Cyclopropane Fatty Acids Research Articles

Occurrence of Cis-11,12-Methylene-Hexadecanoic Acid in the Red Alga Solieria pacifica (Yamada) Yoshida

Fatty acids in marine algae have attracted the attention of natural chemists because of their biological activity. The fatty acid compositions of the Solieriaceae families (Rhodophyceae, Gaigartinales) provide interesting information that unusual cyclic fatty acids have been occasionally found. A survey was conducted to profile the characteristic fatty acid composition of the red alga Solieria pacifica (Yamada) Yoshida using gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy (IR), and proton nuclear magnetic resonance spectroscopy (1H-NMR). In S. pacifica, two cyclopentyl fatty acids, 11-cyclopentylundecanoic acid (7.0%), and 13-cyclopentyltridecanoic acid (4.9%), and a cyclopropane fatty acid, cis-11,12-methylene-hexadecanoic acid (7.9%) contributed significantly to the overall fatty acid profile. In particular, this cyclopropane fatty acid has been primarily found in bacteria, rumen microorganisms or foods of animal origin, and has not previously been found in any other algae. In addition, this alga contains a significant amount of the monoenoic acid cis-11-hexadecenoic acid (9.0%). Therefore, cis-11,12-methylene-hexadecanoic acid in S. pacifica was likely produced by methylene addition to cis-11-hexadecenoic acid.

Effect of high levels of CO2 and O2 on membrane fatty acid profile and membrane physiology of meat spoilage bacteria

The membrane is the major protective barrier separating the cell from the environment and is thus important for bacteria to survive environmental stress. This study investigates changes in membrane lipid compositions and membrane physiology of meat spoiling bacteria in response to high CO2 (30%) and O2 (70%) concentrations, as commonly used for modified atmosphere packaging of meat. Therefore, the fatty acid profile as well as membrane fluidity, permeability and cell surface were determined and correlated to the genomic settings of five meat spoiling bacteria Brochothrix (B.) thermosphacta, Carnobacterium (C.) divergens, C. maltaromaticum, Leuconostoc (L.) gelidum subsp. gelidum and L. gelidum subsp. gasicomitatum cultivated under different gas atmospheres. We identified different genomic potentials for fatty acid adaptations, which were in accordance with actual measured changes in the fatty acid composition for each species in response to CO2 and/or O2, e.g., an increase in saturated, iso and cyclopropane fatty acids. Even though fatty acid changes were species-specific, the general physiological responses were similar, comprising a decreased membrane permeability and fluidity. Thus, we concluded that meat spoiling bacteria facilitate a change in membrane fatty acids upon exposure to O2 and CO2, what leads to alteration of membrane fluidity and permeability. The observed adaptations might contribute to the resistance of meat spoilers against detrimental effects of the gases O2 and CO2 and thus help to explain their ability to grow under different modified atmospheres. Furthermore, this study provides fundamental knowledge regarding the impact of fatty acid changes on important membrane properties of bacteria.

Monitoring degradation of restaurant wastewater by Lysinibacillus sphaericus C3-41

This study investigates the biodegradation abilities of Lysinibacillus sphaericus C3-41 in restaurant wastewater. The extent of biodegradation was monitored using Fourier Transform Infrared Spectroscopy (FTIR) and Fatty Acid Methyl Ester (FAME) analysis. There was significant reduction in mineral content (p < 0.05) of the wastewater after 6 days incubation while the highest growth (1.4 OD600) and lipase production (50mMol/min) was observed on the 5th day. About 2.5% (v/v) oil concentration and pH 8 were found to be optimal conditions for growth and lipase production, while a temperature of 30 °C, and 1% ammonium phosphate and glucose were detected as optimum nitrogen and carbon sources respectively. FAME analyses revealed the presence of some fatty acids such as palmitic acid (11.56 ppm), caprylic (11.22 ppm), caproic (11.19 ppm), lauric acid (9.95 ppm), undecanoic (4.77 ppm) and cyclopropane fatty acid (CFA, 4.34 ppm) in the fresh domestic wastewater. After biodegradation, there was an increase in the concentration of capric (0.39 ppm), lauric (0.33 ppm), palmitoleic (0.78 ppm), trans13 octadecanoic (1.00 ppm) and cyclopropanoic (2.53 ppm) in L. sphaericus C3-41. Also, reduction in concentration of some fatty acids components in the wastewater probably eventually resulted in their bioaccumulation in the bacteria cell. FTIR spectra revealed the changes in functional groups qualitatively and quantitatively with evidence of wastewater biodegradation by L. sphaericus C3-41. The prominent –NH stretching bonds at 3740 cm−1 from the primary and secondary amines completely disappeared after the 6th day, indicating chemical reduction of –NH bonds leading to liberation of NH3(g). This study thus confirms the bioremediation potentials of L. sphaericus C3-41 for domestic and restaurant wastewater.

