Cyclopropane Fatty Acid Content Research Articles

Roseiterribacter gracilis gen. nov., sp. nov., a novel filterable alphaproteobacterium isolated from soil using a gel-filled microwell array device.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-μm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the order Rhodospirillales of Alphaproteobacteria. Strain TMPK1T exhibited small cell sizes (0.08-0.23 μm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from other Rhodospirillales bacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1T and its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1T represents a novel genus and species named Roseiterribacter gracilis gen. nov., sp. nov. (JCM 34627T = KCTC 82790T). We also propose Roseiterribacteraceae fam. nov. to accommodate the genus Roseiterribacter.

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

BackgroundRecent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism’s vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers.ResultsWe utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure.Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates.ConclusionsThis study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Environmental stress stimulates microbial activities as indicated by cyclopropane fatty acid enhancement

Soil microbial responses to environmental stress remain a critical question in microbial ecology. The content of cyclopropane fatty acid (CFA) in cytomembrane has been widely used to evaluate environmental stress on microorganisms. Here, we used CFA to investigate the ecological suitability of microbial communities and found a stimulating impact of CFA on microbial activities during wetland reclamation in Sanjiang Plain, Northeastern China. The seasonality of environmental stress resulted in the fluctuation of CFA content in the soil, which suppressed microbial activities due to nutrient loss upon wetland reclamation. After land conversion, the aggravation of temperature stress to microbes increased the CFA content by 5 % (autumn) to 163 % (winter), which led to the suppression of microbial activities by 7 %–47 %. By contrast, the warmer soil temperature and permeability decreased the CFA content by 3 % to 41 % and consequently aggravated the microbial reduction by 15 %–72 % in spring and summer. Complex microbial communities of 1300 CFA-produced species were identified using a sequencing approach, suggesting that soil nutrients dominated the differentiation in these microbial community structures. Further analysis with structural equation modeling highlighted the important function of CFA content to environmental stress and the stimulating influence of CFA induced by environmental stress on microbial activities. Our study shows the biological mechanisms of seasonal CFA content for microbial adaption to environmental stress under wetland reclamation. It advances our knowledge of microbial physiology affecting soil element cycling caused by anthropogenic activities.

PsrA positively regulates the unsaturated fatty acid synthesis operon fabAB in Azotobacter vinelandii

In Pseudomonas spp. PsrA, a transcriptional activator of the rpoS gene, regulates fatty acid catabolism by repressing the fadBA5 β-oxidation operon. In Azotobacter vinelandii, a soil bacterium closely related to Pseudomonas species, PsrA is also an activator of rpoS expression, although its participation in the regulation of lipid metabolism has not been analyzed. In this work we found that inactivation of psrA had no effect on the expression of β-oxidation genes in this bacterium, but instead decreased expression of the unsaturated fatty acid biosynthetic operon fabAB (3-hydroxydecanoyl-ACP dehydratase/isomerase and 3-ketoacyl-ACP synthase I). This inactivation also reduced the unsaturated fatty acid content, as revealed by the thin-layer chromatographic analysis, and confirmed by gas chromatography; notably, there was also a lower content of cyclopropane fatty acids, which are synthesized from unsaturated fatty acids. The absence of PsrA has no effect on the growth rate, but showed loss of cell viability during long-term growth, in accordance with the role of these unsaturated and cyclopropane fatty acids in the protection of membranes. Finally, an electrophoretic mobility shift assay revealed specific binding of PsrA to the fabA promoter region, where a putative binding site for this regulator was located. Taken together, our data show that PsrA plays an important role in the regulation of unsaturated fatty acids metabolism in A. vinelandii by positively regulating fabAB.

An Overview on Cyclic Fatty Acids as Biomarkers of Quality and Authenticity in the Meat Sector.

A survey was conducted to determine the content of cyclopropane fatty acids (CPFAs) and ω-cyclohexyl fatty acids (CHFAs) by using gas chromatography- mass spectrometry (GC-MS) and proton nuclear magnetic resonance (1H NMR) techniques in various meat samples from different species, including commercial samples and complex and thermally processed products (i.e., Bolognese sauce). The CPFAs concentration (as the sum of two isomers, namely dihydrosterculic acid and lactobacillic acid) in bovine meat fat (ranging between 70 and 465 mg/kg fat) was positively related to a silage-based diet, and therefore, they are potential biomarkers for monitoring the feeding system of cattle. CHFAs, such as 11-cyclohexylundecanoic and 13-cyclohexyltridecanoic acids, were only found in lipid profiles from ruminant species, and a linear trend was observed in their content, together with iso-branched fatty acids (iso-BCFAs) deriving from ruminal fermentation, as a function of bovine meat percentage in both raw and cooked minced meat. Thus, CHFAs are potential biomarkers for the assurance of the meat species and, combined with iso-BCFAs, of the beef/pork ratio even in complex meat matrices. The proposed approaches are valuable novel tools for meat authentication, which is pivotal in the management of meat quality, safety, and traceability.

