- Email: [email protected]
Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module
Journal of Microbiological Methods 114 (2015) 75–77
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module Yoshihiro Ojima a,⁎, Asep Suparman a, Minh Hong Nguyen a,1, Makiko Sakka b, Kazuo Sakka b, Masahito Taya a a b
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan
a r t i c l e
i n f o
Article history: Received 12 April 2015 Received in revised form 11 May 2015 Accepted 11 May 2015 Available online 12 May 2015
a b s t r a c t A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs. © 2015 Elsevier B.V. All rights reserved.
Keywords: Carbohydrate-binding module Exopolysaccharide Escherichia coli Microcolony
Microcolonies are bacterial consortium that can adhere to each other as well as solid surfaces to form matrices enclosing populations (Costerton et al., 1995). Exopolysaccharides (EPShs) are the main component of these matrices, and play a critical role in their structure and virulence (Branda et al., 2005), as well as acting as adhesive polymers (Liu et al., 2004), maintaining the integrity of the microcolonies (Chen and Stewart, 2002) and conferring resistance against antibiotics (Davies, 2003). A variety of chemical methods and microscopic techniques have been developed to evaluate the EPShs present in microcolonies. The chemical methods used to study EPShs are generally categorized as invasive methods because they involve the disruption of the microcolonies (Jahn and Nielsen, 1995; Masuko et al., 2005). In contrast, microscopic techniques allow for the EPShs to be observed using lectins conjugated to a fluorescence material based on its binding specificity without disrupting the integrity of the microcolonies (Strathmann et al., 2002). However, it can be difficult to quantify the EPShs present in microcolonies using microscopic techniques. To date, the quantitative evaluation of EPShs in microcolonies has been limited to methods involving the disruption of the microcolony structure (Burton et al., 2007; Thomas et al., 1997) or the use of indirect measurements based on multiple reactions (Leriche et al., 2000). Carbohydrate binding modules (CBMs) can form strong binding interactions with carbohydrates. CBM3 can bind to a variety of substrates, including ⁎ Corresponding author. E-mail address: [email protected] (Y. Ojima). 1 Present address: Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam.
http://dx.doi.org/10.1016/j.mimet.2015.05.013 0167-7012/© 2015 Elsevier B.V. All rights reserved.
cellulose, xylan, chitin and chitosan. Taking advantages of these properties, we previously reported that a fused protein composed of CBM3 from Paenibacillus curdlanolyticus B-6 (Sakka et al., 2011) and green fluorescence protein (GFP) could be used as a marker for the EPShs present in the biofilms of Escherichia coli cells (Nguyen et al., 2014). In this study, we have developed a new method for the measurement of the EPShs present in E. coli microcolonies using GFP-CBM3 protein fused with a linker. This fused protein can be cleaved by the action of a site-specific protease. The amount of EPShs is therefore evaluated by measuring the fluorescence of the released GFP. Most notably, this method is expected to allow for the quantification of EPShs without the destruction of the microcolonies. E. coli K-12 BW25113 strain was obtained from the National BioResource Project (National Institute of Genetics (NIG), Mishima, Japan). E. coli Rosetta (DE3) pLysS (Novagen, Madison, WI, USA) was used as a host for the production of the recombinant protein. The pET24a-GFP-CBM3 plasmid was constructed according to the procedure described in our previous work (Nguyen et al., 2014). The recombinant GFP-CBM3 protein was expressed and purified according to our previously described procedure (Nguyen et al., 2014) using E. coli Rosetta pLysS cells carrying pET24a-GFP-CBM3. A PreScission cleavage site, which can be cut in a site-specific manner using PreScission Protease (GE Healthcare Japan Corp., Tokyo, Japan), was introduced in the region between the GFP and CBM3 parts to allow for the cleavage of the GFP-CBM3 fused protein (Fig. 1A). After purification, the concentrated GFP-CBM3 protein was quantified using a Bradford assay with bovine serum albumin as a standard. PreScission Protease was added to 100 μl of a 1.0 mg/ml GFP-CBM3 solution (final concentration of 6 U/ml) to
76
Y. Ojima et al. / Journal of Microbiological Methods 114 (2015) 75–77
Fig. 1. (A) Layout of GFP-CBM3 fused protein. (B) SDS-PAGE analysis of purified proteins. Lane M, molecular weight marker; lane 1, GFP-CBM3; lane 2, GFP-CBM3 cut by PreScission Protease.
