Dental amalgam and antibiotic resistance--an association?
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Bacteria↗
Biomedical subjects
R J Rowbury
Publications and source records attributed to R J Rowbury.
Induction of the PhoE porin by NaCl as the basis for salt-induced acid sensitivity in Escherichia coli.
Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol l-1 added NaCl. Salt-induced acid sensitivity only occurs in relA+ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE+ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac- strain carrying pJP102 (phoE-lacZ) produced low levels of beta-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased beta-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA+ strains. Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.
Escherichia coli↗
Exposure of Escherichia coli to acid habituation conditions sensitizes it to alkaline stress.
Escherichia coli transferred from pHo 7.0 to pHo 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response. Alkali sensitization was reduced at pHo 5.0 and virtually abolished at pHo 6.5. The response was triggered by cytoplasmic rather than external or periplasmic acidification and de novo protein synthesis was needed. Alkali sensitivity failed to appear at pHo 5.5 plus DNA gyrase inhibitors and was markedly reduced by himA, himD, hns, ompC and nhaA lesions. A tonB deletion mutant showed alkali sensitivity at pHo 7.0. Alkali sensitivity induction was not subject to catabolite repression nor was it appreciably affected by a relA lesion. Acid-induced cells were more sensitive to alkali damage to both DNA and beta-galactosidase and to alkali inhibition of beta-galactosidase induction. Alkali sensitization induced at pHo 5.5 may involve NhaB loss.
Alkalies↗
Regulatory aspects of alkali tolerance induction in Escherichia coli.
Escherichia coli shifted from external pH (pH(O)) 7.0 to pH(O) 8.5-9.5 rapidly becomes tolerant to pH(O) 10.0-11.5, induction of tolerance (alkali habituation) being dependent on periplasmic or external alkalinization with either NaOH or KOH. Induction needs protein synthesis and makes organisms resistant to DNA damage by alkali and better able to repair any damage that occurs. Induction of tolerance was reduced by glucose (not reversed by cAMP) and by amiloride, was dependent on DNA gyrase and was abolished by fur and himA lesions (the latter suggests IHF involvement). Tolerance induction was not prevented by L-leucine, FeCl3 or FeSO4 nor by hns or relA mutations. Habituation probably involves attachment of IHF upstream of the promoter leading to DNA bending which switches on transcription. Habituation is aberrant in nhaA mutants, so ability to resist alkali damage may only arise if NhaA is induced, with extrusion of Na+ by this antiporter during alkali challenge. In accord with one tolerance component involving NhaA induction, beta-galactosidase formation from nhaA-lacZ fusions at pH(O) 9.0 was inhibited by glucose and amiloride.
DNA Damage↗
Sensitization to acid induced by sodium ions in Escherichia coli: dependence of (p)ppGpp and cAMP and suppression of the relA-associated defect by mutations in envZ.
NaCl-induced acid sensitivity is not a response to high osmotic pressure but is triggered by a high internal sodium ion concentration, evidenced by the finding that conditions which enhance Na+ influx or reduce Na+ efflux, led to increased internal Na+ and allowed induction at lower external sodium ion concentrations and that induction could occur with no osmotic upshock. NaCl-induced acid sensitivity was not observed in Escherichia coli strain MC4100 and its relA lesion was investigated as a possible cause. Transformation of this strain and two other relA mutants to relA+ allowed sensitization by NaCl and whereas strain CF1648 (relA+) was sensitized, its relA deletion derivative was not. Additionally, transduction of two relA+ strains to relA produced derivatives which were not sensitized by salt. Although MC4100 was not sensitized by NaCl, envZ derivatives of it were sensitized. Lesions in fur and tonB did not prevent sensitization by NaCl, although extent of sensitization was slightly increased by fur and considerably increased by tonB. Acetate strongly inhibited sensitization, which was also subject to glucose repression, reversible by cAMP. The possibility was discussed that (p)ppGpp and cAMP both positively affect transcription of the genes encoding the acid sensitization components; in accord with this, high concentrations of cAMP suppressed the effect of the relA lesion of sensitization. Salt-induced organisms are more sensitive to acid damage to DNA, to acid inhibition of enzyme synthesis and DNA transfer and slightly less able to repair acid-damaged transforming DNA.
Bacterial Outer Membrane Proteins↗
Increased resistance of Escherichia coli to acrylic acid and to copper ions after cold-shock.
The effects of cold-shock on the resistance of plasmid-free and plasmid-carrying Escherichia coli to acrylate and copper ions have been tested. Such shock, produced by transfer from 37 to 5 degrees C, with 60 min incubation at the lower temperature, significantly enhanced the resistance of all the tested strains to both inhibitors. Such resistances may have arisen because the inhibitory agents are less able, due to porin changes, to penetrate into the organisms after cold-shock. It is more likely, however, that inhibitor penetration is unaffected but that cold-shocked organisms are better able to repair the damage caused (e.g. to membranes, DNA or cellular enzymes) by the inhibitors.
