Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa
© Tielen et al.; licensee BioMed Central Ltd. 2013
Received: 19 March 2013
Accepted: 8 July 2013
Published: 13 July 2013
As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa.
Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain.
We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.
Pseudomonas aeruginosa is a Gram-negative bacterium which is ubiquitous in water and soil. It is able to produce and secrete several hydrolases which are important for nutrition of the bacterium, for biofilm structure  and, moreover, as virulence factors . As an opportunistic human pathogen, P. aeruginosa can cause severe acute and chronic infections, especially in immuno-compromized patients. In addition to infections of the urinary tract, wounds, middle ear and eyes, P. aeruginosa is well known as the causative agent of chronic lung infections of cystic fibrosis (CF) patients . Most of these infections are biofilm-associated [4, 5]. Biofilms represent a bacterial state of life in which the cells are attached to biotic or abiotic surfaces or to each other. Thereby, they are embedded in a matrix of self-produced extracellular polymeric substances (EPS). Different amounts of polysaccharides, lipids, nucleic acids and proteins can be detected in the EPS matrix of biofilms formed by P. aeruginosa. Part of the proteins show enzyme activities in vitro and in vivo. The expression of several exoenzyme encoding genes was detected in the sputum of infected CF-patients by transcriptome analysis  and the presence of significant levels of extracellular enzyme specific antibodies in sera of infected CF patients is an indirect evidence for the production of extracellular enzymes during infection processes [7, 8]. Therefore, the biofilm matrix of P. aeruginosa was described as a reservoir of enzymes .
The main extracellular enzymes produced by P. aeruginosa are type I and II-secreted hydrolases, including alkaline protease , elastase A (LasA) and B (LasB) , phospholipase C  and lipases [13, 14]. These enzymes alone or synergistically with others are causing cell death, severe tissue damage and necrosis in the human host [2, 15, 16]. The simultaneous production of these exoenzymes and polysaccharides were described for P. aeruginosa[17, 18]. During persistent CF-lung infections the conversion to a mucoid, i.e. an alginate overproducing phenotype is commonly observed . Alginate is a high-molecular weight extracellular copolymer consisting the uronic acid monomers β-D-mannuronate and its C-5 epimer α-L-guluronate, which are linked by 1,4-glycosidic bonds [20, 21]. These components are arranged in homopolymeric blocks of poly-β-D-mannuronate and heteropolymeric sequences with random distribution of mannuronate and guluronate residues . In contrast to the alginate produced by brown algae the alginate from P. aeruginosa contains O-acetyl groups on the C2- and/or C3-position of the β-D-mannuronate residues. This acetylation significantly influences the physico-chemical properties of the polymer, such as the viscosity [23, 24], the ability to bind divalent cations [23, 25] and the water-binding capacity . All of these features are important for the structure and the mechanical stability of the biofilm [24, 27, 28]. The extracellular alginate forms a highly hydrated matrix in which the bacteria cells are embedded. It can protect the cells from dehydration, the activity of antimicrobial substances as antibiotics  and disinfectants  and, moreover, protects the cells from the immune system during the infection process [31, 32]. Several reports described the binding of extracellular enzymes such as lipases to this polysaccharide [33–35], but the type and molecular mechanism of this interaction are still unclear.
Lipases (EC 188.8.131.52) are physiologically and biotechnologically relevant enzymes. In addition to their natural function (hydrolysis of triglycerides), lipases are also able to recognize various substrates and catalyze regio- and enantioselective hydrolysis of many esters. The main extracellular lipase of P. aeruginosa is the 29 kDa lipase LipA , which belongs to the I.1 family of lipases . X-ray studies showed that lipases of this family exhibit an α-helical lid structure, which closes the active centre of the enzyme . The open, active conformation occurs only in contact with the substrate. This complex mechanism is called interfacial activation and can be mediated by a large range of hydrophobic substances, including lipopolysaccharides (LPS) . However, LipA exhibits a lid structure, it does not show an interfacial activation, because interaction with hydrophobic outer membrane components let to a permanent open conformation [13, 38]. Lipase LipA is transported across the cell envelope by the type II secretion system, the main two-step ATP-dependent process of Gram-negative bacteria . It has been reported that mucoid P. aeruginosa strains showed up to 9-fold higher lipase activity than their spontaneous non-mucoid counterparts . The exogenous supplementation of purified bacterial alginate from P. aeruginosa and Azotobacter vinelandii and also algal alginate to the culture media of non-mucoid P. aeruginosa strains increases the release of extracellular lipase from the bacterial cells . It has been hypothesized that this enhanced release of lipase was due to a non-covalent association between lipases and alginate . The co-secretion of LipA and alginate from P. aeruginosa cells may reinforce the synthesis of lipases. Thereby, the removal of the enzyme from the direct cell surface acts as a signal for the bacterial cell . The interaction between lipase and alginate was further used for lipase purification strategies by ethanolic co-precipitation of the two molecules [34, 35]. Beside the biotechnological relevance of this finding, the physiological function of the interaction between extracellular lipase and the polysaccharide alginate for the bacterium remains unclear.
Here, we demonstrate that lipase LipA from P. aeruginosa binds to the extracellular polysaccharide alginate by electrostatic interactions. This interaction localizes the enzyme near the cell surface and enhances the stability of the enzyme against heat and degradation by endogenous proteases.