confirm cleavage of the fused protein, and the resulting solution was stood for 24 h at 4 °C, followed by SDS-PAGE analysis with Coomassie Blue staining. For microcolony formation, the BW25113 cells were cultured in lysogeny broth (LB) medium consisting of 10 g/l polypeptone, 5 g/l yeast extract and 10 g/l NaCl. All of the test cultures were precultured in LB medium for 14 h at 37 °C and then inoculated in fresh LB medium to give an optical density of 0.01 at 660 nm (OD660). Small portions (200 μl) of the resulting suspension were then transferred to the individual wells of a 96-well microtiter plate made from polyvinyl chloride (Corning Inc., Corning, NY, USA). Different concentrations (i.e., 0, 0.1, 0.2, and 0.4 μg/ml) of antibiotic (gentamicin) were then added to the medium to prepare different developing levels of the microcolonies. After being cultured at 37 °C for 24 h, the culture broths containing planktonic cells were removed and microcolonies adhering to the surfaces of the wells were fixed with 2.5% glutaraldehyde before being washed with 10 mM Tris buffer (pH 8.0) and used for the colonization and EPSh assays. For the colonization assay, adhering cells were stained with 200 μl of safranin solution (50 mg/l) for 30 min at room temperature and then washed five times with water. The dye pigmenting cells on the surfaces of the wells were solubilized by adding 200 μl of 20% (v/v) acetone in ethanol. The index of the colonized cells was determined based on the absorbance at 492 nm (A492) using a Chromate-4300 microtiter plate reader (Awareness Technology, Palm City, FL, USA). For the measurement of EPShs using the GFP-CBM3 method, microcolonies in a 96-well microtiter plate were incubated with 200 μl of a GFP-CBM3 solution (1.0 mg/ml) for 30 min at room temperature, followed by washing with phosphate-buffered saline. For the cleavage reaction, a solution (200 μl) containing 6 U/ml PreScission Protease was added to each well, and incubated for 24 h at 4 °C. The fluorescence intensity of released GFP protein (F538) was then measured using an F-1200 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan) with excitation and emission wavelengths of 495 and 538 nm, respectively. The empty wells without E. coli microcolony were treated under the same conditions to use as a control. As a conventional carbohydrate assay, the phenol-sulfuric acid method was conducted according to a general procedure from the literature
(Masuko et al., 2005). Briefly, microcolonies on a 96-well microtiter plate were immersed in the mixture composed of 160 μl of 96% sulfuric acid and 40 μl of 5% phenol solution and heated at 80 °C for 5 min in a water bath. The microtiter plate was then cooled to 25 °C for 5 min and the absorbance of solution in each well was measured at 490 nm using a V-630Bio UV–VIS Spectrophotometer (JASCO Co., Tokyo, Japan). The value from the empty wells with the same treatment was also used as a control in this method. For the conversion of the absorbance value into the amount of EPShs, we evaluated different amounts of Avicel (FMC BioPolymer, Philadelphia, PA, USA), a typical form of microcrystalline cellulose, as a standard. To prepare the samples for observation, E. coli cells were cultured for 24 h at 37 °C with pieces of glass in a 96-well microtiter plate. The microcolonies on the pieces of glass were treated in the same manner as those on the well surfaces. The microcolonies that formed on the glass surface were also stained with a 200 μl GFP-CBM3 solution (1.0 mg/ml) for 30 min before being stained with 200 μl of 10 μM SYTO 60 Red Fluorescent Nucleic Acid Stain (Molecular Probes, Eugene, OR, USA) for 15 min in the dark. After being washed, a solution (200 μl) containing 6 U/ml PreScission Protease was added, and stood for 24 h at 4 °C. The samples were observed using a confocal laser scanning microscope (CLSM; Model FV-300; Olympus, Tokyo, Japan) at 200 × magnification with sequential excitation at wavelengths of 488 and 633 nm. At least, three fields of view were observed at random in the two sets of the experiments. The cleavage of the GFP-CBM3 fused protein with the site-specific protease (PreScission Protease) was confirmed by SDS-PAGE. As shown in Fig. 1B, bands appeared at approximately 27 and 16 kDa after the treatment of fused protein with PreScission Protease (lane 2). These bands were consistent with the molecular weights of the GFP and CBM3 proteins, respectively. This result therefore demonstrated that the fused protein, which had a molecular weight of 43 kDa (lane 1), had been successfully cleaved at the specific linker site. The results of our previous study showed that the GFP-CBM3 protein successfully bound to the EPShs present in E. coli biofilms (Nguyen et al., 2014). One of the key features of this probe is that it allows for the simultaneous detection of cellulose and poly-N-acetyl-1,6-β-glucosamine (PGA), which are both typical polysaccharides found in microcolonies and biofilms (Mika and Hengge, 2013), whereas several other common probes only allow for the detection of specific polysaccharides such as concanavalin A (ConA) for cellulose derivatives, and wheat germ agglutinin (WGA) for PGA (Leriche et al., 2000; Thomas et al., 1997). In this context, the GFP-CBM3 protein developed in the current study was evaluated as a probe for the measurement of the EPShs present in E. coli microcolonies. Fig. 2 shows the representative three-dimensional images of EPShs stained with GFP-CBM3 (green fluorescence) and DNA stained with SYTO 60 dye (red fluorescence) in E. coli microcolonies established on glass surfaces. Green fluorescence was concentrated in the densest regions of the microcolonies, which indicated that the GFP-CBM3 had successfully detected the EPShs present in the microcolonies. No green fluorescence was observed after the protease treatment, which suggested that the GFP had been released from the CBM3 bound to the EPShs in the microcolonies. Furthermore, this result supported the specific binding of the GFP-CBM3 fused protein to EPShs through its CBM3 domain rather its GFP protein. It is noteworthy that red fluorescence still remained on the colonies, which demonstrated that the structure of the microcolonies had not been disturbed by the protease treatment. The non-destructive measurement of the EPShs present in E. coli microcolonies therefore represents a notable advantage for the current method over those previously reported in the literature (Burton et al., 2007; Thomas et al., 1997). To check the sensitivity of the GFP-CBM3 probe for the measurement of EPShs, we prepared different levels of the microcolonies by changing the dose of gentamicin. As expected, the antibiotic reduced the colonization index in a linear manner with increasing concentration. Without gentamicin, the index of the colonized cells (A492) was 0.052
Y. Ojima et al. / Journal of Microbiological Methods 114 (2015) 75–77
77
Fig. 3. Relationship between index of colonized cells of E. coli microcolonies and amount of EPShs evaluated by phenol-sulfuric acid method as cellulose equivalent (closed circle) or GFP-CBM3 method as fluorescence intensity (open circle). Fig. 2. CLSM images of microcolonies formed by E. coli BW25113 cells on glass surfaces cultured for 24 h. The EPShs (green) were stained with GFP-CBM3, and DNA as a whole bacterial cells' indicator (red) was stained with SYTO 60 dye. Scale bar: 20 μm.
