Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility of a starch-utilizing Flavobacterium at low substrate concentrations.

A starch-utilizing yellow-pigmented bacterium, isolated from tap water, was tested for the utilization of 64 natural compounds at a concentration of 1 g/liter by measuring colony growth on agar media. Only 12 carbohydrates and glycerol promoted growth. Growth experiments with the organism in pasteurized tap water supplied with mixtures of substrates at concentrations of 1 or 10 micrograms of C of each substrate per liter, followed by separate experiments with a number of carbohydrates at 10 micrograms of C per liter showed that of these 64 natural compounds only sucrose, maltose, raffinose, starch, and glycerol promoted growth at very low concentrations. Also maltotriose, -tetraose, -pentaose, -hexaose, and stachyose, which were not included in the mixtures, enhanced growth, and generation times of 3 to 5 h at 10 micrograms of C per liter were observed. The organism, which was tentatively identified as a Flavobacterium species, thus appeared to be highly specialized in the utilization of glycerol and a number of oligo- and polysaccharides at very low concentrations.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species.

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Growth of Aeromonas hydrophila at Low Concentrations of Substrates Added to Tap Water

The ability of an Aeromonas hydrophila isolate obtained from filtered river water to grow at low substrate concentrations was studied in batch experiments with tap water supplied with low concentrations of substrates. Growth was assessed by colony count determinations. The isolate only multiplied in the used tap water (2 to 3 mg of dissolved organic carbon per liter) after the addition of a small amount of an assimilable carbon compound. d-Glucose especially caused growth of the organism even at initial concentrations below 10 mug of C per liter. At initial glucose concentrations below the K(s) value (12 mug of C per liter), generation times and yield (colony-forming units per milligram of substrate-C) were nonlinear with 1/initial glucose concentrations and initial glucose concentrations, respectively. From these observations, the maintenance coefficient m was calculated (m = 0.015 mg of glucose per mg [dry wt] per h at 12 degrees C). At initial concentrations below the K(s) value of starch (73 mug of C per liter), no growth was observed, but complete use of starch occurred in these situations after the addition of 10 mug of glucose-C per liter. The results of this study show that information of ecological significance may be obtained by very simple batch experiments. Moreover, the isolate studied may be used in growth experiments to assess the maximum concentration of glucose which might be present in water, particularly tap water.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate Utilization by an Oxalate-Consuming Spirillum Species in Relation to Its Growth in Ozonated Water

The nutritional versatility of a vibrio-shaped, oxalate-utilizing isolate, strain NOX, obtained from tap water supplied with low concentrations of formate, glyoxylate, and oxalate, was determined by growth experiments with low-molecular-weight carbon compounds at high (grams per liter) and very low (micrograms per liter) concentrations. The organism, which was identified as a Spirillum species, appeared to be specialized in the utilization of a number of carboxylic acids. Yields of 2.9 × 10⁶ CFU/μg of oxalate C and 1.2 × 10⁷ CFU/μg of acetate C were obtained from growth experiments in tap water supplied with various low amounts of either oxalate or acetate. A substrate saturation constant of 0.64 μM oxalate was calculated for strain NOX from the relationship between growth rate and concentration of added oxalate. Maximum colony counts of strain NOX grown in ozonated water (dosages of 2.0 to 3.2 mg of O₃ per liter) were 15 to 20 times larger than the maximum colony counts of strain NOX grown in water before ozonation. Based on the nutritional requirements of strain NOX, it was concluded that carboxylic acids were produced by ozonation. Oxalate concentrations were calculated from the maximum colony counts of strain NOX grown in samples of ozonated water in which a non-oxalate-utilizing strain of Pseudomonas fluorescens had already reached maximum growth. The oxalate concentrations obtained by this procedure ranged from 130 to 220 μg of C/liter.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Utilization of Low Concentrations of Starch by a Flavobacterium Species Isolated from Tap Water

Experiments in well-cleaned glass flasks revealed that addition of starch in concentrations of 10 and 25 μg of substrate C per liter to the filtrate of slow sand filters stimulated the development of a yellow-pigmented bacterium which was identified as a Flavobacterium species. The isolate was able to multiply in tap water without substrates added, but addition of starch and glucose in amounts as low as 1 μg of substrate C per liter clearly enhanced growth. The substrate affinities of the Flavobacterium for these compounds were 3.9 μg of starch C and 3.3 μg of glucose C per liter. The results of this study indicate that microorganisms which rapidly utilize starch at a level of a few micrograms per liter commonly occur in water.     

Multiplication of fluorescent pseudomonads at low substrate concentrations in tap water

Two fluorescent pseudomonads, strains P17 and P500, belonging to different biotypes were tested for growth in tap water supplied with different concentrations of acetate and glutamate, low concentrations (10 and 20 g of C per liter) of various other substrates and mixtures of related substrates, the latter being present in amounts of 1 g of C per liter each. Amino acids appeared to be excellent substrates for both isolates, but many other substrates were utilized at very low concentrations as well. Saturation constants (K_s) of P17 with acetate, arginine, aspartate, glutamate, lactate, succinate, malonate, p-hydroxybenzoate and glucose were all below 1 m. The K_s values of strain P500 were about 5 times larger than those of P17. Since especially P17 is able to use a large number of different substrates at low concentrations, assessment of maximal colony counts of this organism by growth experiments in various types of tap water may give information about the concentrations of easily assimilable organic carbon.     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

Effect of unsaturated fatty acids in growth inhibition of some penicillin-resistant and sensitive bacteria

The growth of two penicillin-resistant Gram-positive bacteria, Bacillus licheniformis (749/C, penicillin G-resistant) and Staphylococcus aureus (metR 18, methicillin-resistant) and one Gram-negative strain, Escherichia coli (cloxacillin-resistant) as well as that of their wild counterparts was inhibited by the long-chain unsaturated fatty acids, linoleic, linolenic and arachidonic acid. The minimum inhibitory concentrations (MIC) of all the fatty acids were found to be 4-6 micrograms/ml for Staph. aureus (metR 18 & wild), 8-30 micrograms/ml for B. licheniformis (749/C & wild) and 70-90 micrograms/ml for E. coli (cloxacillin-resistant & wild). The inhibitory activity increased as the number of double bonds in the fatty acids increased. In most instances the concentrations of fatty acids required to inhibit the growth of the penicillin-resistant strains were lower than that required for their sensitive counterparts. This inhibition of growth in the presence of fatty acids may be due to an increase in permeability of the membrane as evidenced by the measurement of the leakage of 260 nm absorbing material and fluidity.     

Intermediate Energy Metabolism of Leptospira

Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B(16) and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B(16) for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for beta-oxidation of fatty acids. Strain B(16) and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B(16) to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids.     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

Involvement of fibronectin type II repeats in the efficient inhibition of gelatinases A and B by long-chain unsaturated fatty acids

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis- and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K(i) values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by long-chain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of long-chain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds.     

Bacterial nutrients in drinking water.

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Bacterial nutrients in drinking water

Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15 degrees C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 micrograms of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to less than 50 micrograms/liter.     

Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.     

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.