Acrylates↗
Tolerance to acid in pH 5.0-grown organisms of potentially pathogenic gram-negative bacteria.
A wide range of potentially pathogenic species of Gram-negative bacteria were far more resistant to extreme acidity (pH 2.0-3.5) when cultured at pH 5.0 (habituated to acid) than after pH 7.0 culture. The differences were particularly great for Citrobacter spp., Enterobacter spp., Klebsiella spp. and for Vibrio parahaemolyticus; substantial habituation was also observed for Proteus mirabilis and Aeromonas formicans but the effect was less marked for Serratia marcescens and Acinetobacter calcoaceticus. Growth at pH 5.0 was substantially poorer than at pH 7.0 for most of the above species and also for Salmonella typhimurium and Salm. enteritidis but phosphate markedly enhanced growth at pH 5.0 for many of these species without affecting growth at pH 7.0.
Buffers↗
An assessment of environmental factors influencing acid tolerance and sensitivity in Escherichia coli, Salmonella spp. and other enterobacteria.
Environmental factors such as temperature, pH and nutrient level affect enterobacterial acid sensitivity, as do the presence of phosphate and Na+ and the extent of aeration. The mechanisms governing these effects are partially understood and the involvement of phoE, fur and atp in acid tolerance, of phoE, envZ, tonB, (p)ppGpp and cAMP in salt-induced acid sensitivity and of rpoS in stationary-phase acid tolerance are of particular interest. It should be noted that surface attachment enhances acid resistance.
Acids↗
Salmonella enteritidis phage type 4 isolates more tolerant of heat, acid, or hydrogen peroxide also survive longer on surfaces.
In a comparative study of different Salmonella enteritidis phage type 4 isolates we found that those isolates with enhanced heat tolerance also survived better than isolates that were heat sensitive either at pH 2.6, in 10 mM H2O2, or on surfaces. Culture to the stationary phase increased the heat tolerance of all isolates and the acid and H2O2 tolerance of heat-tolerant isolates. With heat-sensitive isolates, however, extended culture had no impact on survival in H2O2 and only a marginal impact on acid tolerance. The growth phase had no appreciable impact on the surface survival of any of the isolates.
Animals↗
Sodium chloride induces an NhaA/NhaR-independent acid sensitivity at neutral external pH in Escherichia coli.
Escherichia coli previously grown in low-salt broth, pH 7.0, produced organisms which were markedly more acid sensitive when subsequently cultured in the same broth with 200 mM or more salt (NaCl) added. Induction of acid sensitivity occurred rapidly at both 37 and 30 degrees C, with a substantial effect within 15 min. Sensitization was partially inhibited by chloramphenicol and tetracycline and may depend on both protein synthesis-dependent and -independent physiological changes in the NaCl-induced organisms; sensitization did not result from osmotic shocking on transfer to challenge medium. Induction of acid sensitivity was affected by neither the sodium ion pore inhibitor amiloride nor the DNA synthesis inhibitor nalidixic acid; rifampin had a small effect, similar to that of chloramphenicol. Chlorides of other monovalent cations, especially Li+ and NH4+, also produced sensitization to acid, although CsCl was ineffective but did not interfere with sensitization by NaCl. Other sodium salts were also active as sensitizers, as were chlorides of divalent cations, but although sucrose (but not glycerol) was a good inducer, the results were not fully in accord with triggering of induction solely by the NaCl-associated increase in osmotic pressure. Sensitization was not prevented by deletion of the nhaA, nhaR, or nhaB gene. Acid sensitivity of NaCl-induced cells was slightly reduced after 90 min of growth at 37 degrees C in low-salt broth but was completely lost after 240 min. For NaCl-induced cells, acid killing in challenge media was not inhibited by amiloride. The NaCl-induced sensitization is distinct from the phenomenon of acid sensitivity induction in E. coli at alkaline external pH.
Bacterial Proteins↗
Acid pH responses of Escherichia coli: inhibition of colicin V synthesis and activity at pH 5.0.
On solid medium, streaks of ColV+ Escherichia coli strains produced much larger inhibitory zones on sensitive indicators at pH 7.0 and 9.0 than at pH 5.0. Such an acid pH effect also occurred in liquid medium, with sensitive strains being killed by ColV+ ones at pH 7.0 or 9.0 but not at pH 5.0. Colicin V was responsible for the killing effect. Culture filtrates from PAP1401 (ColV+) were used to examine the basis for the pH effect and it was found that acid pH interfered directly or indirectly with colicin V production but that colicin V was not irreversibly inactivated at pH 5.0. Additionally, colicin V formed at pH 7.0 was less active when tested on sensitive strains at pH 5.0 than at pH 7.0. Neither reduced colicin synthesis nor its reduced activity can be attributed to increased availability of iron at pH 5.0.