per well, and this value was reduced to A492 = 0.015 per well in the presence of 0.4 μg/ml gentamicin. Fig. 3 shows the relationship between the index of the colonized cells and the amount of EPShs measured using the conventional method (phenol-sulfuric acid method) or our newly developed method with the GFP-CBM3 protein. For the conventional method, the amount of EPShs was estimated to be 43 μg cellulose equivalent per well when the A492 value was 0.052 per well. The amount of EPShs then decreased in proportion to the decrease in the amount of colonized cells and reached 20 μg cellulose equivalent per well at A492 = 0.015 per well, which suggested that the production of EPShs was dependent on the amount of colonized cells. In evaluating the amount of EPShs using our newly developed GFPCBM3 protein, the fluorescence intensity (F538) indicated that the EPSh content of the microcolonies corresponded to F538 = 40 per well at A492 = 0.052 per well. The fluorescence intensity also decreased in a linear manner as the amount of microcolonies decreased. These results show that both methods provided similar measurements for the amount of EPShs against the colonized cells, which verifies that the GFP-CBM3 protein could therefore be used for the quantitative evaluation of the EPShs present in E. coli microcolonies. A GFP-CBM3 fused protein was designed in the current study bearing a linker between the two individual domains that could be readily cleaved by a site-specific protease to allow for the quantitative and non-destructive analysis of the EPShs in E. coli microcolonies. The fluorescence intensity of the released GFP can be measured without destroying the structure of the microcolonies. Burton et al. used WGA conjugated to Alexa-fluor 488 to probe the PGA present in biofilms (Burton et al., 2007). In their case, the biofilms were subjected to sonication to allow for the release of the fluorescent material and the measure of the fluorescence intensity. This also led to the destruction of the structure of biofilm. Our GFP-CBM3 method could be used to measure the EPShs present in biofilm in a non-destructive manner, which would allow the biofilms to be subjected to further analysis. However, there is a limitation in the extent to which the GFP-CBM3 fused protein
can diffuse through the multi-layered biofilm, because of its relatively large molecular weight (43 kDa). In conclusion, we have successfully developed a GFP-CBM3 fused protein to measure the EPShs present in E. coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs present in E. coli microcolonies. References Branda, S.S., Vik, Å., Friedman, L., Kolter, R., 2005. Biofilms: the matrix revisited. Trends Microbiol. 13, 20–26. Burton, E., Yakandawala, N., LoVetri, K., Madhyastha, M.S., 2007. A microplate spectrofluorometric assay for bacterial biofilms. J. Ind. Microbiol. Biotechnol. 34, 1–4. Chen, X., Stewart, P.S., 2002. Role of electrostatic interactions in cohesion of bacterial biofilms. Appl. Microbiol. Biotechnol. 59, 718–720. Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber, D.R., Lappin-Scott, H.M., 1995. Microbial biofilms. Annu. Rev. Microbiol. 49, 711–745. Davies, D., 2003. Understanding biofilm resistance to antibacterial agents. Nat. Rev. Drug Discov. 2, 114–122. Jahn, A., Nielsen, P.H., 1995. Extraction of extracellular polymeric substances (EPS) from biofilms using a cation exchange resin. Water Sci. Technol. 32, 157–164. Leriche, V., Sibille, P., Carpentier, B., 2000. Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms. Appl. Environ. Microbiol. 66, 1851–1856. Liu, Y., Yang, S.-F., Li, Y., Xu, H., Qin, L., Tay, J.-H., 2004. The influence of cell and substratum surface hydrophobicities on microbial attachment. J. Biotechnol. 110, 251–256. Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S.-I., Lee, Y.C., 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal. Biochem. 339, 69–72. Mika, F., Hengge, R., 2013. Small regulatory RNAs in the control of motility and biofilm formation in E. coli and Salmonella. Int. J. Mol. Sci. 14, 4560–4579. Nguyen, M.H., Ojima, Y., Sakka, M., Sakka, K., Taya, M., 2014. Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression. J. Biosci. Bioeng. 118, 400–405. Sakka, M., Higashi, Y., Kimura, T., Ratanakhanokchai, K., Sakka, K., 2011. Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a xylanase that contains a family 3 carbohydrate-binding module. Appl. Environ. Microbiol. 77, 4260–4263. Strathmann, M., Wingender, J., Flemming, H.-C., 2002. Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 50, 237–248. Thomas, V.L., Sanford, B.A., Moreno, R., Ramsay, M.A., 1997. Enzyme-linked lectinsorbent assay measures N-acetyl-D-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis. Curr. Microbiol. 35, 249–254.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module Yoshihiro Ojima a,⁎, Asep Suparman a, Minh Hong Nguyen a,1, Makiko Sakka b, Kazuo Sakka b, Masahito Taya a a b
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan
a r t i c l e
i n f o
Article history: Received 12 April 2015 Received in revised form 11 May 2015 Accepted 11 May 2015 Available online 12 May 2015
a b s t r a c t A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs. © 2015 Elsevier B.V. All rights reserved.
