Chilling cells enhances the bactericidal action of fatty acids on Escherichia coli.

Preincubation at 0 C considerably increased the bactericidal action of 0.4% nonanoic and decanoic acids on Escherichia coli K-12 154. This lethal effect seemed to be dependent on the media used to grow the bacteria. Stationary-phase cells were more sensitive than those from exponential cultures. A mutant (FA31) resistant to the bactericidal action of "cold shock" and 0.4% deconoic acid was isolated from E. coli FA23 (AN E. coli 154 derivative able to grow on 0.1% decanoic acid) by a recycling selection procedure. Other E. coli strains tested showed behavior similar to that of strain K-12 154. The chilling of cells as a tool to improve the bactericidal action of fatty acids in foods is discussed.     

β-Oxidation-mediated resistance ofEscherichia coli to inhibition by long-chain fatty acids

Decanoate exerted a stronger bactericidal action onEscherichia coli K76 (-oxidation negative) than on its -oxidation constitutive parent,E. coli K113; both strains had a normal lipopolysaccharide layer.E. coli RC59, a mutant carrying a defective lipopolysaccharide layer, was more sensitive than its parent,E. coli O111B4, to the growth inhibition by decanoate. Oleate did not affectE. coli RC59 viability, but washing of the cells rendered them sensitive.E. coli RC59 was unable to degrade decanoate and oleate. A mutant of this strain was isolated (E. coli ER20) which retained the defective lipopolysaccharide but was able to metabolize oleic acid. When the killing effect of oleate was assayed on washed cells ofE. coli O111B4, RC59, and ER20, a positive correlation between -oxidation activity and the number of surviving viable cells was found. The ability of Gram-negative organisms to withstand the toxic effect of fatty acids was attributed to the presence of a lipolysaccharide (LPS) layer in the cell envelope. An additional mechanism of resistance is suggested by the results in this report which show that the ability to oxidize fatty acids contributes to resistance.     

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

Modulation of cell wall synthesis by DNA replication in Escherichia coli during initiation of cell growth.

Resting cells of Escherichia coli are able to initiate growth and murein biosynthesis in the presence of beta-lactam antibiotics binding to penicillin-binding proteins (PBPs) 1a and 1b (E. J. de la Rosa, M. A. de Pedro, and D. Vázquez, Proc. Natl. Acad. Sci. USA 82:5632-5635, 1985). Under these conditions, cells elongate normally until they approach the first doubling in mass, the time at which cell lysis starts. Assuming that coupling between DNA replication and cell division both in cells starting growth and in growing cells is essentially similar, triggering of the lytic response in the beta-lactam-treated cells coincides with the termination of the first round of DNA replication. This coincidence suggests that both events are interrelated. We investigated this possibility by studying the initiation of growth in cultures of wild-type strains and in cell division mutants treated with beta-lactams inhibiting PBPs 1a and 1b and with the DNA replication inhibitor nalidixic acid. Addition of nalidixic acid, even late in the first cell cycle, prevented the lytic response of the cells to the blockade of PBPs 1a and 1b. The effect of nalidixic acid is more likely due to its action on DNA replication itself than to its indirect inhibitory effect on cell division or to its ability to induce the SOS system of the cell. These observations favor the idea that the cell wall biosynthetic machinery might be modulated by DNA replication at precise periods during cell growth.     

Proximity-dependent inhibition in Escherichia coli isolates from cattle

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-μm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.     

Specific inhibition of cell division by colicin E 2 without degradation of deoxyribonucleic acid in a new colicin sensitivity mutant of Escherichia coli

A new class of colicin sensitivity mutants of Escherichia coli was isolated whose cell division was specifically inhibited by colicin E(2) without detectable degradation of deoxyribonucleic acid (DNA) at 30 C. The mutant could not form colonies in the presence of colicin E(2) but recovered colony-forming ability by trypsin treatment even after prolonged incubation with the colicin. Addition of colicin E(2) to the exponentially growing mutant inhibited cell division completely but did not induce degradation of DNA into cold acid-soluble materials nor any breakage of DNA strands. Synthesis of DNA in the mutant was not inhibited, and long filamentous cells with multiple nuclear bodies were formed by the action of colicin E(2). Degradation of ribosomal ribonucleic acid and development of prophage lambda, both of which were induced by colicin E(2) in the sensitive cells, did not occur in the mutant. At the elevated temperature, however, the mutant was found to undergo colicin-induced degradation of DNA. No differences in ultraviolet light nor drug sensitivities were observed in the mutant compared to the parent E. coli. The data suggested that colicin E(2) had a specific inhibitory effect on cell division of E. coli that was not a consequence of DNA degradation.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: solubilization of Escherichia coli peptidoglycan.

