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.     

Cloning,expression, purification,and characterization of recombinant flagellin isolated from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa as an opportunistic pathogen causes lethal infections in immunocompromised individuals. This bacterium possesses a polar flagellum made up of flagellin subunits. Flagella have important roles in motility, chemotaxis, and establishment of P. aeruginosa in acute phase of infections. Isolation, cloning, and expression of flagellin were aimed at in this study. Flagellin gene (fliC) of P. aeruginosa strain 8821M was isolated by PCR and cloned into a pET expression vector. The recombinant flagellin (46 kDa) was overexpressed as inclusion bodies (IBs). IBs were solubilized in guanidine hydrochloride (GuHCl) followed by affinity-purification and renatured using Ni²⁺-Sepharose resin. Recombinant flagellins reacted with the serum from a rabbit previously immunized with native flagellin. In addition, polyclonal antiserum raised against the recombinant flagellin was shown to significantly inhibit the cell motility of P. aeruginosa strain 8821M in vitro.     

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.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

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.     

Cobalt interference in iron-uptake could inhibit growth in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The present study was undertaken to investigate the possible inhibition of growth in Pseudomonas aeruginosa by interfering with its iron-uptake mechanism. Cobalt was employed as a possible competitive inhibitor of iron-uptake because of its similar size. The results indicate that cobalt competes effectively with iron for uptake by the bacterial cells and interference with iron-uptake could provide an effective means for inhibiting growth in P. aeruginosa.     

Selective loss of <Emphasis Type="Italic">lin</Emphasis> genes from hexachlorocyclohexane-degrading <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ITRC-5 under different growth conditions

The chlorinated insecticide γ-hexachlorocyclohexane (γ-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to β-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on γ-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria–Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by “γ-HCH grown cells” is slower, when compared with “technical-HCH grown cells”, and is completely lost by “LB-grown cells”. The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.     

Palm oil utilization for the simultaneous production of polyhydroxyalkanoates and rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Direct utilization of palm oil for the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids was demonstrated using Pseudomonas aeruginosa IFO3924. By secreted lipase, palm oil was hydrolyzed into glycerol and fatty acids. Fatty acids became favorable carbon sources for cell growth and PHA production via β-oxidation and glycerol for rhamnolipid production via de novo fatty acid synthesis. Both PHA and rhamnolipid syntheses started after the nitrogen source was exhausted and cell growth ceased. PHA synthesis continued until all fatty acids were exhausted, and at that time, PHA content in the cells reached a maximum, but stopped despite the remaining glycerol (<2g/l). In contrast, rhamnolipid synthesis continued until glycerol was exhausted.     

Characterisation of potential virulence markers in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from drinking water

Pseudomonas aeruginosa isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, hemaglutinins, cytotoxins and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics, human serum sensitivity and the survival of P. aeruginosa isolates in a chlorinated environment were also examined. Of the 30 isolates tested, 16 possessed the capacity to adhere to abiotic surfaces, and 28 to adhere to epithelial cells; 30 were capable of producing hemolysins, 27 produced cytotoxins, 9 hemagglutinins, and 18 were classified as serum-resistant. For the lowest concentration of chlorine (0.2 mg/l) tested, no killing of biofilm bacteria could be discerned, even after prolonged exposure to the agent. Although all the drinking water isolates were susceptible to aztreonam, cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, piperacillin-tazobactam, and polymyxin, the P. aeruginosa isolates were resistant to one or more antibiotics. The increasing prevalence of resistance in the isolates from environmental sources may have important therapeutic implications. A notable proportion of the P. aeruginosa isolates from drinking water were able to develop virulence factors, and the incidence of virulence properties was not statistically different among the three sources. A more extensive study of the virulence properties of this bacterium by toxic assays on animals should be explored. Still more interesting would be toxicity assays on immuno-deficient animals with isolates from drinking water in order to better understand the health risk these bacteria may present.     

Cloning,expression and characterization of a Baeyer-Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.     

Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03

Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.     

Evolutionary Analysis of the Two-Component Systems in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Gene organization and functional motif analyses of the 123 two-component system (2CS) genes in Pseudomonas aeruginosa PAO1 were carried out. In addition, NJ and ML trees for the sensor kinases and the response regulators were constructed, and the distances measured and comparatively analyzed. It was apparent that more than half of the sensor-regulator gene pairs, especially the 2CSs with OmpR-like regulators, are derivatives of a common ancestor and have most likely co-evolved through gene pair duplication. Several of the 2CS pairs, especially those with NarL-like regulators, however, appeared to be relatively divergent. This is supportive of the recruitment model, in which a sensor gene and regulator gene with different phylogenetic history are assembled to form a 2CS. Correlation of the classification of sensor kinases and response regulators provides further support for these models. Upon comparison of the phylogenetic trees comprised of sensors and regulators, we have identified six congruent clades, which represent the group of the most recently duplicated 2CS gene pairs. Analyses of the congruent 2CS pairs of each of the clades revealed that certain paralogous 2CS pairs may carry a redundant function even after a gene duplication event. Nevertheless, comparative analysis of the putative promoter regions of the paralogs suggested that functional redundancy could be prevented by a differential control. Both codon usage and G+C content of these 2CS genes were found to be comparable with those of the P. aeruginosa genome, suggesting that they are not newly acquired genes.Reviewing Editor: Dr. Martin Kreitman     