Keywords: Carbohydrate-binding module Exopolysaccharide Escherichia coli Microcolony
Microcolonies are bacterial consortium that can adhere to each other as well as solid surfaces to form matrices enclosing populations (Costerton et al., 1995). Exopolysaccharides (EPShs) are the main component of these matrices, and play a critical role in their structure and virulence (Branda et al., 2005), as well as acting as adhesive polymers (Liu et al., 2004), maintaining the integrity of the microcolonies (Chen and Stewart, 2002) and conferring resistance against antibiotics (Davies, 2003). A variety of chemical methods and microscopic techniques have been developed to evaluate the EPShs present in microcolonies. The chemical methods used to study EPShs are generally categorized as invasive methods because they involve the disruption of the microcolonies (Jahn and Nielsen, 1995; Masuko et al., 2005). In contrast, microscopic techniques allow for the EPShs to be observed using lectins conjugated to a fluorescence material based on its binding specificity without disrupting the integrity of the microcolonies (Strathmann et al., 2002). However, it can be difficult to quantify the EPShs present in microcolonies using microscopic techniques. To date, the quantitative evaluation of EPShs in microcolonies has been limited to methods involving the disruption of the microcolony structure (Burton et al., 2007; Thomas et al., 1997) or the use of indirect measurements based on multiple reactions (Leriche et al., 2000). Carbohydrate binding modules (CBMs) can form strong binding interactions with carbohydrates. CBM3 can bind to a variety of substrates, including ⁎ Corresponding author. E-mail address: [email protected] (Y. Ojima). 1 Present address: Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, Vietnam.
http://dx.doi.org/10.1016/j.mimet.2015.05.013 0167-7012/© 2015 Elsevier B.V. All rights reserved.
cellulose, xylan, chitin and chitosan. Taking advantages of these properties, we previously reported that a fused protein composed of CBM3 from Paenibacillus curdlanolyticus B-6 (Sakka et al., 2011) and green fluorescence protein (GFP) could be used as a marker for the EPShs present in the biofilms of Escherichia coli cells (Nguyen et al., 2014). In this study, we have developed a new method for the measurement of the EPShs present in E. coli microcolonies using GFP-CBM3 protein fused with a linker. This fused protein can be cleaved by the action of a site-specific protease. The amount of EPShs is therefore evaluated by measuring the fluorescence of the released GFP. Most notably, this method is expected to allow for the quantification of EPShs without the destruction of the microcolonies. E. coli K-12 BW25113 strain was obtained from the National BioResource Project (National Institute of Genetics (NIG), Mishima, Japan). E. coli Rosetta (DE3) pLysS (Novagen, Madison, WI, USA) was used as a host for the production of the recombinant protein. The pET24a-GFP-CBM3 plasmid was constructed according to the procedure described in our previous work (Nguyen et al., 2014). The recombinant GFP-CBM3 protein was expressed and purified according to our previously described procedure (Nguyen et al., 2014) using E. coli Rosetta pLysS cells carrying pET24a-GFP-CBM3. A PreScission cleavage site, which can be cut in a site-specific manner using PreScission Protease (GE Healthcare Japan Corp., Tokyo, Japan), was introduced in the region between the GFP and CBM3 parts to allow for the cleavage of the GFP-CBM3 fused protein (Fig. 1A). After purification, the concentrated GFP-CBM3 protein was quantified using a Bradford assay with bovine serum albumin as a standard. PreScission Protease was added to 100 μl of a 1.0 mg/ml GFP-CBM3 solution (final concentration of 6 U/ml) to
76
Y. Ojima et al. / Journal of Microbiological Methods 114 (2015) 75–77
Fig. 1. (A) Layout of GFP-CBM3 fused protein. (B) SDS-PAGE analysis of purified proteins. Lane M, molecular weight marker; lane 1, GFP-CBM3; lane 2, GFP-CBM3 cut by PreScission Protease.
