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1.  Stimulation of adult neural stem cells with a novel glycolipid biosurfactant 
Acta neurologica Belgica  2013;113(4):10.1007/s13760-013-0232-4.
Glycolipids are amphipatic molecules which are highly expressed on cell membranes in skin and brain where they mediate several key cellular processes. Neural stem cells are defined as undifferentiated, proliferative, multipotential cells with extensive self-renewal and are responsive to brain injury. Di-rhamnolipid: α-L-rhamnopyranosyl-(1-2)α-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoic acid, also referred to as di-rhamnolipid BAC-3, is a glycolipid isolated from bacteria Pseudomonas aeruginosa. In the previous studies di-rhamnolipid enhanced dermal tissue healing and regeneration. The present study provides the first assessment of di-rhamnolipid, and glycolipid-biosurfactants in general, on the nervous system. Treatment of neural stem cells isolated from the lateral ventricle of adult mice and cultured in defined media containing growth factors at 0.5 and 1 μg/ml of di-rhamnolipid increased the number of neurospheres (2.7 and 2.8 fold, respectively) compared to controls and this effect remained even after passaging in the absence of di-rhamnolipid. Additionally, neural stem cells treated with di-rhamnolipid at 50 and 100 μg/ml in defined media supplemented with fetal calf serum and without growth factors exhibited increased cell viability indicating an interaction between di-rhamnolipid and serum components in the regulation of neural stem cells and neuroprogenitors. Intracerebroventricular administration of di-rhamnolipid at 300 and 120 ng/day increased the number of neurospheres (1.3 and 1.63 fold, respectively) that could be derived from the anterior lateral ventricles of adult mice. These results indicate that di-rhamnolipid stimulates proliferation of neural stem cells and increases their endogenous pools which may have therapeutic potential in managing neurodegenerative or neuropsychiatric disorders and promoting nervous tissue regeneration following injury.
PMCID: PMC3855528  PMID: 23846482
di-rhamnolipid; Pseudomonas aeruginosa; biosurfactant; neural stem cells; nervous tissue regeneration
2.  Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). 
Applied and Environmental Microbiology  1992;58(10):3276-3282.
A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.
PMCID: PMC183091  PMID: 1444363
3.  HOY1, a homeo gene required for hyphal formation in Yarrowia lipolytica. 
Molecular and Cellular Biology  1997;17(11):6283-6293.
The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source. A visual screening, called FIL (filamentation minus), for Y. lipolytica yeast growth mutants has been developed. The FIL screen was used to identify three Y. lipolytica genes that abolish hypha formation in all media assayed. Y. lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized. HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene. Analysis of the protein predicts a nuclear location. These observations suggest that Hoy1p may function as a transcriptional regulatory protein. In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation. Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb. Expression of this gene was detected when Y. lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae. The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed.
PMCID: PMC232479  PMID: 9343389
4.  Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium 
Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.
PMCID: PMC1087580  PMID: 15870313
5.  Fatty Acid Cosubstrates Provide β-Oxidation Precursors for Rhamnolipid Biosynthesis in Pseudomonas aeruginosa, as Evidenced by Isotope Tracing and Gene Expression Assays 
Applied and Environmental Microbiology  2012;78(24):8611-8622.
Rhamnolipids have multiple potential applications as “green” surfactants for industry, remediation, and medicine. As a result, they have been intensively investigated to add to our understanding of their biosynthesis and improve yields. Several studies have noted that the addition of a fatty acid cosubstrate increases rhamnolipid yields, but a metabolic explanation has not been offered, partly because biosynthesis studies to date have used sugar or sugar derivatives as the carbon source. The objective of this study was to investigate the role of fatty acid cosubstrates in improving rhamnolipid biosynthesis. A combination of stable isotope tracing and gene expression assays was used to identify lipid precursors and potential lipid metabolic pathways used in rhamnolipid synthesis when fatty acid cosubstrates are present. To this end, we compared the rhamnolipids produced and their yields using either glucose alone or glucose and octadecanoic acid-d35 as cosubstrates. Using a combination of sugar and fatty acids, the rhamnolipid yield was significantly higher (i.e., doubled) than when glucose was used alone. Two patterns of deuterium incorporation (either 1 or 15 deuterium atoms) in a single Rha-C10 lipid chain were observed for octadecanoic acid-d35 treatment, indicating that in the presence of a fatty acid cosubstrate, both de novo fatty acid synthesis and β-oxidation are used to provide lipid precursors for rhamnolipids. Gene expression assays showed a 200- to 600-fold increase in the expression of rhlA and rhlB rhamnolipid biosynthesis genes and a more modest increase of 3- to 4-fold of the fadA β-oxidation pathway gene when octadecanoic acid was present. Taken together, these results suggest that the simultaneous use of de novo fatty acid synthesis and β-oxidation pathways allows for higher production of lipid precursors, resulting in increased rhamnolipid yields.
