Changes in fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by growth conditions: consequences of resistance to quaternary ammonium compounds

The effects of changes in the fatty acid composition of Pseudomonas aeruginosa induced by growth conditions on its resistance to two quaternary ammonium compounds (QAC) were investigated. The temperature and growth phase were the most influential parameters affecting the fatty acid composition of this bacterium. Furthermore, the formation of saturated fatty acids and cyclopropane fatty acids was stimulated by increasing the temperature, whereas the proportion of unsaturated fatty acids fell. The degree of saturation and the proportion of cyclopropane fatty acids increased in the course of the exponential and stationary phases. These modifications mostly concerned the inner membrane of the bacterium. Resistance of P. aeruginosa to both QAC tested was not significantly influenced by temperature and growth phase variations. Thus, resistance to the two QAC did not seem to be dependent on modifications of the fatty acid composition of the inner membrane.     

Specific variations of fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by quaternary ammonium compounds and relation with resistance to bactericidal activity

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of Quaternary Ammonium Compounds (QACs) was investigated. The strain was grown in a medium with increasing concentrations of a QAC, benzyldimethyltetradecylammonium chloride (C14) and two non-QACs, sodium dichloroisocyanurate and tri-sodium phosphate. In the presence of C14 only, the strain was able to grow in concentrations higher than the minimal inhibitory concentration. As the strain adapted to C14, resistance to bactericidal activity of the same biocide increased. For the non-QACs, no change was noted when cells were grown in the presence of biocides. The C14-adapted cells showed variations in membrane fatty acid composition. A hierarchical clustering analysis was used to compare all fatty acid compositions of cultures in the presence, or not, of the three biocides used here and another QAC studied previously. The clusters obtained underlined specific variations of membrane fatty acids in response to the presence of QACs. Furthermore, with a simple linear regression analysis, a relationship was shown between the membrane fatty acids and the resistance developed by the strain against the bactericidal activity of C14.     

Characteristics of the fatty acid composition of Pseudomonas aeruginosa cells with different degrees of resistance to dodecyl sulfate

The fatty acid composition of Pseudomonas aeruginosa 1C destroying anionic surfactant alkyl sulfates was studied after its cultivation under different conditions which caused different resistance of the cells against sodium dodecyl sulfate (SDS). The content of monounsaturated fatty acids (in particular, octadecenoic acid) increased while the content of cyclopropane fatty acids decreased in cells resistant against SDS.     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Effect of fatty acid substrate chain length on Pseudomonas aeruginosa ATCC 9027 monorhamnolipid yield and congener distribution

Rhamnolipids are surface-active molecules produced by Pseudomonas aeruginosa as congener mixtures. They are considered “green” alternatives to synthetic surfactants used in industrial, remediation and pharmaceutical applications. Optimizing yield as well as controlling congener distribution are necessary steps for successful commercialization of rhamnolipids. This study used a mixture of glucose and fatty acids of different chain length (C₁₂–C₂₂) and saturation (C_18:1 and C_18:2) to produce monorhamnolipids and determine the effect of fatty acid substrates on rhamnolipid yield, percent carbon conversion and congener distribution. Results show that 1% glucose + 0.25% stearic acid (C₁₈) produced the greatest yield (2.1 g L⁻¹) compared to other glucose–fatty acid combinations (0.8–1.8 g L⁻¹). Various glucose + C₁₈ ratios were then tested to optimize yield and percent substrate carbon conversion to monorhamnolipid. Results revealed a positive linear correlation between the mass percent of C₁₈ used and the percent carbon conversion. A mass percent of 67% C₁₈ was optimal resulting in a 44% carbon conversion and a yield of 13.7 g L⁻¹ monorhamnolipid. For all fatty acid substrates tested, the RhaC₁₀C₁₀ was the most abundant and RhaC₁₀C_12:1 was the least abundant of the four major congeners produced. However, the relative amount of RhaC₁₀C₈ and RhaC₁₀C₁₂ congeners was dependent on several factors: in general, fatty acid substrates with relatively short chain length (C₁₂ and C₁₄), unsaturated fatty acid substrate (_C18:2), and longer cultivation time resulted in a higher RhaC₁₀C₈/RhaC₁₀C₁₂ ratio. These findings will assist in mass production of monorhamnolipids and controlling the specific congeners produced.     