Results and discussion
Expression of lipase in mucoid Pseudomonas aeruginosa biofilms
Cell density, unronic acid (alginate) content and extracellular lipase activity of agar-grown P. aeruginosa biofilms
Bacterial density × 109(cells/cm2)
Uronic acids (μg/cm2)
Lipase activity (nmol/min x cm2)
1.4 ± 0.3
0.22 ± 0.01
0.12 ± 0.01
1.3 ± 0.2
0.23 ± 0.01
0.14 ± 0.01
1.2 ± 0.1
0.22 ± 0.01
0.0 ± 0.0
1.5 ± 0.6
0.23 ± 0.03
6.50 ± 0.1
1.4 ± 0.2
0.23 ± 0.01
63.02 ± 5.2
The biofilms of the five strains revealed comparable cell densities between 1.2 and 1.5 × 109 cells/cm2 (Table 1). Extracellular lipase activity was determined in cell-free supernatants of biofilm suspensions by a photometric assay, using para-nitrophenylpalmitate (pNPP) as a substrate. The parent strain and the vector control strain showed similar levels of extracellular lipase activity, whereas no extracellular lipase activity was detected in biofilms of the lipA mutant. Complementation of lipA in strain SG81ΔlipA::lipA restored lipase activity, and the lipA overexpression strain displayed significantly enhanced lipase activity that was 525-fold higher compared with the parent strain SG81 (Table 1). Uronic acids (alginate) were detected in all biofilms at nearly the same levels, indicating that alginate production was not influenced by the differential expression of lipase activities.
Localization of lipase activity in Pseudomonas aeruginosa biofilms
A heterogeneous distribution of lipase activity within the biofilms was observed (Figure 1). Cellular activity in most of the cells indicated by the yellow colour and extracellular red-coloured regions surrounding the cells could be distinguished. Significantly more extracellular lipase activity was detected in the LipA overproducing strain P. aeruginosa SG81lipA+, indicating that the visualized extracellular lipase activity was mainly based on the activity of LipA. No extracellular but weak cell-associated activity was observed in the lipase mutant P. aeruginosa SG81ΔlipA. This can be explained by the activity of other lipolytic enzymes such as the outer-membrane bound esterase EstA, which is able to degrade palmitate [14, 47]. The second extracellular lipase LipC of P. aeruginosa is unable to degrade palmitate ester substrates (personal communication). Furthermore, a deletion within the foldase gene lipH may also affect folding and activity of LipC . The defect of extracellular lipolytic activity could be complemented by the expression of lipA in trans from the plasmid pBBL7. Accordingly, the complementation strain P. aeruginosa SG81ΔlipA::lipA revealed a level of lipase activity staining of the biofilms similar to the parent strain P. aeruginosa SG81. The biochemical detection of lipase activity in cell-free material from biofilms and the in situ visualization of lipase activity in the intercellular space of biofilms using palmitate-based enzyme substrates indicate that extracellular lipase is expressed in biofilms of mucoid P. aeruginosa, so that the biofilm matrix represents a reservoir of lipase molecules. By the use of strains that differ in their ability of producing LipA indicated that the major activity of extracellular lipase is due to the presence of lipase LipA.
Binding of lipase LipA to alginate
Previous in vitro studies have demonstrated the stimulation of lipase release from non-mucoid wild-type P. aeruginosa by the addition of purified algal and bacterial alginate to cell suspensions. Moreover, the interaction of lipases and algal alginate with a concomitant stabilization of the enzyme against ethanol-induced denaturation was shown in vitro. On the basis of these observations we hypothesized that extracellular lipase in mucoid P. aeruginosa biofilms might be bound to the alginate in the EPS matrix. Therefore, in vitro binding studies in a microtiter plate assay were conducted using purified LipA from P. aeruginosa and bacterial alginate isolated from mucoid P. aeruginosa SG81 biofilms. For comparison, different neutral (dextran, levan) and negatively charged polysaccharides (algal alginate, xanthan) were tested. The immobilization of the polysaccharides on the polystyrene of the microtiter plate was verified by carbohydrate quantification. Binding of polysaccharides to wells of polystyrene microtiter plates in a concentration of 0.01 - 1.0 mg/ml was also shown before .
In summary, the experiments suggest that the binding of lipases to alginate depends on the negative charged monomers of the polysaccharide indicating ionic interactions between the molecules.
Heat stabilization of lipases by polysaccharides
Inactivation temperatures of lipase LipA calculated by extrapolation of the linear gradient of the heat inactivation curves
Tris–HCl buffer (control)
45.0 +/− 2.5
63.8 +/− 1.1
82.7 +/− 2.9
45.1 +/− 3.5
72.2 +/− 2.6
101.7 +/− 2.8
47.3 +/− 2.2
76.7 +/− 1.2
106.2 +/− 3.2
47.9 +/− 2.5
70.3 +/− 3.3
91.0 +/− 3.0
Alginate SG81, deacetylated
49.2 +/− 3.5
78.5 +/− 1.9
109.0 +/− 3.0
54.0 +/− 4.7
68.1 +/− 2.7
87.2 +/− 3.4
46.1 +/− 3.2
66.1 +/− 3.2
86.2 +/− 3.4
47.8 +/− 3.9
74.1 +/− 1.5
95.4 +/− 2.7
The interaction of enzymes with polysaccharides and the influence on the stability of the proteins was described earlier [35, 51, 52]. Heat stabilization effects were also reported for extracellular lipases from P. aeruginosa. According to our results, the residual lipase activity after 60 min at 70°C in the presence of algal alginate was 15% of the initial activity. Also the stabilization of other bacterial extracellular enzymes by non-covalent associations with exopolysaccharides from the same bacterial species has been described before [53, 54]. This thermostabilizing effect might be relevant for survival of biofilm grown P. aeruginosa cells in environmental habitats under conditions of elevated temperatures as for example sun-shined soil or heated water bodies.
