Production of 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from olive oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.     

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301–306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25°C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.     

Production of 7, 10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from triolein via lipase induction by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.     

Production of a value-added hydroxy fatty acid, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid,from high oleic safflower Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally obtained in small amounts from plant systems, are good examples of structurally modified lipids, and they render special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial potential in a wide range of applications. Recently, various microbial strains were tested for the production of HFAs from different unsaturated fatty acids since HFA production is limited to plant systems. Among the microbial strains tested, Pseudomonas aeruginosa PR3 has been well studied for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) from oleic acid. Previously, we reported that strain PR3 could utilize triolein instead of oleic acid as a substrate for the production of DOD (Appl. Microbiol. Biotechnol. 2007, 74: 301–306). In this study, we focused on utilization of vegetable oil as a substrate for DOD production by PR3. Consequently, strain PR3 efficiently utilized high oleic safflower oil as a substrate for DOD production. Optimal initial medium pH and incubation time were pH 8.0 and 72 h, respectively. Optimal carbon and nitrogen sources were fructose and glutamine, respectively. Results from this study demonstrate that normal vegetable oils could be used as efficient substrates for the production of value-added HFAs by microbial bioconversion.     

Production and identification of a novel compound, 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-hexadecenoic acid from palmitoleic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids are considered as important value-added product for industrial application because of their special properties such as higher viscosity and reactivity. Microbial production of the hydroxy fatty acids from various fatty acid substrates have been actively studied using several microorganisms. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were produced from oleic acid and ricinoleic acid, respectively. Based on the postulated common metabolic pathway involved in DOD and TOD formation by PR3, it was assumed that palmitoleic acid containing a singular 9-cis double bond, common structural property sharing with oleic acid and ricinoleic acid, could be utilized by PR3 to produce hydroxy fatty acid. In this study, we tried to use palmitoleic acid as substrate for production of hydroxy fatty acid by PR3 and firstly confirmed that PR3 could produce 7,10-dihydroxy-8(E)-hexadecenoic acid (DHD) with 23% yield from palmitoleic acid. DHD production was peaked at 72 h after the substrate was added to the 24-h-culture.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

Cloning,expression and characterization of a lipase gene (<Emphasis Type="Italic">lip3</Emphasis>) from<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> LST-03

A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0–35°C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.Communicated by H. Ikeda     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.     

Biocidal effect of (<Emphasis Type="Italic">E</Emphasis>)-anethole on the cyanobacterium <Emphasis Type="Italic">Aphanizomenon gracile</Emphasis> Lemmermann

Biocidal natural substances of botanical origin offer a promising ecofriendly option for controlling toxic cyanobacteria. Herein, we study 11 essential oils and some of their major components for their activity on Aphanizomenon gracile. On the basis of our results we support that Origanum vulgare and O. dictamnus, Ocimum basilicum, Eucalyptus meliodora, Melissa officinalis, and Pimpinella anisum exhibited the strongest activities, and the IC_50/1d values of the extracts were calculated to be between 168.43 and 241.97 μg mL^?1. When the major components of the biocidal essential oils were tested individually, (E)-anethole was found active, exhibiting an IC_50/1d value of 71.35 μg mL^?1. On the other hand, the half-life (t _1/2 ) of (E)-anethole was calculated at 1 h. A preliminary attempt of (E)-anethole microencapsulation was conducted, in order to slowly release this biocidal agent, increasing the residual life under open air conditions and thus the biological activity. Results were promising since the microencapsulated product exhibited better activity than did the non-formulated (E)-anethole. This is a first report on the biocidal activity of EOs and (E)-anethole on A. gracile and a preliminary indication of the microencapsulated (E)-anethole potential use as a natural biocidal in fresh waters.

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Graphical abstract Filaments of Aphanizomenon gracile capable of bloom forming (strain SAG 31.79, in a batch culture) and (E)-anethole

     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

Antibiotic synergy against biofilm-forming <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Eight antibiotics (aztreonam, ceftazidim, cefoperazon, cefepim, netilmicin, amikacin, ofloxacin and ciprofloxacin) exhibited antimicrobial activity individually and/or in combinations against 20 wild-type biofilm-forming strains of Pseudomonas aeruginosa. The strains were less susceptible in biofilm; in 10 strains antibiotic synergy was observed for the combination of aztreonam and ciprofloxacin. Synergy was also demonstrated in the case of β-lactams and aminoglycosides, β-lactams and fluoroquinolones, aminoglycosides and fluoroquinolones, and for monobactams and β-lactams although the strains were resistant to the individual antibiotics. Synergism or partial synergism was found with one or more antibiotic combinations against 32.4% of isolates.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

N-acetylcysteine inhibit biofilms produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Pseudomonas aeruginosa is a common pathogen in chronic respiratory tract infections. It typically makes a biofilm, which makes treatment of these infections difficult. In this study, we investigated the inhibitory effects of N-acetylcysteine (NAC) on biofilms produced by P. aeruginosa.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Evaluation of the toxicity of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis> extract

In this study, the methanol extract of Arthrospira (Spirulina) platensis was examined for acute and subchronic toxicities. The extract did not produce any sign of toxicity within 7 days after feeding it at a single high dose of 6 g kg⁻¹ body weight to female and male Swiss mice. For the subchronic toxicity test, the extract at doses of 6, 12, and 24 mg kg⁻¹ body weight was orally administered to six male and six female Wistar rats daily for 12 weeks. Throughout the study period, we did not observe any abnormalities on behavior, food and water intakes, and health status among the treated animals. The hematology and clinical chemistry parameters of treated groups did not significantly differ from those of the controls in both sexes. Postmortem examination of the test groups also showed no abnormalities in both gross and histological findings. These results thus suggest that the methanol extract of A. platensis did not cause acute or subchronic toxicity in our experimental animals.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.