confirm cleavage of the fused protein, and the resulting solution was stood for 24 h at 4 °C, followed by SDS-PAGE analysis with Coomassie Blue staining. For microcolony formation, the BW25113 cells were cultured in lysogeny broth (LB) medium consisting of 10 g/l polypeptone, 5 g/l yeast extract and 10 g/l NaCl. All of the test cultures were precultured in LB medium for 14 h at 37 °C and then inoculated in fresh LB medium to give an optical density of 0.01 at 660 nm (OD660). Small portions (200 μl) of the resulting suspension were then transferred to the individual wells of a 96-well microtiter plate made from polyvinyl chloride (Corning Inc., Corning, NY, USA). Different concentrations (i.e., 0, 0.1, 0.2, and 0.4 μg/ml) of antibiotic (gentamicin) were then added to the medium to prepare different developing levels of the microcolonies. After being cultured at 37 °C for 24 h, the culture broths containing planktonic cells were removed and microcolonies adhering to the surfaces of the wells were fixed with 2.5% glutaraldehyde before being washed with 10 mM Tris buffer (pH 8.0) and used for the colonization and EPSh assays. For the colonization assay, adhering cells were stained with 200 μl of safranin solution (50 mg/l) for 30 min at room temperature and then washed five times with water. The dye pigmenting cells on the surfaces of the wells were solubilized by adding 200 μl of 20% (v/v) acetone in ethanol. The index of the colonized cells was determined based on the absorbance at 492 nm (A492) using a Chromate-4300 microtiter plate reader (Awareness Technology, Palm City, FL, USA). For the measurement of EPShs using the GFP-CBM3 method, microcolonies in a 96-well microtiter plate were incubated with 200 μl of a GFP-CBM3 solution (1.0 mg/ml) for 30 min at room temperature, followed by washing with phosphate-buffered saline. For the cleavage reaction, a solution (200 μl) containing 6 U/ml PreScission Protease was added to each well, and incubated for 24 h at 4 °C. The fluorescence intensity of released GFP protein (F538) was then measured using an F-1200 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan) with excitation and emission wavelengths of 495 and 538 nm, respectively. The empty wells without E. coli microcolony were treated under the same conditions to use as a control. As a conventional carbohydrate assay, the phenol-sulfuric acid method was conducted according to a general procedure from the literature
(Masuko et al., 2005). Briefly, microcolonies on a 96-well microtiter plate were immersed in the mixture composed of 160 μl of 96% sulfuric acid and 40 μl of 5% phenol solution and heated at 80 °C for 5 min in a water bath. The microtiter plate was then cooled to 25 °C for 5 min and the absorbance of solution in each well was measured at 490 nm using a V-630Bio UV–VIS Spectrophotometer (JASCO Co., Tokyo, Japan). The value from the empty wells with the same treatment was also used as a control in this method. For the conversion of the absorbance value into the amount of EPShs, we evaluated different amounts of Avicel (FMC BioPolymer, Philadelphia, PA, USA), a typical form of microcrystalline cellulose, as a standard. To prepare the samples for observation, E. coli cells were cultured for 24 h at 37 °C with pieces of glass in a 96-well microtiter plate. The microcolonies on the pieces of glass were treated in the same manner as those on the well surfaces. The microcolonies that formed on the glass surface were also stained with a 200 μl GFP-CBM3 solution (1.0 mg/ml) for 30 min before being stained with 200 μl of 10 μM SYTO 60 Red Fluorescent Nucleic Acid Stain (Molecular Probes, Eugene, OR, USA) for 15 min in the dark. After being washed, a solution (200 μl) containing 6 U/ml PreScission Protease was added, and stood for 24 h at 4 °C. The samples were observed using a confocal laser scanning microscope (CLSM; Model FV-300; Olympus, Tokyo, Japan) at 200 × magnification