Protection of lipase from proteolytic degradation
A significantly decreased lipase activity was detected after 24 h incubation with LasB in the absence of alginate (p = 7.9 × 10-6). In contrast, no activity was lost in the presence of alginate. Moreover, the experiment again clearly showed that the addition of alginate did not stimulate the lipase activity, since the activity was similar in presence and absence of alginate. A stimulation of lipase activity would be a hint on conformational changes of the lipase protein. However, this seemed not to be happened. Lipase activity was found similar in the presence and in the absence of alginate without proteolytic treatment. Furthermore, no interfacial activation of the lipase was observed. This was expected as discussed above. However, elastase activity measured at the end of the experiment revealed constant over time. These results led to the suggestions that i) LasB is able to degrade the lipase LipA and ii) alginate protects the lipase molecule from degradation, possibly by covering of cleavage sites.
Elastase LasB has been described as one of the major extracellular proteases of P. aeruginosa. The influence of LasB on the biofilm structure of mucoid P. aeruginosa was shown recently . It was hypothesized that the proteolytic degradation of extracellular proteins mediated by LasB changes the physico-chemical properties of the EPS of P. aeruginosa and thereby, influences the structure of the biofilm . Accordingly, a post-translational degradation of extracellular proteins during P. aeruginosa biofilm maturation was shown by proteome analysis . Thereby, LasB has been identified as one of the enzymes involved . Post-translational proteolytic processing cascades of extracellular proteins have also been found in other organisms [57, 58].
Modeling of interaction between lipase and polysaccharide alginate
The interaction between alginate and lipases was hypothesized previously to be predominantly polar and non-specific, since addition of NaCl impaired co-precipitation, whereas Triton X-100 did not [34, 41]. In a number of other studies the formation of complexes of alginate with various proteins such as trypsin, α-chymotrypsin, albumins, human leukocyte elastase and myoglobin has been demonstrated [41, 59, 60] underlining the non-specific binding of alginate to proteins.
Interestingly, the positively charged amino acids are localized on the surface of the protein mainly opposite of the active centre. This resulted in an immobilisation of the protein, while the reactive part of the biocatalyst remains unaffected and is directed to the surrounding environment and the substrate-containing reaction room.
We demonstrate a binding of extracellular lipase LipA to the endogenous exopolysaccharide alginate from P. aeruginosa based on electrostatic interactions. This interaction has important biological advantages for the bacterium in biofilms. First, it prevents extracellular lipases from being rapidly diluted into the surrounding environment - the lipase accumulates and is immobilized near the cells within the alginate matrix, which facilitates the uptake of fatty acids released by the action of lipases. Moreover, the interaction between alginate and the backbone of the protein helps to direct the catalytic site of the enzyme to its substrate and therefore, can enhance the activity level.
A stabilization of the conformation of the enzyme by the interaction with the polysaccharide can be proposed. An evidence for this is the protection against proteolytic degradation and the enhanced heat tolerance of the enzyme. This gives an essential advantage for survival of P. aeruginosa under adverse environmental conditions.
Bacterial strains and cultivation
Bacterial strains and plasmids used in this study
Relevant genotype/ phenotype
E. coli S17-1
thi pro hsdR-M+, chromosomally integrated [RP4-2 Tc::Mu:Kmr::Tn7, Tra+ Trir Strr]
Overexpression of lipA and lipH from pUCPL6A
Mucoid ΔmucA22 CF-lung isolate
ΔalgJ5-mutant derived from FRD1, defect in O-acetylation of alginate
Mucoid biofilm isolate from technical water system
Vector control pBBR1MCS
Δ(2/3 lipA 1/5 lipH)::Ω-Gmr , deletion of lipA and lipH
Deletion of lipA and lipH complemented in trans from pBBL7
Expression of lipA and lipH in trans from pBBL7
lacZ α Cmr mob Plac, PT7
2.8 kbp Xmn I/Sma I fragment with lipA/H operon in pBBR1MCS under Plac control
2.06 kbp Kpn I/Xba I-fragment with Δ(2/3 lipA 1/5 lipH) in pME3087
1.6 kbp Sma I-fragment with Ω-Gmr from pBSL142 in pMEΔAH11
Biofilm cultures were grown for 24 h at 36°C on Pseudomonas Isolation Agar (PIA; Difco) in the form of confluent mucoid lawns. Cell numbers of biofilms, which were scraped from the agar surface and suspended in 0.14 M NaCl, were determined microscopically using a Thoma counting chamber. Cell-free EPS solutions prepared from the biofilm suspensions according to Tielen et al.  were used to measure uronic acid (alginate) concentration and lipase activity as described below. For CLSM analysis, biofilms were grown on membrane-filters (polycarbonate, size: 2.5 cm, pore size: 0.4 μm; Millipore, Billerica, Massachusetts) placed on PIA supplemented with 0.1 M CaCl2 for stabilization of the biofilm matrix as described previously .
