the cells to synthesize murein. The tripeptide was used directly, without degradation, to form UDP-N-acetylmuramyl-L-Ala-D-Glu-A2pm. The tetrapeptide lost its carboxy-terminal D-Ala, apparently in the periplasm, before being used. The dipeptide was degraded to D-Ala and A2pm before uptake.     

Formation of ethylene by Escherichia coli.

Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.     

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.     

Induction of Shiga toxin-converting prophage in Escherichia coli by high hydrostatic pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20 degrees C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Anaerobic iron uptake by Escherichia coli.

Assimilation and uptake of iron in anaerobic cultures of Escherichia coli were supported by iron supplied as ferrienterobactin, ferrichrome, and ferrous ascorbate; however, as in the aerobic cultures, ferrichrome A was a poor iron source. Albomycin inhibited both aerobically and anaerobically grown cells. The siderophore outer membrane receptor proteins FepA and FhuA were produced under anaerobic iron-deficient conditions. Anaerobic transport of ferrienterobactin and ferrichrome was inhibited by KCN and dinitrophenol. The Km for ferrienterobactin uptake in anaerobically grown cells was 0.8 microM, and the Vmax was 38 pmol/min per mg, compared with 0.1 microM and 80 pmol/min per mg, respectively, in aerobically grown cells.     

Predation of Escherichia coli by Colpoda steinii.

A study of single-stage chemostat cultures of Colpoda steinii. Escherichia coli, and glucose is reported here. Two levels of glucose were fed as the limiting nutrient to the chemostat cultures. The cultures were studied at three holding times. Oscillations developed at short holding time and damped oscillations developed a long-residence times that approached steady-state conditions of populations of C. steinii and E. coli and concentrations of glucose. The experimental data are fitted to and compared with Jost's model.     

Biphasic inactivation kinetics of Escherichia coli in liquid whole egg by high hydrostatic pressure treatments.

Kinetic studies on the isothermal high hydrostatic pressure (HHP) inactivation of Escherichia coli in liquid whole egg (LWE) were performed at 5 and 25 degrees C in the pressure range of 250-400 MPa. The characteristic tailing inactivation curves were described by a first-order biphasic model. As compared to a previous rheological study, it is suggested that the phase change of LWE during pressure treatment affects the inactivation rate of E. coli. Within the processing criteria where the rheological properties of LWE were still comparable to those of fresh LWE, HHP treatments at 5 degrees C induced more E. coli inactivations than those at 25 degrees C. From the results of approximately 3 log reductions of E. coli and over 5 log reductions of Pseudomonas and Paenibacillus, HHP treatment of LWE at 5 degrees C is regarded to be as effective as conventional thermal pasteurization. However, no post-process contamination and the consistency of temperature during preparation, HHP treatment, and storage provide clear processing advantages.