Chemical and Fatty Acid Compositions of Crude and Purified Extracts Obtained from Datura innoxia Seeds Extracted with Different Solvents.

Oils play a key role as raw materials in a variety of industries. The aim of this study was to evaluate the potential of Datura innoxia seed oil cultivated in Saudi Arabia for industrial purpose and to study the effects of hexane, chloroform, and isopropanol as extraction solvents on the compositions of the extracts. The results showed that the hexane and chloroform extracts were mainly neutral oils which were rich in linoleic (≈46%) and oleic (≈31%) acids. However, the isopropanol extract contained large amount of neutral oil and organic acids. Neutral oil contained mainly palmitic acid (40.2%) and some important and valuable epoxy (15.4%) and cyclopropane (13.2%) fatty acids. Analysis of the sterol and tocopherol levels of the crude and purified oil extracted revealed that they were significantly affected by the extraction solvent used.

Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties.

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.

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.

Are endoplasmic reticulum subdomains shaped by asymmetric distribution of phospholipids? Evidence from a C. elegans model system.

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein-protein and protein-lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER-LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage-like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin-positive peri-LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.

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.

Diclofenac Degradation-Enzymes, Genetic Background and Cellular Alterations Triggered in Diclofenac-Metabolizing Strain Pseudomonas moorei KB4.

Diclofenac (DCF) constitutes one of the most significant ecopollutants detected in various environmental matrices. Biological clean-up technologies that rely on xenobiotics-degrading microorganisms are considered as a valuable alternative for chemical oxidation methods. Up to now, the knowledge about DCF multi-level influence on bacterial cells is fragmentary. In this study, we evaluate the degradation potential and impact of DCF on Pseudomonas moorei KB4 strain. In mono-substrate culture KB4 metabolized 0.5 mg L−1 of DCF, but supplementation with glucose (Glc) and sodium acetate (SA) increased degraded doses up to 1 mg L−1 within 12 days. For all established conditions, 4′-OH-DCF and DCF-lactam were identified. Gene expression analysis revealed the up-regulation of selected genes encoding biotransformation enzymes in the presence of DCF, in both mono-substrate and co-metabolic conditions. The multifactorial analysis of KB4 cell exposure to DCF showed a decrease in the zeta-potential with a simultaneous increase in the cell wall hydrophobicity. Magnified membrane permeability was coupled with the significant increase in the branched (19:0 anteiso) and cyclopropane (17:0 cyclo) fatty acid accompanied with reduced amounts of unsaturated ones. DCF injures the cells which is expressed by raised activities of acid and alkaline phosphatases as well as formation of lipids peroxidation products (LPX). The elevated activity of superoxide dismutase (SOD) and catalase (CAT) testified that DCF induced oxidative stress.

Virtual screening of potentially endocrine-disrupting chemicals against nuclear receptors and its application to identify PPAR\u03b3-bound fatty acids