Visualization of lipase activity in situ
For visualization of lipase activity in biofilms of P. aeruginosa strains, ELF® 97 palmitate (Molecular Probes, Invitrogen GmbH, Karlsruhe, Germany) was used as a substrate. This enzyme substrate is cleaved by lipases to the water-insoluble ELF® 97 alcohol, which precipitates directly at the site of enzymatic hydrolysis, thus reporting the location of lipase enzyme activity, when visualized by fluorescence microscopy . ELF® 97 palmitate obtained as a solid was dissolved at a concentration of 5 mM in isopropanol under weak heating. 1 ml substrate solution was mixed with 9 ml Sørensen phosphate buffer (pH 8.0) containing 20.7 mg sodium desoxycholate and 10 mg gum arabic. This substrate emulsion was stored in the dark for maximally 1 h.
24 h-old biofilms on membrane filters cultivated on calcium-amended PIA as described above were covered with 50 μl of the substrate emulsion. After incubation for 3 h at 30°C in the dark, lipase activities were detected by fluorescence microscopy using a LSM 510 confocal laser scanning microscope (Zeiss, Jena, Germany) with an excitation wavelength of 351 nm and emission long pass filter LP 505 nm or wide pass filter 505–550. In parallel, the biofilm cells were stained with SYTO 9 (Molecular Probes, Invitrogen GmbH, Karlsruhe, Germany) by adding 100 μl of SYTO 9 solution (1.5 μl SYTO added to 1 ml 0.9% (w/v) NaCl). After 15 min of incubation the fluorescence was recorded at an excitation wavelength of 488 nm by use of an argon laser in combination with an emission long pass filter LP 505 nm. Images were obtained with a Zeiss LD Achroplan 40x/0.60 NA objective. Digital image acquisition and analysis of the CLSM optical thin sections were performed with the Zeiss LSM software (version 3.2). For better visibility the fluorescence signals were stained with two different colors for imaging.
Purification of extracellular lipase from P. aeruginosa
Lipase protein was purified by a two-step chromatographic procedure as described earlier . In brief: lipase protein was produced in larger amounts by growing P. aeruginosa PABST7.1/pUCPL6A in 10 ml of double strength Luria Broth (2 × LB) containing 200 μg/ml carbenicillin and 50 μg/ml tetracycline in a 100 ml Erlenmeyer flask after inoculation with a single colony. Cells were grown overnight at 30°C, lipase gene expression was induced by addition of 0.4 mM IPTG and cells were further grown for 24 h.
Lipase expression cultures of recombinant P. aeruginosa were centrifuged; the culture supernatant was sterile filtered and concentrated by ultrafiltration by a factor of 15. One ml of the concentrated culture supernatant was mixed with 1 ml 10 mM Tris–HCl (pH 8.0), 100 mM NaCl and loaded onto a Fractogel EMD Bio SEC-chromatography column (length: 500 mm, inner diameter: 15 mm; Merck, Darmstadt, Germany) at room temperature. Proteins were eluted at 1 ml/min using the same buffer. Fractions containing the highest lipase activity (usually 15–20 fractions) were pooled and loaded onto an Uno-Q1 column (Bio-Rad, Munich, Germany), pre-equilibrated with buffer A (20 mM Tris–HCl pH 8.0, 100 mM NaCl) and connected to an FPLC unit (Pharmacia, Sweden). Proteins were eluted at 0.5 ml/min with the following NaCl gradient: 0–7 min with buffer A, 8–17 min from 100 mM to 400 mM NaCl in buffer A, 18–27 min from 400 mM to 1 M NaCl in buffer A, 28–32 min 1 M NaCl, 33–37 min from 1 M to 2 M NaCl in buffer A.
Purification of bacterial alginate and other polysaccharides
Bacterial alginate was isolated and purified from biofilm cultures of P. aeruginosa SG81 (PIA, 36°C, 24 h) as described before . Additionally, the bacterial polysaccharides dextran from Leuconostoc mesenteroides (Sigma-Aldrich, Munich, Germany), xanthan from Xanthomonas campestris (Sigma-Aldrich, Munich, Germany), levan from Erwinia herbicola (Fluka, Munich, Germany) and alginate (sodium salt) produced by brown algae (Manucol LHF, Nutra Sweet Kelco Company, Chicago, USA) were used. For further purification of dextran and algal alginate, 2 g of the polysaccharides were dissolved in 100 ml deionized water. After centrifugation of the solutions at 40,000 × g for 30 min the supernatants were collected, again centrifuged at 40,000 × g for 30 min and dialyzed (exclusion size: 12–14 kDa) twice against 5 l deionized water overnight. Finally, the polysaccharides were recovered by lyophilization. For further purification of xanthan and levan, the polysaccharides were dissolved in a concentration of 2.5 mg/ml in 50 mM Tris–HCl buffer (pH 7.5) containing 2 mM MgCl2. After addition of Benzonase (Merck, Darmstadt, Germany; final concentration 5 U/ml) and incubation for 4 h at 36°C, proteinase K (Sigma-Aldrich, Munich, Germany) was added (final concentration 5 μg/ml) followed by incubation at 36°C for 24 h. After centrifugation at 20,000 × g for 30 min, the supernatants were dialyzed (exclusion size: 12–14 kDa) twice against 5 l deionized water overnight and finally lyophilized.
Chemical deacetylation of bacterial alginate
Deacetylation of bacterial alginates was performed as described before . For complete deacetylation 25 mg purified alginate from P. aeruginosa SG81 was dissolved in 5 ml deionized water. After addition of 2.5 ml 0.3 M NaOH and incubation for 1 h at room temperature the pH was adjusted to 8.0 with 0.5 M HCl. Finally, the solution was dialyzed (exclusion size: 12–14 kDa) twice against 5 l deionized water overnight and lyophilized.
