Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments

Acid adaptation of Streptococcus mutans UA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of the S. mutans acid-adaptation arsenal.     

Low pH-induced membrane fatty acid alterations in oral bacteria

Four oral bacterial strains, of which two are considered aciduric and two are considered acid-sensitive, were grown under glucose-limiting conditions in chemostats to determine whether their membrane fatty acid profiles were altered in response to environmental acidification. Streptococcus gordonii DL1, as well as the aciduric strains S. salivarius 57.I, and Lactobacillus casei 4646 increased the levels of mono-unsaturated membrane fatty acids. The non-aciduric strain S. sanguis 10904 did not alter its membrane composition in response to pH values examined here. Thus, in response to low pH, aciduric oral bacteria alter their membrane composition to contain increased levels of long-chained, mono-unsaturated fatty acids. This suggests that membrane fatty acid adaptation is a common mechanism utilized by bacteria to withstand environmental stress.     

Shifts in the Membrane Fatty Acid Profile of Streptococcus mutans Enhance Survival in Acidic Environments

Acid adaptation of Streptococcus mutans UA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of the S. mutans acid-adaptation arsenal.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

S-Adenosylmethionine,cyclopropane fatty acid synthase,and the production of lactobacillic acid in Lactobacillus plantarum

S-Adenosylmethionine (AdoMet) levels in Lactobacillus plantarum were found to increase concomitantly with the production of membrane cyclopropane fatty acids under normal growth conditions. This increase in AdoMet did not occur when the pH of the culture medium (initially pH 6.5) was not allowed to fall (pH 4 or lower) during growth. When the culture medium was maintained at pH 6.5, cyclopropane fatty acid synthesis also remained low. While the activity of cyclopropane fatty acid synthase is increased as the pH decreases, the activity of AdoMet synthetase is largely unaffected by the variation of pH of the culture medium. The production of cyclopropane fatty acids is also dependent upon continued protein synthesis; in the presence of chloramphenicol cyclopropane fatty acid synthase activity is decreased, resulting in a lowered production of cyclopropane fatty acids. A dramatic increase in AdoMet levels occurs in the presence of chloramphenicol. It is proposed that AdoMet levels, in conjunction with cyclopropane fatty acid synthase activities, regulate cyclopropane fatty acid synthesis in L. plantarum.     

Fatty acid transport and metabolism in HepG2 cells

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Physicochemical Effects of Long Chain Fatty Acids on Bacterial Cells and their Protoplasts

S ummary . Fatty acids of chain length > C₁₀ induced lysis of protoplasts at pH 7·4 when the concentration was nearly bactericidal. At pH 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysis was produced by the same acids below bactericidal concentrations. The lysis was immediate at pH 8, but at pH 6 the effect was preceded by contraction of protoplasts. At pH 7·4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplasts. Reversal agents antagonized the lysis of protoplasts by fatty acids. A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed.     

Effects of pyrazinamide on fatty acid synthesis by whole mycobacterial cells and purified fatty acid synthase I

The effects of low extracellular pH and intracellular accumulation of weak organic acids were compared with respect to fatty acid synthesis by whole cells of Mycobacterium tuberculosis and Mycobacterium smegmatis. The profile of fatty acids synthesized during exposure to benzoic, nicotinic, or pyrazinoic acids, as well as that observed during intracellular hydrolysis of the corresponding amides, was not a direct consequence of modulation of fatty acid synthesis by these compounds but reflected the response to inorganic acid stress. Analysis of fatty acid synthesis in crude mycobacterial cell extracts demonstrated that pyrazinoic acid failed to directly modulate the fatty acid synthase activity catalyzed by fatty acid synthase I (FAS-I). However, fatty acid synthesis was irreversibly inhibited by 5-chloro-pyrazinamide in a time-dependent fashion. Moreover, we demonstrate that pyrazinoic acid does not inhibit purified mycobacterial FAS-I, suggesting that this enzyme is not the immediate target of pyrazinamide.     

Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport.

The maximum specific growth rate of Streptococcus lactis and Streptococcus cremoris on synthetic medium containing glutamate but no glutamine decreases rapidly above pH 7. Growth of these organisms is extended to pH values in excess of 8 in the presence of glutamine. These results can be explained by the kinetic properties of glutamate and glutamine transport (B. Poolman, E. J. Smid, and W. N. Konings, J. Bacteriol. 169:2755-2761, 1987). At alkaline pH the rate of growth in the absence of glutamine is limited by the capacity to accumulate glutamate due to the decreased availability of glutamic acid, the transported species of the glutamate-glutamine transport system. Kinetic analysis of leucine and valine transport shows that the maximal rate of uptake of these amino acids by the branched-chain amino acid transport system is 10 times higher in S. lactis cells grown on synthetic medium containing amino acids than in cells grown in complex broth. For cells grown on synthetic medium, the maximal rate of transport exceeds by about 5 times the requirements at maximum specific growth rates for leucine, isoleucine, and valine (on the basis of the amino acid composition of the cell). The maximal rate of phenylalanine uptake by the aromatic amino acid transport system is in small excess of the requirement for this amino acid at maximum specific growth rates. Analysis of the internal amino acid pools of chemostat-grown cells indicates that passive influx of (some) aromatic amino acids may contribute to the net uptake at high dilution rates.     

Patterns of fatty acid release from endogenous substrates by human platelet homogenates and membranes.