Nuclear receptors (NRs) are key regulators of energy homeostasis, body development, and sexual reproduction. Xenobiotics binding to NRs may disrupt natural hormonal systems and induce undesired adverse effects in the body. However, many chemicals of concerns have limited or no experimental data on their potential or lack-of-potential endocrine-disrupting effects. Here, we propose a virtual screening method based on molecular docking for predicting potential endocrine-disrupting chemicals (EDCs) that bind to NRs. For 12 NRs, we systematically analyzed how multiple crystal structures can be used to distinguish actives and inactives found in previous high-throughput experiments. Our method is based on (i) consensus docking scores from multiple structures at a single functional state (agonist-bound or antagonist-bound), (ii) multiple functional states (agonist-bound and antagonist-bound), and (iii) multiple pockets (orthosteric site and alternative sites) of these NRs. We found that the consensus enrichment from multiple structures is better than or comparable to the best enrichment from a single structure. The discriminating power of this consensus strategy was further enhanced by a chemical similarity-weighted scoring scheme, yielding better or comparable enrichment for all studied NRs. Applying this optimized method, we screened 252 fatty acids against peroxisome proliferator-activated receptor gamma (PPARγ) and successfully identified 3 previously unknown fatty acids with Kd = 100–250 μM including two furan fatty acids: furannonanoic acid (FNA) and furanundecanoic acid (FUA), and one cyclopropane fatty acid: phytomonic acid (PTA). These results suggested that the proposed method can be used to rapidly screen and prioritize potential EDCs for further experimental evaluations.

Nisin and acid resistance in Salmonella is enhanced by N-dodecanoyl-homoserine lactone

Salmonella is a foodborne pathogen that can develop resistance to different stresses, which is essential for successful infection of the host. Some genes directly related to acid resistance are also involved in cationic peptide resistance in Gram-negative bacteria and could be under the control of quorum sensing (QS) mediated by autoinducer 1, known as acyl-homoserine lactone. Here, we investigated the influence of autoinducer 1, N-dodecanoyl-homoserine lactone (C12-HSL) on the resistance of Salmonella enterica subspecies enterica serovar Enteritidis to nisin and acid stress. Salmonella cells growing in anaerobic tryptic soy agar (TSB) at a pH of 7.0 for 7 h were submitted to acid stress at a pH of 4.5 in the presence and absence of nisin and were either supplemented or not with C12-HSL. Viable cell counts, gene expression, membrane charge alterations, fatty acid composition, and intracellular content leakage were observed. The autoinducer C12-HSL increased nisin resistance and survival at a pH of 4.5 in Salmonella. Also, C12-HSL increased the expression of the genes, phoP, phoQ, pmrA, and pmrB, which are involved with antimicrobial and acid resistance. The positive charge on the cell surface and concentration of cyclopropane fatty acid of the cellular membrane were increased in the presence of C12-HSL under acidic conditions, whereas membrane fluidity decreased. The loss of K+ and NADPH, promoted by nisin, was reduced in the presence of C12-HSL at a pH of 4.5. Taken together, these findings suggest that quorum sensing plays an important role in enhanced nisin and acid resistance in Salmonella.

Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06g L-1) after 72h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56g L-1). After 165h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3mg L-1 h-1) was maximal at 72.5h of cultivation.

Effect of the anti-inflammatory drug diclofenac on lipid composition of bacterial strain Raoultella sp. KDF8.

The strain Raoultella sp. KDF8 was cultivated on three sources of carbon and energy, glycerol, ethanol and diclofenac, for periods of time ranging from 24 to 72h. Using thin-layer chromatography, nine classes of phospholipids were detected and the amount of phosphatidylethanolamine (PtdEtn) decreased with increasing cultivation time. Conversely, the ratio of phospholipids having three or four acyls (acyl-phosphatidylglycerol (APtdGro), N-acyl-PtdEtn (NAPtdEtn) and cardiolipin (Ptd2Gro) increased during cultivation. GC-MS analysis showed that the percentage of fatty acids containing a cyclopropane ring increased almost tenfold whereas the amount of fatty acids bearing even-numbered chains dropped to less than one-third after 24h and 72h in cultures on glycerol and diclofenac, respectively. Shotgun analysis showed significant changes in the representation of molecular species of phospholipids. For instance, there was a 36-fold change in the ratio of 16:1/16:1/16:1-APtdGro to c17:0/c17:0/c17:0-APtdGro and a 12-fold ratio change for 16:1/16:1/16:1-NAPtdEtn to c17:0/c17:0/c17:0-NAPtdEtn; the Ptd2Gro ratio of 16:1 to c17:0 acids equalled 1750. Our results show that the bacteria overcome destabilization of the inner cytoplasmic cell membrane and a bacterial outer membrane by altering the geometric arrangement of acyl chains, i.e. switching from monounsaturated to cyclopropane fatty acids (16:1 versus c17:0).