During penetration of Bdellovibrio bacteriovorus into Escherchia coli, two enzymatic activities, a glycanase and a peptidase, rapidly solubilized some 10 to 15% of the E. coli peptidoglycan. The glycanase activity, which solubilizes peptidoglycan amino sugars, came to a sharp halt with completion of the penetration process. Peptidase activity, which cleaves diaminopimelic acid residues from the peptidoglycan, continued, but at a decreasing rate. By 90 min after bdellovibrio attack, some 30% of the initial E. coli diaminopimelic acid residues were solubilized and present in the culture fluid as free diaminopimelic acid. During bdellovibrio penetration some 25% of the lipopolysaccharide glucosamine was also solubilized by an as yet undefined enzymatic activity that yielded products having molecular weights below 2,000. The solubilization of E. coli lipopolysaccharide glucosamine also terminated at completion of bdellovibrio penetration. At the end of bdellovibrio growth, a second period of rapid solubilization of bdelloplast peptidoglycan began which resulted in lysis of the bdelloplast and complete solubilization of the peptidoglycan amino sugars and diaminopimelic acid. The final lytic enzyme(s) was synthesized just before the time of lysis.     

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

The beta-oxidation systems of Escherichia coli and Salmonella enterica are not functionally equivalent

Based on its genome sequence, the pathway of beta-oxidative fatty acid degradation in Salmonella enterica serovar Typhimurium LT2 has been thought to be identical to the well-characterized Escherichia coli K-12 system. We report that wild-type strains of S. enterica grow on decanoic acid, whereas wild-type E. coli strains cannot. Mutant strains (carrying fadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. The S. enterica fadR strains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast, E. coli fadR strains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions, E. coli failed to grow, whereas S. enterica grew well. Exchange of the wild-type fadR genes between the two organisms showed this to be a property of S. enterica rather than of the FadR proteins per se. This difference in growth was attributed to S. enterica having higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid. S. enterica cleanly converted all of the acid to acetyl-CoA, whereas E. coli accumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that the S. enterica FadE and FadBA enzymes were responsible for the greater efficiency of beta-oxidation relative to that of E. coli.     

Inhibitory effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina, on growth of Escherichia coli

The effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly), on Escherichia coli was investigated. Sarcotoxin IIA was found to have a bacterial effect on growing bacteria, but little on non-growing bacteria. At a concentration of 25 micrograms/ml, it induced significant morphological change of growing E. coli cells. In its presence, growing cells became greatly elongated, and spheroplast-like bulges and projections appeared on their surface. A rough mutant strain of E. coli with a defect in the structure of lipopolysaccharide was more sensitive than the parent strain to sarcotoxin IIA. These results suggest that the main effect of sarcotoxin IIA is to inhibit cell wall synthesis, including septum formation.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.     

Alteration of Escherichia coli murein during amino acid starvation.

We have studied the mechanisms by which amino acid starvation of Escherichia coli induces resistance against the lytic and bactericidal effects of penicillin. Starvation of E. coli strain W7 of the amino acids lysine or methionine resulted in the rapid development of resistance to autolytic cell wall degradation, which may be effectively triggered in growing bacteria by a number of chemical or physical treatments. The mechanism of this effect in the amino acid-starved cells involved the production of a murein relatively resistant to the hydrolytic action of crude murein hydrolase extracts prepared from normally growing E. coli. Resistance to the autolysins was not due to the covalently linked lipoprotein. Resistance to murein hydrolase developed most rapidly and most extensively in the portion of cell wall synthesized after the onset of amino acid starvation. Lysozymes digests of the autolysin-resistant murein synthesized during the first 10 min of lysine starvation yielded (in addition to the characteristic degradation products) a high-molecular-weight material that was absent from the lysozyme-digests of control cell wall preparations. It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of murein hydrolase molecules is inhibited. The mechanism may involve some aspects of the relaxed control system since protection against penicillin-induced lysis developed much slower in amino acid-starved relaxed controlled (relA) cells than in isogenic stringently controlled (relA+) bacteria.     

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.     