We describe a method for measuring the release of fatty acids from endogenous substrates of human platelet homogenates and membranes. The method depends on the availability of lipids whose fatty acids are odd-chained and therefore suitable as internal reference compounds that, at the time of lipid extraction, can be added to an incubation to permit subsequent quantification of the content of free fatty acids or fatty acids esterified to specific lipids. We found four types of lipolytic activities in human platelets. In homogenates at pH 4.0 a triglyceride lipase operated as shown by the synchrony of triglyceride degradation and release of glycerol and those fatty acids that are the predominant constituents of triglycerides. However, enough arachidonic acid was released at this pH level to suggest some phospholipid breakdown, since triglycerides hold relatively small amounts of this acid. With membranous preparations, in the alkaline pH range there were two peaks of fatty acid release with accompanying degradation of phospholipids. At pH 8.5, where release of the saturated acids, palmitic and stearic, predominated, their sum was 3.5 times that of arachidonic acid. At pH 9.5 the release of palmitic and stearic acids was only slightly below their peak values; however, the release of arachidonic acid nearly equaled the sum of the saturated acids. Linoleic acid was not released in representative amounts by those reactions that released arachidonic acid, despite the overwhelming propensity of both to be esterified at the 2-position of phospholipids. Pertinently, the choline phospholipids are linoleic-rich and the non-choline phospholipids linoleic-poor, while both have a generous endowment of arachidonic acid. With this in mind, we raise the possibility that the phospholipase A2 of human platelets is an endoenzyme because of its tendency to act on those phospholipids that are thought to comprise the inner layer of the cell membrane.     

Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium

Willett, Norman P. (University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pa.), and Guy E. Morse. Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium. J. Bacteriol. 91:2245-2250. 1966.-A chemically defined medium was developed for Streptococcus agalactiae which supported growth comparable to that obtained in complex medium. The effects of long-chain fatty acids on growth of the organisms were determined turbidimetrically. The order of activity of the fatty acids was dependent upon whether complete inhibition or median response (50% inhibition point) was used as a parameter of activity. When complete inhibition of growth was used as a measure, the degree of unsaturation of C(18) acids enhanced antimicrobial activity. However, when the median response was used as an index, this order was reversed. Increase in carbon chain from C(12) to C(18) did not correlate with either complete inhibition or median response points. Antimicrobial activity of unsaturated and saturated fatty acids was reversed by bovine serum albumin and other compounds, suggesting a bacteriostatic action.     

Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

Incorporation of deoxycytidine into deoxyribonucleic acid deoxycytidylate in Lactobacillus acidophilus R-26.

A mutant of Bacillus subtilis synthesizes a variety of omega-alicyclic fatty acids when fed with the respective alicyclic carboxylic acids. These fatty acids are: omega-cyclopropane, omega-cyclobutane, omega-cyclopentane, omega-cyclohexane, and omega-cyclohexene fatty acids. These unusual fatty acids did not lead to an inhibition of growth at 37 degrees C and pH 7. The selective advantage of these fatty acids under extrene conditions was studied in comparison with the acidophilic, thermophilic bacterium B. acidocaldarius, which normally contains a high proportion of omega-cyclohexane fatty acids.     

Adaptive acid tolerance response of Streptococcus sobrinus

Streptococcus mutans and Streptococcus sobrinus are the bacteria most commonly associated with human dental caries. A major virulence attribute of these and other cariogenic bacteria is acid tolerance. The acid tolerance mechanisms of S. mutans have begun to be investigated in detail, including the adaptive acid tolerance response (ATR), but this is not the case for S. sobrinus. An analysis of the ATR of two S. sobrinus strains was conducted with cells grown to steady state in continuous chemostat cultures. Compared with cells grown at neutral pH, S. sobrinus cells grown at pH 5.0 showed an increased resistance to acid killing and were able to drive down the pH through glycolysis to lower values. Unlike what is found for S. mutans, the enhanced acid tolerance and glycolytic capacities of acid-adapted S. sobrinus were not due to increased F-ATPase activities. Interestingly though, S. sobrinus cells grown at pH 5.0 had twofold more glucose phosphoenolpyruvate:sugar phosphotransferase system (PTS) activity than cells grown at pH 7.0. In contrast, glucose PTS activity was actually higher in S. mutans grown at pH 7.0 than in cells grown at pH 5.0. Silver staining of two-dimensional gels of whole-cell lysates of S. sobrinus 6715 revealed that at least 9 proteins were up-regulated and 22 proteins were down-regulated in pH 5.0-grown cells compared with cells grown at pH 7.0. Our results demonstrate that S. sobrinus is capable of mounting an ATR but that there are critical differences between the mechanisms of acid adaptation used by S. sobrinus and S. mutans.     

Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. II. Comparison of coryneform bacteria, Micrococcaceae and some lactic acid bacteria starters

Various strains of coryneform bacteria, Micrococcaceae and commercial starters of Lactococcus lactis and Leuconostoc were compared for their aptitude to form S-methyl thioesters. Resting cells were incubated with methanethiol alone at pH 7 and in conjunction with a mixture of straight, branched and hydroxy short-chain fatty acids up to C₆ at pH 7 and 5. Results showed that all the strains synthesized at least S-methyl thioacetate, with strains that were low and high producers in each group. This is the only thioester formed in small amount by Leuconostoc. Brevibacterium linens (six strains) and Micrococcaceae (five strains) were able to form branched-chain thioesters especially from their intracellular fatty acids at neutral pH, and straight-chain thioesters mostly from exogenous fatty acids at acid pH. Coryneform bacteria other than B. linens (four strains) and L. lactis (four starters) synthesized thioesters up to S-methyl thiobutyrate from endogenous or exogenous fatty acids but not branched-chain ones, except for one starter which formed a very little thioisovalerate. Some particular effects of pH and added fatty acids revealed differences between species or strains in their specific enzymatic systems. Received: 7 April 1997 / Received revision: 5 June 1997 / Accepted: 7 June 1997     

Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.