Changes in membrane fluidity and fatty acid composition of Pseudomonas putida CN-T19 in response to toluene

A bacterial isolate, Pseudomonas putida CN-T19, could grow in a two-phase medium with toluene up to 50% (v/v). Changes in fatty acid composition and membrane fluidity of the isolate were investigated to understand how this microorganism responds toluene. The changes in the ratios of unsaturated to saturated fatty acids were insignificant between cells grown with and without toluene. The changes in the ratio of cis- to trans-fatty acids of C16:1 and C18:1 was, however, significantly lower in cells grown with toluene than cells grown without toluene, giving approximately 1.3 and 9.7, respectively. Toluene had a fluidizing effect on the membrane of cells grown without toluene, resulting in decrease in membrane polarization ratio. Less fluidizing effect of toluene on the membrane of cells grown with toluene was observed, giving 11% of polarization percentage, which was significantly lower than 53% in cells grown without toluene. These results suggest that cis/trans isomeration of C16:1 and C18:1 makes cell membranes more rigid to respond toluene, and is an adaptive strategy allowing P. putida CN-T19 to grow in the presence of organic solvent.     

A moderate change in temperature induces changes in fatty acid composition of storage and membrane lipids in a soil arthropod

A moderate change in ambient temperature can lead to vital physiological and biochemical adjustments in ectotherms, one of which is a change in fatty acid composition. When temperature decreases, the composition of membrane lipids (phospholipid fatty acids) is expected to become more unsaturated to be able to maintain homeoviscosity. Although different in function, storage lipids (triacylglycerol fatty acids) are expected to respond to temperature changes in a similar way. Age-specific differences, however, could influence this temperature response between different life stages. Here, we investigate if fatty acid composition of membrane and storage lipids responds similarly to temperature changes for two different life stages of Orchesella cincta. Juveniles and adults were cold acclimated (15 °C → 5 °C) for 28 days and then re-acclimated (5 °C → 15 °C) for another 28 days. We found adult membranes had a more unsaturated fatty acid composition than juveniles. Membrane lipids became more unsaturated during cold acclimation, and a reversed response occurred during warm acclimation. Membrane lipids, however, showed no warm acclimation, possibly due to the moderate temperature change. The ability to adjust storage lipid composition to moderate changes in ambient temperature may be an underestimated fitness component of temperature adaptation because fluidity of storage lipids permits accessibility of enzymes to energy reserves.     

Effect of titanium ion and resistance encoding plasmid of Pseudomonas aeruginosa ATCC 10145

Titanium and its alloys are technically superior and cost-effective materials, with a wide variety of aerospace, industrial, marine, and commercial applications. In this study, the effects of titanium ions on bacterial growth were evaluated. Six strains of bacteria known to be resistant to both metal ions and antibiotics were used in this study. Two strains, Escherichia coli ATCC 15489, and Pseudomonas aeruginosa ATCC 10145, proved to be resistant to titanium ions. Plasmid-cured P. aeruginosa resulted in the loss of one or more resistance markers, indicating plasmid-encoded resistance. The plasmid profile of P. aeruginosa revealed the presence of a 23-kb plasmid. The plasmid was isolated and transformed into DH5alpha. Interestingly, the untransformed DH5alpha did not grow in 300 mg/l titanium ions, but the transformed DH5alpha grew quite well under such conditions. The survival rate of the transformed DH5alpha also increased more than 3-fold compared to that of untransformed DH5alpha.     

Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes.

Pseudomonas putida S12 was more tolerant to ethanol when preadapted to supersaturating concentrations of toluene. Cellular reactions at the membrane level to the toxicities of both compounds were different. In growing cells of P. putida S12, sublethal concentrations of toluene resulted in an increase in the degree of saturation of the membrane fatty acids, whereas toxically equivalent concentrations of ethanol led to a decrease in this value. Contrary to this, cells also reacted to both substances with a strong increase of the trans unsaturated fatty acids and a corresponding decrease of the cis unsaturated fatty acids under conditions where growth and other cellular membrane reactions were totally inhibited. While the isomerization of cis to trans unsaturated fatty acids compensates for the fluidizing effect caused by ethanol, a decrease in the degree of saturation is antagonistic with respect to the chemo-physical properties of the membrane. Consequently, the results support the hypothesis that the decrease in the degree of saturation induced by ethanol is not an adaptation mechanism but is caused by an inhibitory effect of the compound on the biosynthesis of saturated fatty acids.     