Comparison of the glutamate-, arginine- and lysine-dependent acid resistance systems in Escherichia coli O157:H7

AIMS: The objective of this study was to investigate the effect of growing conditions on the glutamate-, arginine- and lysine-dependent acid resistance (AR) systems of Escherichia coli O157:H7. METHODS AND RESULTS: Seven E. coli O157:H7 strains were grown in five different media at neutral or acidic pH under aerobic or anaerobic conditions, and the survival rate after acid shocks (pH 2.0, 1 h, 37 degrees C) in the presence of glutamate, arginine and lysine was determined. Six strains induced the glutamate-dependent AR at stationary phase, and maximal survival were observed (> or =10%) when grown in pH 5- Luria-Bertani media with glucose (LBG) and in pH 4.5-anaerobic media. The arginine- and lysine-dependent systems were also present, but were only induced if cells had grown in LBG. For strain ATCC 43895, the minimum glutamate concentration that resulted in at least 10% survival rate was 10 micromol l(-1), but it required at least 10-fold more arginine and lysine. CONCLUSIONS: The lysine-dependent AR system could be as important as the arginine-mediated one, but the contribution of both systems to E. coli O157:H7 overall AR response might be minor compared with the glutamate-dependent system. SIGNIFICANCE AND IMPACT OF THE STUDY: Under typical environmental conditions, the glutamate-dependent AR system might be solely responsible for protecting cells against acidic pH.     

Bacteriolytic effect of cessation of glucosamine supply, induced by specific inhibition of glucosamine-6-phosphate synthetase

The antibiotic tetaine (bacilysin) and its C-terminal epoxyaminoacid--anticapsin--are powerful inhibitors of glucosamine-6-phosphate synthetase (EC 5.3.1.19.) in cell-free extracts of Escherichia coli K-12. Tetaine acts on growing cells as a bactericidal agent. This bactericidal action, measured from 10 to 160 muM concentration, is a consequence of the induction of lysis of growing cells. The induction of lysis by tetaine is compared with the lytic action of some beta-lactams. Hypertonic medium, destruction of the antibiotic, presence of chloramphenicol or the addition of N-acetylglucosamine protect E. coli K-12 cells against lysis induced by tetaine. These effects are compared with those observed in the presence of penicillin G. The results indicate that inhibition of early or late stages of peptidoglycan synthesis all result in more or less the same consequence, i.e. death via cell lysis.     

Lipopolysaccharide Layer Protection of Gram-Negative Bacteria Against Inhibition by Long-Chain Fatty Acids

Growth, amino acid transport, and oxygen consumption of Escherichia coli and Salmonella typhimurium are inhibited by short-chain (C(2)-C(6)) but not by medium or long-chain fatty acids (C(10)-C(18)) at concentrations at which these processes are completely inhibited in Bacillus subtilis. The resistance of gram-negative organisms is not correlated with their ability to metabolize fatty acids, since an E. coli mutant unable to transport oleic acid is still resistant. However, mutants of both E. coli and S. typhimurium in which the lipopolysaccharide layer does not contain the residues beyond the 2-keto-3-deoxyoctonate core are inhibited by medium (C(10)) but not by long-chain (C(18)) fatty acids. Furthermore, removal of a portion of the lipopolysaccharide layer by ethylenediaminetetraacetate treatment renders the organisms sensitive to medium and partially sensitive to long-chain fatty acids. The intact lipopolysaccharide layer of gram-negative organisms apparently screens the cells against medium and long-chain fatty acids and prevents their accumulation on the inner cell membrane (site of amino acid transport) at inhibitory concentrations. These results are relevant to the use of antimicrobial food additives, and they allow the characterization of gram-positive versus gram-negative bacteria and their lipopolysaccharide mutants.     

Induction of complement sensitivity in Escherichia coli by citric acid and low pH

AIMS: The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested. METHODS AND RESULTS: E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics. CONCLUSION: Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The elucidation of the mechanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies.     

Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, identified as a compatible solute in moderately halophilic bacteria. Exogenously provided ectoine was found to stimulate growth of Escherichia coli in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte or nonelectrolyte. It is accumulated in E. coli cells proportionally to the osmotic strength of the medium, and it is not metabolized. Its osmoprotective ability was as potent as that of glycine betaine. The ProP and ProU systems are both involved in ectoine uptake and accumulation in E. coli. ProP being the main system for ectoine transport. The intracellular ectoine pool is regulated by both influx and efflux systems.