Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A

Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low μM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1× MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

X-Ray and Ultraviolet Sensitivity of Synchronously Dividing Escherichia coli

A paper pile filtration technique was used to obtain synchronously dividing populations of E. coli strains B and B/r from cultures in the exponential growth phase. Three generations of highly phased cell division were obtained by rapid pressure filtration which selected approximately 1 per cent of the exponentially growing culture. The sensitivity of E. coli strain B to x-ray and UV inactivation as a function of the cell division cycle was determined on synchronous populations. E. coli strain B showed a sharp decrease in sensitivity to inactivation by both radiations in the middle of the division cycle, and a further decrease near the end of the cycle. The sensitivity of E. coli strain B/r to x-irradiation was also investigated. Only the mid-cycle decrease in sensitivity was found during the division cycle of this strain. It was concluded that the repetition of the observed sensitivity patterns in both strains through the first three cycles after synchronization indicates that the same basic sensitivity patterns are probably also present in the individual cells of an exponential phase culture.     

The activity of ribosome modulation factor during growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.     

Variation in Resistance of Natural Isolates of Escherichia coli O157 to High Hydrostatic Pressure, Mild Heat, and Other Stresses

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.     

Intracellular Steady-State Concentration of Integron Recombination Products Varies with Integrase Level and Growth Phase

IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attI × attI than attC × attI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.     

Inactivation of Escherichia coli and Listeria innocua in Milk by Combined Treatment with High Hydrostatic Pressure and the Lactoperoxidase System

We have studied inactivation of four strains each of Escherichia coli and Listeria innocua in milk by the combined use of high hydrostatic pressure and the lactoperoxidase-thiocyanate-hydrogen peroxide system as a potential mild food preservation method. The lactoperoxidase system alone exerted a bacteriostatic effect on both species for at least 24 h at room temperature, but none of the strains was inactivated. Upon high-pressure treatment in the presence of the lactoperoxidase system, different results were obtained for E. coli and L. innocua. For none of the E. coli strains did the lactoperoxidase system increase the inactivation compared to a treatment with high pressure alone. However, a strong synergistic interaction of both treatments was observed for L. innocua. Inactivation exceeding 7 decades was achieved for all strains with a mild treatment (400 MPa, 15 min, 20°C), which in the absence of the lactoperoxidase system caused only 2 to 5 decades of inactivation depending on the strain. Milk as a substrate was found to have a considerable effect protecting E. coli and L. innocua against pressure inactivation and reducing the effectiveness of the lactoperoxidase system under pressure on L. innocua. Time course experiments showed that L. innocua counts continued to decrease in the first hours after pressure treatment in the presence of the lactoperoxidase system. E. coli counts remained constant for at least 24 h, except after treatment at the highest pressure level (600 MPa, 15 min, 20°C), in which case, in the presence of the lactoperoxidase system, a transient decrease was observed, indicating sublethal injury rather than true inactivation.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.     

Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.     

The Indole Pulse: A New Perspective on Indole Signalling in Escherichia coli

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5–1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Influence of Apple Cultivars on Inactivation of Different Strains of Escherichia coli O157:H7 in Apple Cider by UV Irradiation

This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10⁶ to 10⁷ CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm². Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (α ≤ 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (α ≤ 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of °Brix, pH, and malic acid content failed to show any statistically significant relationship (R² ≥ 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested.     

Degradation of trichloroethylene by a growth-arrestedPseudomonas putida

A toluene-oxidizing strain ofPseudomonas mendocina KR1 containing toluene-4-mono-oxygenase (TMO) completely degrades TCE with the addition of toluene as a co-substrate in aerobic condition. In order to constructin situ bioremediation system for TCE degradation without any growth-stimulating nutrients or toxic inducers such as toluene, we used the carbon-starvation promoter ofPseudomonas putida MK1 (Kim, Y.et al., J. bacteriol., 1995). Upon entry into the stationary phase due to the deprivation of nutrients, this promoter is strongly induced without further cell growth. The TMO gene cluster (4.5 kb) was spliced downstream of the carbon starvation promoter ofPseudomona putida MK1, already cloned in pUC19. TMO under the carbon starvation promoter was not expressed inE. coli cells either in stationary phase or exponential phase. For TMO expression inPseudomonas strains,tmo and carbon starvation promoter region were recloned into a modified broad-host range vector pMMB67HES which was made from pMMB67HE (8.9 kb) by deletion oftac promoter andlacI ^q (about 1.5 kb). Indigo was produced by TMO under the carbon starvation promoter in aPseudomonas strain of post-exponential phase on M9 (0.2% glucose and 1mM indole) or LB. 18% of TCE was degraded in 14 hours after entering the stationary phase at the initial concentration of 6.6μ M in liquid phase.     

Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy

A small container of several to a few hundred µm³ (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of ~80 nm was found. In addition to this ‘80 nm fiber’, a thinner ‘40 nm fiber’ and a higher order ‘loop’ structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a ‘coral reef structure’ that also possessed the 80 nm fiber units, and, finally, into a ‘tightly compacted nucleoid’ that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.     

Effects of growth phase and repair capacity on rejoining of ethyl methanesulfonate-induced DNA breaks in Escherichia coli

The effects of growth phase and DNA repair capacity on the production and rejoining of ethyl methanesulfonate (EMS)-induced single-strand breaks were studied in 4 strains of E. coli. DNAs from logarithmic and stationary phase cells of the DNA polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇recA, and from the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃rec⁺ were examined by sedimentation in alkaline sucrose gradients.In both parental strains, stationary phase cells exhibited enhanced strand rejoining. In the mutants, alkylated DNA was repaired to some extent in both growth phases, but it contained a greater proportion of small DNA fragments compared to the parental strains. Some DNA breakdown occured in all four strains but this was most extensive in stationary phase cells of the repair-deficient mutants.These results indicate that the four strains can rejoin EMS-induced DNA strand breaks with varying efficiency depending on the physiological state and the genetic capacity for repair.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H₂O₂ compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Survival of Campylobacter jejuni during Stationary Phase: Evidence for the Absence of a Phenotypic Stationary-Phase Response

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39°C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50°C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.     

Stationary-Phase Quorum-Sensing Signals Affect Autoinducer-2 and Gene Expression in Escherichia coli

Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5α. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5α stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold ± 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.