Quantification of lipase activity
Lipase activity was measured with para-nitrophenyl palmitate (pNPP) as a substrate as described before . An absorbance at 410 nm of 1.0 per 15 min corresponds to a lipase activity of 48.3 nmol/min x ml solution.
Quantification of polysaccharides
Total carbohydrate and uronic acid (alginate) concentrations were determined with the phenol-sulfuric acid method  and the hydroxydiphenyl assay , respectively, using purified alginate from P. aeruginosa SG81 as a standard.
Interaction of lipase with polysaccharides
For the investigation of interactions between lipase and polysaccharides a microtiter plate (polystyrene, Nalgene Nunc, Roskilde, Denmark) binding assay was applied. Purified polysaccharides were dissolved in 0.9% (w/v) NaCl solution and incubated for 15 min at 90°C to inactivate possibly remained enzymes. After cooling on ice, 100 μl of these polysaccharides in various concentrations (0–5 mg/ml) were immobilized by drying in the wells of microtiter plates at 30°C overnight, followed by a washing step with 200 μl distilled water (A. dest.) to remove NaCl. Afterwards 20 μl of purified lipase LipA from P. aeruginosa PABST7.1/pUCPL6A (72 ng/ml A. dest.) was added and incubated at 30°C for 30 min. Non-bound lipase was removed by two washing steps with 100 μl A. dest. each. Bounded lipase was detected via activity measurement in the microtiter plate using pNPP as substrate. The cleavage of the substrate was monitored at 405 nm in a microtiter plate reader. All experiments were performed in duplicates and repeated three times.
Heat treatment of lipase
The stabilization of lipases through the interaction with alginate was investigated by heat treatment of purified lipase in presence and absence of polysaccharides. One volume purified lipase LipA from P. aeruginosa PABST7.1/pUCPL6A (36 μg/ml A. dest.) was mixed with one volume purified polysaccharides (2 mg/ml in 100 mM Tris–HCl buffer, pH 7.5), which were previously heated (15 min, 90°C) and afterwards cooled on ice to room temperature. After pre-incubation for 30 min at room temperature the samples were incubated for 0–60 min at 70°C to determine lipase inactivation kinetics. Moreover, the samples were incubated for 20 min at different temperatures (37°C; 50°C; 60°C; 70°C; 80°C; 90°C) to determine T50. T50 represents the temperature at which incubation for 20 minutes reduces the enzymes activity by half. Every 10 min the residual lipase activities were detected after cooling on ice, using pNPP as substrate. All experiments were performed in duplicates and repeated three times.
Degradation of lipase by proteases
The protection of lipase from proteolytic degradation through the interaction with alginate was studied by using purified elastase LasB from P. aeruginosa (EMD4Biosciences, San Diego, USA). Briefly, 0.5 ml purified lipase LipA from P. aeruginosa PABST7.1/pUCPL6A (36 μg/ml A. dest.) was mixed with 0.5 ml purified polysaccharides (2 mg/ml in 100 mM Tris–HCl buffer, pH 7.5) previously heated for 15 min and 90°C and afterwards cooled on ice to room temperature. After pre-incubation for 30 min at room temperature, 20 μl purified elastase (0.1 mg/ml with 25 U/ml in A. dest.) were added. After 24 h incubation at 37°C the residual lipase activity was detected, using pNPP as a substrate. All experiments were performed in duplicates and repeated three times.
Modeling of lipase-alginate interaction
The protein structure was based on the crystal structure of the lipase protein resulting from the X-ray diffraction analysis of a lipase protein . The crystal structure was obtained from the RCSB protein data bank . The hydrogens of the amino acids were adjusted according to the pKs values of the amino acids at a pH value of 7.0. Therefore, the resulting net charge of the protein was in accordance to an aqueous solution of pH = 7.0. Inter- and intramolecular interactions were calculated by a molecular mechanics force field approach. The here used atom types and atomic partial charges of the CHARMM27 force field were described before .
In order to calculate the lipase – alginate interaction a modified docking procedure was applied including a water shell around the protein and alginate chain. In this procedure the lipase and alginate atoms were randomly moved. This resulted in a slight rotation and translation of the molecules. In consequence the potential energy of the resulting structure was minimized and saved. The step of changing the atomic positions was repeated several thousand times and the potential energy differences between all collected structures were checked. The structures with the lowest potential energies were extracted.
The significance of the data were analysed using the two-sample t-test and one way analyses of variance (ANOVA; ). A significant difference was considered to be p < 0.05.
Financial support from the “Deutsche Forschungsgemeinschaft” (DFG grant WI 831/3-1) is gratefully acknowledged.