PMCID: PMC3502905  PMID: 23042167
6.  Timing and Localization of Rhamnolipid Synthesis Gene Expression in Pseudomonas aeruginosa Biofilms 
Journal of Bacteriology  2005;187(1):37-44.
Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 μm have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.
PMCID: PMC538809  PMID: 15601686
7.  Biotreatment of oily wastewater by rhamnolipids in aerated active sludge system*  
Oily wastewater generated by various industries creates a major ecological problem throughout the world. The traditional methods for the oily wastewater treatment are inefficient and costly. Surfactants can promote the biodegradation of petroleum hydrocarbons by dispersing oil into aqueous environment. In the present study, we applied rhamnolipid-containing cell-free culture broth to enhance the biodegradation of crude oil and lubricating oil in a conventional aerobically-activated sludge system. At 20 °C, rhamnolipids (11.2 mg/L) increased the removal efficiency of crude oil from 17.7% (in the absence of rhamnolipids) to 63%. At 25 °C, the removal efficiency of crude oil was over 80% with the presence of rhamnolipids compared with 22.3% in the absence of rhamnolipids. Similarly, rhamnolipid treatment (22.5 mg/L) for 24 h at 20 °C significantly increased the removal rate of lubricating oil to 92% compared with 24% in the absence of rhamnolipids. The enhanced removal of hydrocarbons was mainly attributed to the improved solubility and the reduced interfacial tension by rhamnolipids. We conclude that a direct application of the crude rhamnolipid solution from cell culture is effective and economic in removing oily contaminants from wastewater.
PMCID: PMC2772891  PMID: 19882761
Oily wastewater; Rhamnolipid; Aerated active sludge system; Biodegradation
8.  Flagellin Delivery by Pseudomonas aeruginosa Rhamnolipids Induces the Antimicrobial Protein Psoriasin in Human Skin 
PLoS ONE  2011;6(1):e16433.
The opportunistic pathogen Pseudomonas aeruginosa can cause severe infections in patients suffering from disruption or disorder of the skin barrier as in burns, chronic wounds, and after surgery. On healthy skin P. aeruginosa causes rarely infections. To gain insight into the interaction of the ubiquitous bacterium P. aeruginosa and healthy human skin, the induction of the antimicrobial protein psoriasin by P. aeruginosa grown on an ex vivo skin model was analyzed. We show that presence of the P. aeruginosa derived biosurfactant rhamnolipid was indispensable for flagellin-induced psoriasin expression in human skin, contrary to in vitro conditions. The importance of the bacterial virulence factor flagellin as the major inducing factor of psoriasin expression in skin was demonstrated by use of a flagellin-deficient mutant. Rhamnolipid mediated shuttle across the outer skin barrier was not restricted to flagellin since rhamnolipids enable psoriasin expression by the cytokines IL-17 and IL-22 after topical application on human skin. Rhamnolipid production was detected for several clinical strains and the formation of vesicles was observed under skin physiological conditions. In conclusion we demonstrate herein that rhamnolipids enable the induction of the antimicrobial protein psoriasin by flagellin in human skin without direct contact of bacteria and responding cells. Hereby, human skin might control the microflora to prevent colonization of unwanted microbes in the earliest steps before potential pathogens can develop strategies to subvert the immune response.
PMCID: PMC3026827  PMID: 21283546
9.  The Autotransporter Esterase EstA of Pseudomonas aeruginosa Is Required for Rhamnolipid Production, Cell Motility, and Biofilm Formation▿  
Journal of Bacteriology  2007;189(18):6695-6703.
Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.
PMCID: PMC2045186  PMID: 17631636
10.  Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440 
Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.
Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.
A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.
PMCID: PMC3258213  PMID: 21999513
flux analysis; quantitative physiology; metabolic network; biodetergent; non-pathogenic Pseudomonas; biosurfactants; rhamnolipids; off-gas analysis; 13C labeling; BlueSens
11.  Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane. 