Transcriptional and Translational Leakiness in Expression of a Fungal Cyclopropane Fatty Acid Synthase in the Escherichia coli pET-vector Expression System

We expressed the putative cyclopropane synthase Cfs1 from the mushroom Coprinopsis cinerea and the native bacterial Cfa enzyme in Escherichia coli as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the lacUV5 promoter. In lower amounts, Cfs1 and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on Cfs1 from proteomics suggest that the sequence at the pET-16b-cfs1 fusion point CTCGAGGAT CCTATG (BamHI cloning site in italic, native cfs1 ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native cfs1 start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native Cfs1 protein was found in E. coli which is only possible if the normal cfs1 ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native cfs1 start codon features a partial Shine-Dalgarno (SD) sequence.

Producing Cyclopropane Fatty Acid in Plant Leafy Biomass via Expression of Bacterial and Plant Cyclopropane Fatty Acid Synthases.

Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.

Enhanced tolerance to inhibitors of Escherichia coli by heterologous expression of cyclopropane-fatty acid-acyl-phospholipid synthase (cfa) from Halomonas socia.

Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.

Metabolic transcriptional analysis on copper tolerance in moderate thermophilic bioleaching microorganism Acidithiobacillus caldus

Bioleaching, an alternative environmental smelting technology, typically uses high concentrations of heavy metal ions, especially in the subsequent phase, due to metal ion accumulation from the mineral. In this study, we analyzed the overall response of the bioleaching microorganism Acidithiobacillus caldus to copper stress through physiological and transcriptomic analyses. Scanning electron microscopy results showed higher extracellular polymeric substances secretion and cell aggregation under copper stress. Intracellular levels of glutamic acid, glycine and cysteine increased, favoring the synthesis of glutathione for maintenance of the oxidation-reduction state. GSH, during copper stress conditions, the activity of GSH-PX and CAT increased, resulting in reduced oxidative damage while maintaining stable intracellular pH. Higher unsaturated and cyclopropane fatty acid levels resulted in increased membrane fluidity and compactness and decreased ATP levels to support the energy requirements for stress resistance. Initially, H+-ATPase activity increased to provide energy for proton output and decreased later at higher copper ion stress. From transcriptome analysis, 140 genes were differentially expressed under low copper stress (1g/L), while 250 genes exhibited altered transcriptional levels at higher copper stress (3g/L). These differentially expressed genes were involved primarily in metabolic pathways such as energy metabolism, two-component systems, amino acid metabolism, and signal transduction. The Sox family cluster gene cluster involved in the conversion of thiosulfate to sulfate was upregulated in the sulfur metabolism pathway. In the oxidative phosphorylation pathway, genes participating in the synthesis of NADH oxidoreductase and cytochrome c oxidase, nuoL, cyoABD (cyoA, cyoB and cyoD) and cydAB (cydA and cydB), were downregulated. The TCS element ompR, closely associated with the osmotic pressure, exhibited active response, while Cu2+ efflux system gene cusRS was upregulated. In the amino acid metabolism, the glnA involved in nitrogen fixation was upregulated and promoted the synthesis of glutamine synthetase for reducing excessive oxidative stress. This study provides new insights into the mechanism underlying A. caldus response to heavy-metal ion stress under harsh bioleaching conditions.

Characterizing how probiotic Lactobacillus reuteri 6475 and lactobacillic acid mediate suppression of osteoclast differentiation

Osteoporosis is a disease that impacts over 200 million people worldwide. Taking into consideration the side effects stemming from medications used to treat this illness, investigators have increased their efforts to develop novel therapeutics for osteoporosis. In a previous study, we demonstrated that ovariectomy-induced bone loss in mice was prevented by treatment with the probiotic bacterium Lactobacillus reuteri 6475 (L. reuteri), an effect that correlated with reduced osteoclastogenesis in the bone marrow of L. reuteri treated mice. We also demonstrated that L. reuteri directly inhibited osteoclastogenesis in vitro. To better understand how L. reuteri impacts osteoclast formation, we used additional in vitro analyses to identify that conditioned supernatant from L. reuteri inhibited osteoclastogenesis at the intermediate stage of fused polykaryons. To elucidate the effect of L. reuteri treatment on host cell physiology, we performed RNAseq at multiple time points during in vitro osteoclastogenesis and established that L. reuteri downregulated several KEGG pathways including osteoclast differentiation as well as TNF-α, NF-κB, and MAP kinase signaling. These results were consistent with Western Blot data demonstrating that NF-κB and p38 activation were decreased by L. reuteri treatment. We further identified that lactobacillic acid (LA), a cyclopropane fatty acid produced by L. reuteri, contributed significantly to the suppression of osteoclastogenesis. Additionally, we demonstrated that L. reuteri is signaling through the long chain fatty acid receptor, GPR120, to impact osteoclastogenesis. Overall, these studies provide both bacterial and host mechanisms by which L. reuteri impacts osteoclastogenesis and suggest that long chain fatty acid receptors could be targets for preventing osteoclastogenesis.