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia

Fluid transport across cultures of bovine tracheal epitheliumwas measured with a capacitance probe technique. Baseline fluid absorption (J_v)across bovine cells of 3.2 µl · cm² · h¹was inhibited by ~78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with aconcomitant decrease in transepithelial potential (TEP) and increase intransepithelial resistance(R_t). Effectsof P. aeruginosa were blocked byamiloride, which decreased J_v by 112% frombaseline of 2.35 ± 1.25 µl · cm² · h¹,increased R_t by101% from baseline of 610 ± 257  · cm², anddecreased TEP by 91% from baseline of 55 ± 18.5 mV.Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption weredue in part to a block of basolateral membrane K channels. In thepresence of Cl transport inhibitors[5-nitro-2-(3-phenylpropylamino)-benzoic acid,H₂-DIDS, and bumetanide],P. aeruginosa induced a fluid secretion of ~2.5 ± 0.4 µl · cm² · h¹and decreased R_twithout changing TEP. However, these changes were abolished when thetransport inhibitors were used in a medium in which Cl was replaced byan impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect onJ_v, TEP, orR_t. Mutantslacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encodinga  factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial saltand water transport induced by P. aeruginosa may alter viscosity and ionic composition ofairway secretions so as to foster further bacterial colonization.

     

Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

Plasmid-determined resistance to chromate in Pseudomonas aeruginosa

Abstract Resistance to chromate in five independent Pseudomonas aeruginosa clinical isolates was transferred by conjugation to P. aeruginosa strain PU21. All chromate-resistant transconjugants contained large plasmids that also conferred resistance to inorganic mercury. One of these plasmids, pUM505, increased the resistance to CrO₄²⁻ and decreased the accumulation of intracellular ⁵¹CrO₄²⁻ by the host cells as compared to the plasmidless strain PU21.     

Adaptation of aerobically growing Pseudomonas aeruginosa to copper starvation

Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.     

Alpha-tocopherol and gamma-tocopherol attenuate ethanol-induced changes in membrane fatty acid composition in embryonic chick brains

BACKGROUND: This project investigated whether or not EtOH-induced reductions in the levels of long-chain polyunsaturated membrane fatty acids could be attenuated by exogenous exposure to either alpha-tocopherol, gamma-tocopherol, or diallyl sulfide (DAS). METHODS: At 0 days of development, fertile chicken eggs were injected with a single dose of either saline supplemented with various concentrations of EtOH, alpha- or gamma-tocopherol and EtOH, or DAS and EtOH. At 18 days of development, brains were isolated and subjected to membrane analyses. RESULTS: When exposed to EtOH, concentrations ranging from 0-60.50 microm/Kg egg, dose-dependent decreases in the levels of brain 18:0, 18:1 (n-9), 18:2 (n-6), 18:3 (n-3), and 20:4 (n-6) were observed. These ethanol-induced changes in membrane fatty acid composition correlated with ethanol-induced reductions in brain mass, brain protein levels, acetylcholine esterase (AChE) activities and correlated with increased lipid hydroperoxide levels. Exposure to either 2.5 microm alpha-tocopherol/Kg egg and 6.050 mm EtOH/Kg egg, or 2.5 microm alpha-tocopherol/ Kg egg and 6.050 mm EtOH/Kg egg attenuated EtOH-induced changes in membrane fatty acid composition, brain mass, brain protein levels, AChE activities, and lipid hydroperoxide levels. Embryonic exposure to the cytochrome p450-2E1 inhibitor, diallyl sulfide (DAS), also attenuated EtOH-induced decreases in long-chain, unsaturated membrane fatty acids. However, embryonic exposure to DAS promoted abnormally low brain mass. CONCLUSION: EtOH-induced reductions in the levels of brain long-chain polyunsaturated fatty acid are caused by lipid peroxidation.