In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC201607  PMID: 8031099
12.  Pseudomonas aeruginosa Exopolysaccharide Psl Promotes Resistance to the Biofilm Inhibitor Polysorbate 80 
Polysorbate 80 (PS80) is a nonionic surfactant and detergent that inhibits biofilm formation by Pseudomonas aeruginosa at concentrations as low as 0.001% and is well tolerated in human tissues. However, certain clinical and laboratory strains (PAO1) of P. aeruginosa are able to form biofilms in the presence of PS80. To better understand this resistance, we performed transposon mutagenesis with a PS80-resistant clinical isolate, PA738. This revealed that mutation of algC rendered PA738 sensitive to PS80 biofilm inhibition. AlgC contributes to the biosynthesis of the exopolysaccharides Psl and alginate, as well as lipopolysaccharide and rhamnolipid. Analysis of mutations downstream of AlgC in these biosynthetic pathways established that disruption of the psl operon was sufficient to render the PA738 and PAO1 strains sensitive to PS80-mediated biofilm inhibition. Increased levels of Psl production in the presence of arabinose in a strain with an arabinose-inducible psl promoter were correlated with increased biofilm formation in PS80. In P. aeruginosa strains MJK8 and ZK2870, known to produce both Pel and Psl, disruption of genes in the psl but not the pel operon conferred susceptibility to PS80-mediated biofilm inhibition. The laboratory strain PA14 does not produce Psl and does not form biofilms in PS80. However, when PA14 was transformed with a cosmid containing the psl operon, it formed biofilms in the presence of PS80. Taken together, these data suggest that production of the exopolysaccharide Psl by P. aeruginosa promotes resistance to the biofilm inhibitor PS80.
PMCID: PMC3421584  PMID: 22585230
13.  Rhamnolipid-Induced Removal of Lipopolysaccharide from Pseudomonas aeruginosa: Effect on Cell Surface Properties and Interaction with Hydrophobic Substrates 
Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.
PMCID: PMC92143  PMID: 10919779
14.  Increase in Rhamnolipid Synthesis under Iron-Limiting Conditions Influences Surface Motility and Biofilm Formation in Pseudomonas aeruginosa▿ † 
Journal of Bacteriology  2010;192(12):2973-2980.
Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.
PMCID: PMC2901684  PMID: 20154129
15.  Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype†  
Eukaryotic Cell  2005;4(2):356-364.
We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.
PMCID: PMC549329  PMID: 15701798
16.  Analysis of biosurfactants from industrially viable Pseudomonas strain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity 
Rhamnolipid biosurfactants produced mainly by Pseudomonas sp. had been reported to possess a wide range of potential industrial application. These biosurfactants are produced as monorhamnolipid (MRL) and di-rhamnolipid (DRL) congeners. The present study deals with rhamnolipid biosurfactants produced by three bacterial isolates from crude oil. Biosurfactants produced by one of the strains (named as IMP67) was found to be very efficacious based on its critical micelle concentration value and hydrocarbon emulsification property. Strikingly, antimicrobial, and anti-biofilm potential of this biosurfactant were higher than biosurfactants produced by other two strains. Thin layer chromatography analysis and rhamnose quantification showed that the rhamnolipids of IMP67 had more MRL congeners than biosurfactants of the other two strains. Emulsification and antimicrobial actions were affected by manual change of MRL and DRL congener proportions. Increase of MRL proportion enhanced emulsification index and antimicrobial property to Gram negative bacteria. This result indicated that the ratio of MRL and DRL affected the emulsification potentials of rhamnolipids, and suggested that high emulsification potentials might enhance rhamnolipids to penetrate the cell wall of Gram negative bacteria. In line with this finding, rhamnolipids of IMP67 also reduced the MIC of some antibiotics against bacteria, suggesting their synergistic role with the antibiotics.