Investigation of lipid profile in Acetobacter pasteurianus Ab3 against acetic acid stress during vinegar production.

Elucidation of the acetic acid resistance (AAR) mechanisms of Acetobacter pasteurianus is significant for vinegar production. In this study, cell membrane lipid profile of A. pasteurianus Ab3 was investigated by gas chromatography-mass spectrometer (GC-MS) and high performance liquid chromatography-electrospray ionization (HPLC-ESI) combined with high resolution accurate mass/mass spectrometry (HRAM/MS). We observed that cell remodeled the membrane physical state by decreasing the ratio of saturated fatty acids (SFAs)/unsaturated fatty acids (UFAs), and increasing the chain length of fatty acids (FAs) and the content of cyclopropane FAs in response to extreme acid stress. Noticeably, the content of octadecadienoic acid (C18:2) elevated remarkably. Moreover, a continuous reduction in cell membrane fluidity and a "V-type" variance in permeability were discovered. The content of glycerophospholipid and ceramide increased significantly in cells harvested from culture with acidity of 75g/L and 95g/L compared to that with acidity of 30g/L. Among the identified lipid species, the content of phosphatidylcholine (e.g. PC 19:0/18:2 and 19:1/18:0), ceramide (e.g. Cer d18:0/16:1 and d18:0/16:1 + O), and dimethylphosphatidylethanolamine (e.g. dMePE 19:1/16:1) increased notably with increasing acidity. Collectively, these findings refresh our current understanding of the AAR mechanisms in A. pasteurianus Ab3, and should direct future strain breeding and vinegar fermentation.

Transcriptional regulation of fatty acid cis-trans isomerization in the solvent-tolerant soil bacterium, Pseudomonas putida F1.

One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis-trans isomerase (Cti) an enzyme that converts the cis-unsaturated fatty acids (FAs) of the membrane lipids to their trans-isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent-tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti-279 and cti-77) were identified with cti-279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic-AMP receptor protein (Crp) and repression required the cyclic-AMP ligand of Crp. Production of trans-unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans-unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.

Presence of cyclopropane fatty acids in foods and estimation of dietary intake in the Italian population

Cyclopropane fatty acids (CPFAs) are the most abundant cyclic fatty acids in microorganisms with unknown role(s) regarding their dietary relevance and biological effects in humans. This work was aimed to draw up a list of CPFAs-containing foods for estimating their dietary intake in the Italian population to provide a basis for evaluating their nutritional relevance and potential health-related effects. The CPFAs content of more than 500 food items was investigated and a preliminary dietary intake was assessed (12.0 ± 6.0 mg/day), based on the data reported by the Italian National Food Consumption Survey INRAN-SCAI 2005–06. CPFAs should be considered of dietary relevance in view of their potential physiological activity in humans and their presence in significant amounts in dairy products, as Grana Padano cheese (9.0–30.0 mg/100 g), and in bovine meat (0.7–4.0 mg/100 g). Future studies should elucidate whether this uncommon class of fatty acids has a biological role in human health.

Determination of Cyclopropane Fatty Acids in Food of Animal Origin by 1H NMR.

Cyclopropane fatty acids (CPFAs) are unusual fatty acids of microbial origin, recently detected in milk and dairy products. CPFAs have been demonstrated to be interesting molecular markers for authentication of dairy products obtained without ensiled feeds. Moreover, they can also be recognized as a new secondary component of human diet. Information is lacking on the presence of cyclic fatty acids in other food sources. Cyclopropane fatty acids have been detected by GC-MS analysis in cheese and other animal fats in concentration ranging from 200 to 1000 mg/kg fat, but in some cases, the complex fatty acid profile and the possible presence of interfering peaks make the separation not straightforward and the quantification uneasy. Therefore, a new reliable 1H NMR method was developed to detect and measure CPFA content in different foods of animal origin, based on the detection of the characteristic signals of cyclopropane ring. The 1H NMR (600 MHz) method showed detection limits comparable with those of full scan GC-MS, and it allowed the identification and quantitation of the cyclopropane fatty acids in different foods.