- Tielen P, Rosenau F, Wilhelm S, Jaeger KE, Flemming HC, Wingender J: Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa. Microbiology. 2010, 156 (Pt 7): 2239-2252.PubMedView ArticleGoogle Scholar
- Van Delden C: Virulence factors in Pseudomonas aeruginosa. Pseudomonas. Edited by: Ramos JL. 2004, New York: Kluwer Academics/Plenum Publisher, 3-46.View ArticleGoogle Scholar
- Pier GB: Pseudomonas aeruginosa: a key problem in cystic fibrosis. ASM News. 1998, 64: 339-347.Google Scholar
- Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science. 1999, 284 (5418): 1318-1322. 10.1126/science.284.5418.1318.PubMedView ArticleGoogle Scholar
- Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, Birrer P, Bellon G, Berger J, Weiss T, et al.: Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest. 2002, 109 (3): 317-325.PubMedPubMed CentralView ArticleGoogle Scholar
- Son MS, Matthews WJ, Kang Y, Nguyen DT, Hoang TT: In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect Immun. 2007, 75 (11): 5313-5324. 10.1128/IAI.01807-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Granstrom M, Ericsson A, Strandvik B, Wretlind B, Pavlovskis OR, Berka R, Vasil ML: Relation between antibody response to Pseudomonas aeruginosa exoproteins and colonization/infection in patients with cystic fibrosis. Acta Paediatr Scand. 1984, 73 (6): 772-777. 10.1111/j.1651-2227.1984.tb17774.x.PubMedView ArticleGoogle Scholar
- Döring G, Goldstein W, Roll A, Schiotz PO, Hoiby N, Botzenhart K: Role of Pseudomonas aeruginosa exoenzymes in lung infections of patients with cystic fibrosis. Infect Immun. 1985, 49 (3): 557-562.PubMedPubMed CentralGoogle Scholar
- Wingender J, Flemming HC: The biofilm matrix. Nature Rev Microbiol. 2010, 8: 623-633.Google Scholar
- Cryz SJ, Iglewski BH: Production of alkaline protease by Pseudomonas aeruginosa. J Clin Microbiol. 1980, 12 (1): 131-133.PubMedPubMed CentralGoogle Scholar
- Bever RA, Iglewski BH: Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol. 1988, 170 (9): 4309-4314.PubMedPubMed CentralGoogle Scholar
- Ostroff RM, Vasil ML: Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa. J Bacteriol. 1987, 169 (10): 4597-4601.PubMedPubMed CentralGoogle Scholar
- Stuer W, Jaeger KE, Winkler UK: Purification of extracellular lipase from Pseudomonas aeruginosa. J Bacteriol. 1986, 168 (3): 1070-1074.PubMedPubMed CentralGoogle Scholar
- Martinez A, Ostrovsky P, Nunn DN: LipC, a second lipase of Pseudomonas aeruginosa, is LipB and Xcp dependent and is transcriptionally regulated by pilus biogenesis components. Mol Microbiol. 1999, 34 (2): 317-326. 10.1046/j.1365-2958.1999.01601.x.PubMedView ArticleGoogle Scholar
- Galloway DR: Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments. Mol Microbiol. 1991, 5 (10): 2315-2321. 10.1111/j.1365-2958.1991.tb02076.x.PubMedView ArticleGoogle Scholar
- König B, Jaeger KE, Sage AE, Vasil ML, König W: Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes. Infect Immun. 1996, 64 (8): 3252-3258.PubMedPubMed CentralGoogle Scholar
- Pier GB: Cystic fibrosis and Pseudomonas infections. Lancet. 1983, 2 (8353): 794-PubMedView ArticleGoogle Scholar
- Sherbrock-Cox V, Russell NJ, Gacesa P: The purification and chemical characterisation of the alginate present in extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydr Res. 1984, 135 (1): 147-154. 10.1016/0008-6215(84)85012-0.PubMedView ArticleGoogle Scholar
- Govan JR: Characteristics of mucoid Pseudomonas aeruginosa in vitro and in vivo. Pseudomonas infection and alginates - Biochemistry, genetics and pathology. Edited by: Gacesa P, Russell NJ. 1990, London/New York/Tokyo: Chapman and Hall, 50-75.View ArticleGoogle Scholar
- Evans LR, Linker A: Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol. 1973, 116 (2): 915-924.PubMedPubMed CentralGoogle Scholar
- Chitnis CE, Ohman DE: Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J Bacteriol. 1990, 172 (6): 2894-2900.PubMedPubMed CentralGoogle Scholar
- Skjar-Braek G, Grasgalen H, Larsen B: Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydr Res. 1986, 154: 239-250. 10.1016/S0008-6215(00)90036-3.View ArticleGoogle Scholar
- Lee JW, Ashby RD, Day DF: Role of acetylation on metal induced precipitation of alginates. Carbohydr Polym. 1996, 29: 337-345. 10.1016/S0144-8617(96)00017-3.View ArticleGoogle Scholar
- Tielen P, Strathmann M, Jaeger KE, Flemming HC, Wingender J: Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa. Microbiol Res. 2005, 160 (2): 165-176. 10.1016/j.micres.2004.11.003.PubMedView ArticleGoogle Scholar
- Lattner D, Flemming HC, Mayer C: 13C-NMR study of the interaction of bacterial alginate with bivalent cations. Int J Biol Macromol. 2003, 33 (1–3): 81-88.PubMedView ArticleGoogle Scholar
- Skjar-Braek G, Zanetti FSP: Effect of acetylation on some solution and gelling properties of alginates. Carbohydr Res. 1989, 185: 131-138. 10.1016/0008-6215(89)84028-5.View ArticleGoogle Scholar
- Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002, 15 (2): 167-193. 10.1128/CMR.15.2.167-193.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Körstgens V, Flemming HC, Wingender J, Borchard W: Uniaxial compression measurement device for investigation of the mechanical stability of biofilms. J Microbiol Methods. 2001, 46 (1): 9-17. 10.1016/S0167-7012(01)00248-2.PubMedView ArticleGoogle Scholar
- Allison DG, Matthews MJ: Effect of polysaccharide interactions on antibiotic susceptibility of Pseudomonas aeruginosa. J Appl Bacteriol. 1992, 73 (6): 484-488. 10.1111/j.1365-2672.1992.tb05009.x.PubMedView ArticleGoogle Scholar
- Grobe S, Wingender J, Flemming HC: Capability of mucoid Pseudomonas aeruginosa to survive in chlorinated water. Int J Hyg Environ Health. 2001, 204 (2–3): 139-142.PubMedView ArticleGoogle Scholar
- Pier GB, Coleman F, Grout M, Franklin M, Ohman DE: Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun. 2001, 69 (3): 1895-1901. 10.1128/IAI.69.3.1895-1901.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK: The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing. J Immunol. 2005, 175 (11): 7512-7518.PubMedView ArticleGoogle Scholar
- Wingender J, Winkler U: A novel biological function of algiante in Pseudomonas aeruginosa and its mucoid mutants: stimulation of exolipase. FEMS Microbiol Lett. 1984, 21: 63-69. 10.1111/j.1574-6968.1984.tb00187.x.View ArticleGoogle Scholar
- Wingender J, Volz S, Winkler UK: Interaction of extracellular Pseudomonas lipase with alginate and its potential use in biotechnology. Appl Microbiol Biotechnol. 1987, 27: 139-145.View ArticleGoogle Scholar
- Sharma S, Gupta MN: Alginate as a macroaffinity ligand and an additive for enhanced activity and thermostability of lipases. Biotechnol Appl Biochem. 2001, 33 (Pt 3): 161-165.PubMedView ArticleGoogle Scholar
- Arpigny JL, Jaeger KE: Bacterial lipolytic enzymes: classification and properties. Biochem J. 1999, 343 (Pt 1): 177-183.PubMedPubMed CentralView ArticleGoogle Scholar
- Nardini M, Lang DA, Liebeton K, Jaeger KE, Dijkstra BW: Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J Biol Chem. 2000, 275 (40): 31219-31225.PubMedView ArticleGoogle Scholar
- Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger KE: Directed evolution of an enantioselective lipase. Chem Biol. 2000, 7 (9): 709-718. 10.1016/S1074-5521(00)00015-6.PubMedView ArticleGoogle Scholar
- Rosenau F, Jaeger K: Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie. 2000, 82 (11): 1023-1032. 10.1016/S0300-9084(00)01182-2.PubMedView ArticleGoogle Scholar
- Wingender J, Jaeger KE, Flemming HC: Interaction between extracellular polysaccharides and enzymes. Microbial extracellular polymeric substances. Edited by: Wingender J, Neu T, Flemming HC. 1999, Berlin/Heidelberg/New York: Springer Verlag, 231-247.View ArticleGoogle Scholar
- Wingender J: Interactions of alginate with exoenzymes. Pseudomonas infection and alginates - Biochemistry, genetics and pathology. Edited by: Gacesa P, Russell NJ. 1990, London/New York/Tokyo: Chapman and Hall, 160-180.View ArticleGoogle Scholar
- Borriello G, Richards L, Ehrlich GD, Stewart PS: Arginine or nitrate enhances antibiotic susceptibility of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother. 2006, 50 (1): 382-384. 10.1128/AAC.50.1.382-384.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Auerbach ID, Sorensen C, Hansma HG, Holden PA: Physical morphology and surface properties of unsaturated Pseudomonas putida biofilms. J Bacteriol. 2000, 182 (13): 3809-3815. 10.1128/JB.182.13.3809-3815.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Steinberger RE, Allen AR, Hansa HG, Holden PA: Elongation correlates with nutrient deprivation in Pseudomonas aeruginosa-unsaturates biofilms. Microb Ecol. 2002, 43 (4): 416-423. 10.1007/s00248-001-1063-z.PubMedView ArticleGoogle Scholar
- Winkler UK, Stuckmann M: Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol. 1979, 138 (3): 663-670.PubMedPubMed CentralGoogle Scholar
- Körstgens V, Flemming HC, Wingender J, Borchard W: Influence of calcium ions on the mechanical properties of a model biofilm of mucoid Pseudomonas aeruginosa. Water Sci Technol. 2001, 43 (6): 49-57.PubMedGoogle Scholar
- Rosenau F, Isenhardt S, Gdynia A, Tielker D, Schmidt E, Tielen P, Schobert M, Jahn D, Wilhelm S, Jaeger KE: Lipase LipC affects motility, biofilm formation and rhamnolipid production in Pseudomonas aeruginosa. FEMS Microbiol Lett. 2010, 309 (1): 25-34.PubMedGoogle Scholar
- Strathmann M, Wingender J, Flemming HC: Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J Microbiol Methods. 2002, 50 (3): 237-248. 10.1016/S0167-7012(02)00032-5.PubMedView ArticleGoogle Scholar
- Schürks N, Wingender J, Flemming HC, Mayer C: Monomer composition and sequence of alginates from Pseudomonas aeruginosa. Int J Biol Macromol. 2002, 30 (2): 105-111. 10.1016/S0141-8130(02)00002-8.PubMedView ArticleGoogle Scholar