PMCID: PMC4273660  PMID: 25566212
biosurfactant; rhamnolipid congeners; emulsification; thin layer chromatography; MRL-DRL proportion; antimicrobial action; biofilm disruption
17.  Effects of azithromycin on Pseudomonas aeruginosa isolates from catheter-associated urinary tract infection 
Pseudomonas aeruginosa is a common pathogenic bacterium in urinary tract infections (UTIs), particularly catheter-associated UTIs. The aim of this study was to investigate the effect of azithromycin (AZM) on P. aeruginosa isolated from UTIs. Isolates were identified by biochemical assays and the Vitek system. Antimicrobial susceptibility was determined using the disk diffusion assay. Biofilm formation and adhesion were assayed using a crystal violet staining method. The swimming motility was assayed on agar plates. The elastase activity and rhamnolipid production were determined by the elastin-Congo red method and orcinol reaction, respectively. A total of 32 bacterial isolates were collected from 159 urinary catheters and eight of them were P. aeruginosa isolates. The results showed that the P. aeruginosa isolates had stronger biofilm formation capability and the biofilms were thicker than those of P. aeruginosa PAO1. AZM inhibited biofilm formation and adhesion on urinary catheters, and also decreased swimming motility and the production of virulence factors. The results of this study indicated that AZM is potentially a good choice for use in the treatment of UTIs.
PMCID: PMC4280936  PMID: 25574236
Pseudomonas aeruginosa; azithromycin; urinary tract infection; biofilm; swimming; virulence factor
18.  Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core 
PLoS ONE  2012;7(6):e39976.
Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
PMCID: PMC3386995  PMID: 22768188
19.  Simultaneous Inhibition of Rhamnolipid and Polyhydroxyalkanoic Acid Synthesis and Biofilm Formation in Pseudomonas aeruginosa by 2-Bromoalkanoic Acids: Effect of Inhibitor Alkyl-Chain-Length 
PLoS ONE  2013;8(9):e73986.
Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.
PMCID: PMC3762805  PMID: 24023921
20.  Disruption of Contact Lens–Associated Pseudomonas aeruginosa Biofilms Formed in the Presence of Neutrophils 
This study identifies a mechanism for neutrophil-enhanced early biofilm development on contact lens surfaces and indicates a potential new strategy in the prevention of pathogenic biofilm formation during contact lens wear.
To evaluate the capacity of neutrophils to enhance biofilm formation on contact lenses by an infectious Pseudomonas aeruginosa (PA) corneal isolate. Agents that target F-actin and DNA were tested as a therapeutic strategy for disrupting biofilms formed in the setting of neutrophils in vitro and for limiting the infectious bioburden in vivo.
Biofilm formation by infectious PA strain 6294 was assessed in the presence of neutrophils on a static biofilm plate and on unworn etafilcon A soft contact lenses. A d-isomer of poly(aspartic acid) was used alone and with DNase to reduce biofilm formation on test contact lenses. The gentamicin survival assay was used to determine the effectiveness of the test compound in reducing subsequent intracellular bacterial load in the corneal epithelium in a contact lens infection model in the rabbit.
In a static reactor and on hydrogel lenses, PA biofilm density was enhanced 30-fold at 24 hours in the presence of neutrophils (P < 0.0001). The combination of DNase and anionic poly(aspartic acid) reduced the PA biofilms formed in the presence of activated neutrophils by 79.2% on hydrogel contact lenses (P < 0.001). An identical treatment resulted in a 41% reduction in internalized PA in the rabbit corneal epithelium after 24 hours (P = 0.03).
These results demonstrate that PA can exploit the presence of neutrophils to form biofilm on contact lenses within a short time. Incorporation of F-actin and DNA represent a mechanism for neutrophil-induced biofilm enhancement and are targets for available agents to disrupt pathogenic biofilms formed on contact lenses and as a treatment for established corneal infections.
PMCID: PMC3088567  PMID: 21245396
21.  Salmonella enterica Serovar Typhimurium Swarming Mutants with Altered Biofilm-Forming Abilities: Surfactin Inhibits Biofilm Formation 
Journal of Bacteriology  2001;183(20):5848-5854.
Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation. We report that surfactin inhibits biofilm formation of wild-type S. enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested had similar effects.
PMCID: PMC99661  PMID: 11566982
22.  Maggot Excretions Inhibit Biofilm Formation on Biomaterials 
Biofilm-associated infections in trauma surgery are difficult to treat with conventional therapies. Therefore, it is important to develop new treatment modalities. Maggots in captured bags, which are permeable for larval excretions/secretions, aid in healing severe, infected wounds, suspect for biofilm formation. Therefore we presumed maggot excretions/secretions would reduce biofilm formation.
We studied biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae on polyethylene, titanium, and stainless steel. We compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times. Maggot excretions/secretions were added to existing biofilms to examine their effect.