Metabolic adaptability shifts of cell membrane fatty acids of Komagataeibacter hansenii HDM1-3 improve acid stress resistance and survival in acidic environments.

Komagataeibacter hansenii HDM1-3 (K. hansenii HDM1-3) has been widely applied for producing bacterial cellulose (BC). The yield of BC has been frequently limited by the acidification during sugar metabolism, due to the generation of organic acids such as acetic acid. In this study, the acid resistance mechanism of K. hansenii HDM1-3 has been investigated from the aspect of metabolic adaptability of cell membrane fatty acids. Firstly, we observed that the survival rate of K. hansenii HDM1-3 was decreased with lowered pH values (adjusted with acetic acids), accompanied by increased leakage rate. Secondly, the cell membrane adaptability in response to acid stress was evaluated, including the variations of cell membrane fluidity and fatty acid composition. The proportion of unsaturated fatty acids was increased (especially, C18-1w9c and C19-Cyc), unsaturation degree and chain length of fatty acids were also increased. Thirdly, the potential molecular regulation mechanism was further elucidated. Under acid stress, the fatty acid synthesis pathway was involved in the structure and composition variations of fatty acids, which was proved by the activation of both fatty acid dehydrogenase (des) and cyclopropane fatty acid synthase (cfa) genes, as well as the addition of exogenous fatty acids. The fatty acid synthesis of K. hansenii HDM1-3 may be mediated by the activation of two-component sensor signaling pathways in response to the acid stress. The acid resistance mechanism of K. hansenii HDM1-3 adds to our knowledge of the acid stress adaptation, which may facilitate the development of new strategies for improving the industrial performance of this species under acid stress.

The Cyclopropane Fatty Acid Synthase Mediates Antibiotic Resistance and Gastric Colonization of Helicobacter pylori.

Cyclopropane fatty acids (CFAs) are synthetized by the addition of a methylene group from S-adenosyl-l-methionine across the carbon-carbon double bonds of unsaturated fatty acid chains of membrane phospholipids. This fatty acid cyclopropanation, catalyzed by the CFA synthase (CfaS) enzyme, occurs in many bacteria, including the human pathogen Helicobacter pylori Although the cyclopropane modification was reported to play a key role in the adaptation in response to environmental stress, its role in H. pylori remains unknown. In this study, we showed that H. pylori HP0416 encodes a functional CfaS. The enzyme was demonstrated to be required for acid resistance, antibiotic resistance, intracellular survival and mouse gastric colonization, and cell membrane integrity. Moreover, the tool compound dioctylamine, which acts as a substrate mimic, directly inhibits the CfaS function of H. pylori, resulting into sensitivity to acid stress, increased antibiotic susceptibility, and attenuated abilities to avoid macrophage killing and to colonize mouse stomachs. These results validate CfaS as a promising antibiotic target and provide new potentials for this recognized target in future anti-H. pylori drug discovery efforts.IMPORTANCE The increasing prevalence of multidrug-resistant Helicobacter pylori strains has created an urgent need for alternative therapeutic regimens that complement the current antibiotic treatment strategies for H. pylori eradication; however, this is greatly hampered due to a lack of "druggable" targets. Although the CFAs are present in H. pylori cytoplasmic membranes at high levels, their physiological role has not been established. In this report, deletion of the CFA synthase CfaS was shown to attenuate acid and drug resistance, immune escape, and gastric colonization of H. pylori These findings were validated by inhibition of the CfaS activity with the tool compound dioctylamine. These studies identify this enzyme as an attractive target for further drug discovery efforts against H. pylori.