Acid Response of Bifidobacterium longum subsp. longum BBMN68 Is Accompanied by Modification of the Cell Membrane Fatty Acid Composition.

The acid response of Bifidobacterium longum subsp. longum BBMN68 has been studied in our previous study. The fab gene, which is supposed to be involved in membrane fatty acid biosynthesis, was demonstrated to be induced in acid response. In order to investigate the relationship between acid response and cell membrane fatty acid composition, the acid adaptation of BBMN68 was assessed and the membrane fatty acid composition at different adaptation conditions was identified. Indeed, the fatty acid composition was influenced by acid adaptation. Our results showed that the effective acid adaptations were accompanied with decrease in the unsaturated to saturated fatty acids ratio (UFA/SFA) and increase in cyclopropane fatty acid (CFA) content, which corresponded to previous studies. Moreover, both effective and non-effective acid adaptation conditions resulted in decrease in the C18:1 cis-9/C18:1 trans-9 ratio, indicating that the C18:1 cis-9/C18:1 trans-9 ratio is associated with acid tolerance response but not with acid adaptation response. Taken together, this study indicated that the UFA/SFA and CFA content of BBMN68 were involved in acid adaptation and the C18:1 cis-9/C18:1 trans-9 ratio was involved in acid tolerance response.

An overview on the presence of cyclopropane fatty acids in milk and dairy products.

A survey was carried out to determine the presence of cyclopropane fatty acids (CPFA) in various dairy products. CPFA such as lactobacillic acid and dihydrosterculic acid are components of bacterial membranes and have been recently detected in milk from cows fed with maize silage. In this paper about 200 dairy samples comprising cow, sheep, and goat milk, cheese, yogurt/fermented milk, and butter were analyzed. Results showed that cow milks were generally positive to CPFA (0.014-0.105% of total fatty acids), while goat, yak, and sheep milks were negative. Experimental yogurt and fermented milks showed the same CPFA content of the starting milk. Positive to CPFA were also the majority of samples of commercial butter and cheeses, except some PDO cheeses as Parmigiano-Reggiano and Fontina, cheeses from mountain regions, and goat and sheep cheeses. These data suggest that the presence of CPFA in dairy products could be used as a marker of silage feeding.

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage

Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic.

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

FAMEs profiles of phenol-degrading Pseudomonas stutzeri introduced into soil

Fatty acid composition during phenol biodegradation by Pseudomonas stutzeri introduced into three sterile soils differed in organic matter content was investigated. It was found that type of soil strongly influenced the rate of phenol removal and survival of bacteria. In soil (MF) with the highest amount of organic matter (37.30%) the complete phenol biodegradation proceeded within 44 days, in soil (WM) with intermediate organic matter content (18%) lasted 68 days, whereas in sandy soil (PF) (2.10% of organic matter) phenol degradation was not observed. In phenol-polluted soil the number of introduced P. stutzeri cells decreased during experiment period from an initial density of 2.5 × 10 9 CFU/g soil to 1.73 × 10 5 CFU/g soil and to 9.7 × 10 3 CFU/g soil in MF and WM samples, respectively. Under phenol exposure changes in FAMEs profiles isolated from soils were detected. The most significant alterations were connected with the occurrence and content of cyclopropane fatty acids. Fatty acid 17:0 cy was detected only in uncontaminated soil whereas 19:0 cy ω8 c fatty acid was synthesised by introduced bacteria in phenol amended soils when its concentration decreased significantly. Other noticeable changes included the increase of branched and straight-chain fatty acid contents, resulted in the higher level of fatty acids saturation in bacteria isolated from phenol-polluted soils. Our results show that fatty acid composition and the occurrence of specific fatty acids can be used for monitoring the progress of bioremediation process in phenol-polluted environment.

Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

The long-term response of the broad-salt growing halophile Chromohalobacter salexigens DSM 3043 T to salt stress has been investigated with respect to adaptive changes in membrane lipid composition. This study included the wild-type and three salt-sensitive, ectoine-deficient strains: CHR62 ( ectA::Tn 1732, unable to grow above 0.75 M NaCl), CHR63 ( ectC::Tn 1732, unable to grow above 1.5 M NaCl), and CHR64, which was able to grow in minimal medium M63 up to 2.5 M NaCl, but its growth was slower than the wild-type strain at salinities above 1.5 M NaCl. This mutant accumulated ectoine and hydroxyectoine as major compatible solutes, but also the ectoine precursor, N- γ-acetyldiaminobutyric acid, and was found to be affected in the ectoine synthase gene ectC. The main phospholipids of the wild-type strain were phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL). Major fatty acids were detected as 16:0, 18:1, and 16:1, including significant amounts of cyc-19:0, and cyc-17:0. CL and cyclopropane fatty acids (CFA) levels were elevated when the wild-type strain was grown at high salinity (2.5 M NaCl). Membranes of the most salt-sensitive trains CHR62 and CHR63, but not of the less salt-sensitive strain CHR64, contained lower levels of CL. The proportion of cyc-19:0 in CHR64 was three-fold (at 2.0 M NaCl) and 2.5-fold (at 2.5 M NaCl) lower than that of the wild type, suggesting that this mutant has a limited capacity to incorporate CFA into phospholipids at high salt. The addition of 1 mM ectoine to cultures of the wild-type strain increased the ratio PG/CL from 1.8 to 3.3 at 0.75 M NaCl, and from 1 to 6.5 at 2.5 M NaCl, and led to a slight decrease in CFA content. Addition of 1 mM ectoine to the mutants restored the steady-state levels of CL and CFA found in the wild-type strain supplemented with ectoine. These findings suggest that exogenous ectoine might attenuate the osmostress response involving changes in membrane lipids.

Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824.

The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.

Effect of Heat Shock or Cold Shock Treatment on the Resistance of Lactococcus lactis to Freezing and Lyophilization

This study investigated the effect of heat shock or cold shock treatment on the resistance of commercial Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris cheese starter bacteria to freezing and freeze-drying. The ability to withstand freezing at −60°C for 24 h was variable among lactococci, but could be significantly (P < 0.05) improved in most strains by a 25-min heat shock at 42°C (L. lactis ssp. lactis) or 39°C (L. lactis ssp. cremoris) or by a 2-h cold shock at 10°C. In addition, stress treatments that improved lactococcal cryotolerance significantly (P < 0.05) enhanced the resistance of most strains to lyophilization. Greater resistance to freezing or lyophilization was not detected when stress treatments were performed in broth that contained erythromycin, which indicated that stress-induced proteins were required for cell protection. Membrane fatty acid analysis of stress-treated cells suggested that enhanced resistance to freezing and lyophilization may be related to changes in cell membrane lipid composition. Heat-shocked cells had a higher 19:0 cyclopropane fatty acid content than control cells, and cold-shocked cells contained a lower ratio of saturated to unsaturated fatty acids. Other factors must also be involved in cell protection, however, because similar changes in membrane lipid composition were detected in strains whose resistance to freezing and lyophilization was not improved by heat or cold shock treatment.

Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli.

Cyclopropane fatty acid (CFA) formation is a post-synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid. The synthesis of CFA depends in part on the RpoS sigma factor, but the role of RpoS in resistance to acid shock involves additional factors because strains with null mutations in both cfa and rpoS are more sensitive to acid shock than either single mutant strain. Exponential phase cultures of E. coli are much more sensitive to acid shock than stationary phase cultures, but survival is greatly increased if the exponential phase cultures are exposed to moderately acid conditions (pH 5) before shift to pH 3. We show that exposure to moderately acid conditions gives a marked increase in cfa transcription. The efficiency of the survival of acid shock is extremely strain dependent, even among putative wild-type strains. Much, but not all, of this variability can be explained by the partially or totally defective RpoS alleles carried by many strains.

Acid habituation of Escherichia coli and the potential role of cyclopropane fatty acids in low pH tolerance

A reversible adaptive tolerance to low pH termed `acid habituation' is demonstrated for five strains of Escherichia coli. Superimposed upon the intrinsic acid tolerance of individual strains, acid habituation significantly enhances the survival of exponential phase cultures exposed to a lethal acid challenge (pH 3.0), and minimises inter-strain variability in acid tolerance. The fatty acid composition of acid habituated, non-habituated, and de-habituated exponential phase cultures is also reported. During acid habituation, monounsaturated fatty acids (16:1 ω7c and 18:1 ω7c) present in the phospholipids of E. coli are either converted to their cyclopropane derivatives (cy17:0 and cy19:0), or replaced by saturated fatty acids. The acid tolerance of individual strains of E. coli appears to be correlated with membrane cyclopropane fatty acid content and, thus, it is postulated that increased levels of cyclopropane fatty acids may enhance the survival of microbial cells exposed to low pH. The results presented illustrate the remarkable capacity of E. coli to adapt to environmental challenges, and have significant implications for the survival of spoilage and pathogenic bacteria, and hence for food safety.

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.