Iron dictates the virulence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in urinary tract infections

Iron-limiting conditions have been reported to be prevalent in the milieu of urinary tract. In the present investigation, effect of iron on virulence of uropathogenic Pseudomonas aeruginosa in planktonic and biofilm cell mode was studied. Significant enhancement in elaboration of all the virulence traits along with increased adherence to uroepithelial cells and decreased phagocytosis of P. aeruginosa was observed following growth in iron-deplete medium. On the contrary, decrease in all these parameters except phagocytosis was observed when P. aeruginosa was grown in iron-rich medium. In vivo, P. aeruginosa grown in iron-deplete medium showed increased renal bacterial load and tissue pathology in a mouse model of ascending urinary tract infection compared with organisms grown in iron-replete medium. The results of the present study may help in understanding host–parasite interaction and in developing alternative preventive approach against P. aeruginosa induced urinary tract infections.     

Concentration dependent growth/non-growth linked kinetics of endosulfan biodegradation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The rates of biodegradation of endosulfan by P. aeruginosa were determined with different initial endosulfan concentrations (10, 50, 100, 150, 200 and 250 mg l⁻¹) and different growth linked kinetic models were fitted at these concentrations. At 10 mg endosulfan l⁻¹, Monod no growth model was well fitted. Monod with growth model described the biodegradation pattern at an initial concentration of 50, 100 and 150 mg endosulfan l⁻¹. Significant increases of P. aeruginosa MN2B14 density in broth culture during incubation further support this result. Conversely, zero order kinetic model was well fitted into the biodegradation data if initial endosulfan concentration was ≥200 mg endosulfan l⁻¹. The kinetics of endosulfan biodegradation by P. aeruginosa MN2B14 in liquid broth was highly dependent upon its initial concentration. The results of this study could be employed for predicting the persistence of endosulfan in water environment containing P. aeruginosa as an endosulfan degrading bacterium.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mucoid strain 8830 binds glycans containing the sialyl-Lewis x epitope

Pseudomonas aeruginosa infection of patients with cystic fibrosis (CF) is a leading cause of their morbidity and mortality. Pathogenesis is initiated in part by molecular interactions of P. aeruginosa with carbohydrate residues in airway mucins that accumulate in the lungs of patients with this disease. To explore the nature of the glycans recognized by a stable, mucoid, alginate-producing strain P. aeruginosa 8830 we generated a genetically modified Pa8830 expressing green fluorescent protein (Pa3380-GFP). We tested its binding to a panel of glycolipids and neoglycolipids in which selected glycans were covalently attached to dipalmitoyl phosphatidylethanolamine and analyzed on silica gel surfaces. Among all glycans tested, Pa8830-GFP bound best to sialyl-Le^x-containing glycan NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R and bound weakly to H-type blood group Fucα1-2Galβ1-4GlcNAc-R, sialyl-lactose, and Le^x, and exhibited little binding toward non-fucosylated derivatives. Interestingly, while Pa8830-GFP bound to the glycosphingolipid asialoGM1, it did not appear to bind to a wide variety of other glycosphingolipids including GM1, GM2, asialoGM2, and sulfatide. These results indicate that P. aeruginosa 8830 has preferential binding to sialyl-Le^x-containing glycans and has weak recognition of related fucose- and sialic acid-containing glycans. The finding that Pa8830 binds sialyl-Le^x-containing glycans, which occur at increased levels in mucins from CF patients, is consistent with studies of other strains of P. aeruginosa and further suggests that such glycans on CF mucins contribute to disease pathogenesis. Invited Submission from Dr. Subhash Basu, from the 7th International Symposium on Biochemical Roles of Eukaryotic Cell Surface Macromolecules in Puri, India, January, 2005.     

Enhanced crude oil biodegradability of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ZJU after preservation in crude oil-containing medium

This study investigated the enhanced crude oil biodegradability of Pseudomonas aeruginosa ZJU, a strain isolated from the Shengli oil field (Shandong Province, China), after preservation in a crude oil-containing medium. This strain previously could not emulsify crude oil during preservation, but after switching to a subculture in a glycerol medium for passages, it expressed increased biodegradation of crude oil within the first six passages and this biodegradation sharply decreased after the seventh passage. It is noticed that about 70% of crude oil was degraded by Pseudomonas aeruginosa ZJU in the third passage while this biodegradability was less than 19% in the seventh passage. Similar to the trend on biodegradation of crude oil, rhamnolipid production increased during the first six passages and later sharply decreased. Thus, it seems that biodegradability was proportionally related to the rhamnolipid productivity in each passage in glycerol medium. Interestingly, both rhamnolipid production and crude oil biodegradation were maintained if this strain was continuously preserved in crude oil and could be retrieved if this strain was then re-preserved in crude oil-containing medium for seven days after the significant decline in these two characteristics previously observed in the seventh passage.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.