Comb-like models of the biomaterials, made to fit in a 96-well microtiter plate, were incubated with bacterial suspension. The formed biofilms were stained in crystal violet, which was eluted in ethanol. The optical density (at 595 nm) of the eluate was determined to quantify biofilm formation. Maggot excretions/secretions were pipetted in different concentrations to (nonstained) 7-day-old biofilms, incubated 24 hours, and finally measured.
The strongest biofilms were formed by S. aureus and S. epidermidis on polyethylene and the weakest on titanium. The highest quantity of biofilm formation was reached within 7 days for both bacteria. The presence of excretions/secretions reduced biofilm formation on all biomaterials. A maximum of 92% of biofilm reduction was measured.
Our observations suggest maggot excretions/secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials.
PMCID: PMC2939353  PMID: 20309656
23.  Maggot Excretions Inhibit Biofilm Formation on Biomaterials 
Biofilm-associated infections in trauma surgery are difficult to treat with conventional therapies. Therefore, it is important to develop new treatment modalities. Maggots in captured bags, which are permeable for larval excretions/secretions, aid in healing severe, infected wounds, suspect for biofilm formation. Therefore we presumed maggot excretions/secretions would reduce biofilm formation.
We studied biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae on polyethylene, titanium, and stainless steel. We compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times. Maggot excretions/secretions were added to existing biofilms to examine their effect.
Comb-like models of the biomaterials, made to fit in a 96-well microtiter plate, were incubated with bacterial suspension. The formed biofilms were stained in crystal violet, which was eluted in ethanol. The optical density (at 595 nm) of the eluate was determined to quantify biofilm formation. Maggot excretions/secretions were pipetted in different concentrations to (nonstained) 7-day-old biofilms, incubated 24 hours, and finally measured.
The strongest biofilms were formed by S. aureus and S. epidermidis on polyethylene and the weakest on titanium. The highest quantity of biofilm formation was reached within 7 days for both bacteria. The presence of excretions/secretions reduced biofilm formation on all biomaterials. A maximum of 92% of biofilm reduction was measured.
Our observations suggest maggot excretions/secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials.
PMCID: PMC2939353  PMID: 20309656
24.  Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. 
Journal of Bacteriology  1994;176(7):2044-2054.
A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.
PMCID: PMC205310  PMID: 8144472
25.  Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureus strains with different biofilm-forming abilities 
PeerJ  2014;2:e326.
Chronic wounds are a major global health problem. Their management is difficult and costly, and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative wound treatments. Honey is now being revisited as an alternative treatment due to its broad-spectrum antibacterial activity and the inability of bacteria to develop resistance to it. Many previous antibacterial studies have used honeys that are not well characterized, even in terms of quantifying the levels of the major antibacterial components present, making it difficult to build an evidence base for the efficacy of honey as an antibiofilm agent in chronic wound treatment. Here we show that a range of well-characterized New Zealand manuka-type honeys, in which two principle antibacterial components, methylglyoxal and hydrogen peroxide, were quantified, can eradicate biofilms of a range of Staphylococcus aureus strains that differ widely in their biofilm-forming abilities. Using crystal violet and viability assays, along with confocal laser scanning imaging, we demonstrate that in all S. aureus strains, including methicillin-resistant strains, the manuka-type honeys showed significantly higher anti-biofilm activity than clover honey and an isotonic sugar solution. We observed higher anti-biofilm activity as the proportion of manuka-derived honey, and thus methylglyoxal, in a honey blend increased. However, methylglyoxal on its own, or with sugar, was not able to effectively eradicate S. aureus biofilms. We also demonstrate that honey was able to penetrate through the biofilm matrix and kill the embedded cells in some cases. As has been reported for antibiotics, sub-inhibitory concentrations of honey improved biofilm formation by some S. aureus strains, however, biofilm cell suspensions recovered after honey treatment did not develop resistance towards manuka-type honeys. New Zealand manuka-type honeys, at the concentrations they can be applied in wound dressings are highly active in both preventing S. aureus biofilm formation and in their eradication, and do not result in bacteria becoming resistant. Methylglyoxal requires other components in manuka-type honeys for this anti-biofilm activity. Our findings support the use of well-defined manuka-type honeys as a topical anti-biofilm treatment for the effective management of wound healing.
PMCID: PMC3970805  PMID: 24711974
Staphylococcus aureus; Biofilm; Honey; Antibacterial; Wounds; Methylglyoxal

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