- Sikorski P, Mo F, Skjak-Braek G, Stokke BT: Evidence for egg-box-compatible interactions in calcium-alginate gels from fiber X-ray diffraction. Biomacromolecules. 2007, 8 (7): 2098-2103. 10.1021/bm0701503.PubMedView ArticleGoogle Scholar
- Kemmling A, Kämper M, Flies C, Schieweck O, Hoppert M: Biofilms and extracellular matrices on geomaterils. Environ Geol. 2004, 46: 429-435.View ArticleGoogle Scholar
- Leza A, Plameros B, Garcia JO, Galindo E, Sobéron-Chávez G: Xanthomonas campstris as a host for the production of recombinant Pseudomonas aeruginosa lipase. J Ind Microbiol. 1996, 16: 22-28. 10.1007/BF01569917.View ArticleGoogle Scholar
- Duckworth M, Turvey JR: An extracellular agarase from a Cytophaga species. Biochem J. 1969, 113: 139-142.PubMedPubMed CentralView ArticleGoogle Scholar
- Kuwabara S, Lloyd PH: Protein and carbohydrate moieties of a preparation of ß-lactamase II. Biochem J. 1971, 124: 215-220.PubMedPubMed CentralView ArticleGoogle Scholar
- Nouwens AS, Beatson SA, Whitchurch CB, Walsh BJ, Schweizer HP, Mattick JS, Cordwell SJ: Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1. Microbiology. 2003, 149 (Pt 5): 1311-1322.PubMedView ArticleGoogle Scholar
- Folders J, Tommassen J, Van Loon LC, Bitter W: Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa. J Bacteriol. 2000, 182 (5): 1257-1263. 10.1128/JB.182.5.1257-1263.2000.PubMedPubMed CentralView ArticleGoogle Scholar
- Vu TH, Werb Z: Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 2000, 14 (17): 2123-2133. 10.1101/gad.815400.PubMedView ArticleGoogle Scholar
- Kearns DB, Bonner PJ, Smith DR, Shimkets LJ: An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus. J Bacteriol. 2002, 184 (6): 1678-1684. 10.1128/JB.184.6.1678-1684.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Imeson AP, Ledward DA, Mitchell JR: On the nature of the interaction between some anionic polysaccharides and proteins. Sci Food Agric. 1977, 28: 661-668. 10.1002/jsfa.2740280802.View ArticleGoogle Scholar
- Ying QL, Kemme M, Simon SR: Alginate, the slime exopolysaccharide of Pseudomonas aeruginosa, binds human leukocyte elastase, retards inhibition by alpha 1-proteinase inhibitor, and accelerates inhibition by secretory leukoprotease inhibitor. Am J Respir Cell Mol Biol. 1996, 15 (2): 283-291. 10.1165/ajrcmb.15.2.8703486.PubMedView ArticleGoogle Scholar
- Franklin MJ, Ohman DE: Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation. J Bacteriol. 1993, 175 (16): 5057-5065.PubMedPubMed CentralGoogle Scholar
- Franklin MJ, Ohman DE: Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation. J Bacteriol. 1996, 178 (8): 2186-2195.PubMedPubMed CentralGoogle Scholar
- Wilhelm S, Rosenau F, Becker S, Buest S, Hausmann S, Kolmar H, Jaeger KE: Functional cell-surface display of a lipase-specific chaperone. Chem Bio Chem. 2007, 8 (1): 55-60. 10.1002/cbic.200600203.PubMedView ArticleGoogle Scholar
- Kovach ME, Phillips RW, Elzer PH, Roop RM, Peterson KM: pBBR1MCS: a broad-host-range cloning vector. Biotechniques. 1994, 16 (5): 800-802.PubMedGoogle Scholar
- Simon R, Priefer U, Pühler A: A broad host range mobilization system for in vitro genetic engeneering: transposon mutagenesis in Gram-negative bacteria. Biological Technology. 1983, 1: 784-791. 10.1038/nbt1183-784.View ArticleGoogle Scholar
- Ohman DE, Chakrabarty AM: Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun. 1981, 33 (1): 142-148.PubMedPubMed CentralGoogle Scholar
- Grobe S, Wingender J, Truper HG: Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems. J Appl Bacteriol. 1995, 79 (1): 94-102. 10.1111/j.1365-2672.1995.tb03129.x.PubMedView ArticleGoogle Scholar
- Wingender J, Strathmann M, Rode A, Leis A, Flemming HC: Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa. Methods Enzymol. 2001, 336: 302-314.PubMedView ArticleGoogle Scholar
- Singer VL, Paragas VB, Larison KD, Wells KS, Fox CJ, Haugland RP: Fluorescence-based signal amplification technology. Am Biotechnol Lab. 1994, 12 (11): 55-56. 58PubMedGoogle Scholar
- Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F: A colorimetric method for the determination of sugars. Nature. 1951, 168 (4265): 167-PubMedView ArticleGoogle Scholar
- Blumenkrantz N, Asboe-Hansen G: New method for quantitative determination of uronic acids. Anal Biochem. 1973, 54 (2): 484-489. 10.1016/0003-2697(73)90377-1.PubMedView ArticleGoogle Scholar
- Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE: The protein data bank. Nucleic Acids Res. 2000, 28 (1): 235-242. 10.1093/nar/28.1.235.PubMedPubMed CentralView ArticleGoogle Scholar
- MacKerell JAD, Bashford D, Bellott M, Dunbrack JRL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, et al.: All-atomempirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem. 1998, 102: 3586-3616. 10.1021/jp973084f.View ArticleGoogle Scholar
- Fagerland MW, Sandvik L: Performance of five two-sample location tests for skewed distributions with unequal variances. Contemp Clin Trials. 2009, 30 (5): 490-496. 10.1016/j.cct.2009.06.007.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.