Colicins↗
PhoE porin of Escherichia coli and phosphate reversal of acid damage and killing and of acid induction of the CadA gene product.
The lethal effects of inorganic acid on phoE+ Escherichia coli strains, grown at neutral pHo, were enhanced by chloramphenicol, apparently because some organisms acquire acid tolerance (habituate) during challenge and chloramphenicol stops this. Phosphate (and/or polyphosphate) present during challenge prevented killing and damage by acid to outer membranes, DNA and cellular enzymes but did not prevent acid pHo enhancing novobiocin activity. To reverse acid effects, phosphate must interact with or cross the outer membrane but need not enter the cytoplasm; it is probable that it competes with H+ (or protonated anions) for passage through the PhoE pore. Phosphate also prevented induction of beta-galactosidase in a strain with the cadA promoter fused to lacZ. Four unc mutants showed essentially normal acid sensitivity and habituation; the same was true for strains with lesions in fur, oxyR, katF, phoP, cadA and hycB. In contrast, deletion of rpoH led to slightly increased acid sensitivity for cells grown at pHo 7.0, although habituation was relatively normal.
Acids↗
Effects of temperature shift on acid and heat tolerance in Salmonella enteritidis phage type 4.
The transfer of cells of Salmonella enteritidis phage type 4 from 20 to 37-46 degrees C resulted in marked increases in acid and heat tolerance. The former was maximized within 5 to 15 min of the shift and was largely independent of protein synthesis. In contrast, induction of increased heat tolerance was slower, requiring more than 60 min to be completed, and was prevented by inhibition of protein synthesis. When cells were transferred to medium at temperatures between 47 and 50 degrees C, the kinetics of induction of heat tolerance were essentially the same as at the lower temperatures. In contrast, the cells became more acid sensitive. The results of these studies clearly show that although both acid and heat resistance can be enhanced by preexposure to high incubation temperatures, the mechanisms involved are different.
Acids↗
The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli.
Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0. Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH. The habituation sensor does not detect increased delta pH (or decreased delta psi) nor an increased difference between pHo and periplasmic pH but probably detects a fall in either external or periplasmic pH. Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions. Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant. Strains derepressed for phoE, in contrast, showed increased acid sensitivity. These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions. Stimulation by phosphate of the poor growth of E. coli at pH 5.0 is in accord with the above. Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pHi near to neutrality at an acidic pHo level.
Adaptation, Physiological↗
Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli.
A study of the conjugal transfer of ColV,I-K94 tn 10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.
Conjugation, Genetic↗
Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.
Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.
Adaptation, Physiological↗
RecA-independent resistance to irradiation with u.v. light in acid-habituated Escherichia coli.
Growth of Escherichia coli 1829 ColV, I-K94 at pH 5.0 led to an increase in u.v. resistance compared with cells grown at pH 7.0. This was due to a phenotypic change, since organisms grown at pH 7.0 showed increased resistance after only 2.5-5.0 min incubation at the mildly acid pH. Other E. coli K12 derivatives became more u.v.-resistant at pH 5.0 including uvrA, recA and polA1 mutants. Organisms grown at pH 5.0 also showed increased Weigle reactivation of u.v.-irradiated lambda phage and this applied to the repair-deficient mutants as well as the parent strains. Both the increased u.v. resistance of acid-habituated cells and their increased ability to bring about Weigle reactivation appear to involve RecA-independent processes and are presumably, therefore, independent of the SOS response.
DNA Repair↗
Suppression by the ColV,I-K94 plasmid of inhibitor sensitivities in ompA mutants of Escherichia coli.
A series of ompA mutants derived from Escherichia coli K12 strains showed increased sensitivity (compared with the ompA+ parents) to aminoglycoside antibiotics and to other cationic agents including polymyxin B. One tested mutant also showed increased sensitivity to nafcillin and fusidic acid, but not to the hydrophilic ampicillin. All these inhibitor sensitivities in the ompA mutants were suppressed by ColV, I-K94 and by certain other ColV plasmids, but not by any of the other tested large plasmids. Suppression correlated with the production of the VmpA protein, but transfer and colicin components were not needed for suppression. Further comparison of the ompA and vmpA genes and their products was made and it indicated that there is little if any homology between the genes, that the synthesis of their products is regulated by quite different mechanisms, and that regions of these gene products exposed at the cell surface show different susceptibility to protease attack after denaturation.
Bacterial Outer Membrane Proteins↗