with sequential excitation at wavelengths of 488 and 633 nm. At least, three fields of view were observed at random in the two sets of the experiments. The cleavage of the GFP-CBM3 fused protein with the site-specific protease (PreScission Protease) was confirmed by SDS-PAGE. As shown in Fig. 1B, bands appeared at approximately 27 and 16 kDa after the treatment of fused protein with PreScission Protease (lane 2). These bands were consistent with the molecular weights of the GFP and CBM3 proteins, respectively. This result therefore demonstrated that the fused protein, which had a molecular weight of 43 kDa (lane 1), had been successfully cleaved at the specific linker site. The results of our previous study showed that the GFP-CBM3 protein successfully bound to the EPShs present in E. coli biofilms (Nguyen et al., 2014). One of the key features of this probe is that it allows for the simultaneous detection of cellulose and poly-N-acetyl-1,6-β-glucosamine (PGA), which are both typical polysaccharides found in microcolonies and biofilms (Mika and Hengge, 2013), whereas several other common probes only allow for the detection of specific polysaccharides such as concanavalin A (ConA) for cellulose derivatives, and wheat germ agglutinin (WGA) for PGA (Leriche et al., 2000; Thomas et al., 1997). In this context, the GFP-CBM3 protein developed in the current study was evaluated as a probe for the measurement of the EPShs present in E. coli microcolonies. Fig. 2 shows the representative three-dimensional images of EPShs stained with GFP-CBM3 (green fluorescence) and DNA stained with SYTO 60 dye (red fluorescence) in E. coli microcolonies established on glass surfaces. Green fluorescence was concentrated in the densest regions of the microcolonies, which indicated that the GFP-CBM3 had successfully detected the EPShs present in the microcolonies. No green fluorescence was observed after the protease treatment, which suggested that the GFP had been released from the CBM3 bound to the EPShs in the microcolonies. Furthermore, this result supported the specific binding of the GFP-CBM3 fused protein to EPShs through its CBM3 domain rather its GFP protein. It is noteworthy that red fluorescence still remained on the colonies, which demonstrated that the structure of the microcolonies had not been disturbed by the protease treatment. The non-destructive measurement of the EPShs present in E. coli microcolonies therefore represents a notable advantage for the current method over those previously reported in the literature (Burton et al., 2007; Thomas et al., 1997). To check the sensitivity of the GFP-CBM3 probe for the measurement of EPShs, we prepared different levels of the microcolonies by changing the dose of gentamicin. As expected, the antibiotic reduced the colonization index in a linear manner with increasing concentration. Without gentamicin, the index of the colonized cells (A492) was 0.052
Y. Ojima et al. / Journal of Microbiological Methods 114 (2015) 75–77
77
Fig. 3. Relationship between index of colonized cells of E. coli microcolonies and amount of EPShs evaluated by phenol-sulfuric acid method as cellulose equivalent (closed circle) or GFP-CBM3 method as fluorescence intensity (open circle). Fig. 2. CLSM images of microcolonies formed by E. coli BW25113 cells on glass surfaces cultured for 24 h. The EPShs (green) were stained with GFP-CBM3, and DNA as a whole bacterial cells' indicator (red) was stained with SYTO 60 dye. Scale bar: 20 μm.
per well, and this value was reduced to A492 = 0.015 per well in the presence of 0.4 μg/ml gentamicin. Fig. 3 shows the relationship between the index of the colonized cells and the amount of EPShs measured using the conventional method (phenol-sulfuric acid method) or our newly developed method with the GFP-CBM3 protein. For the conventional method, the amount of EPShs was estimated to be 43 μg cellulose equivalent per well when the A492 value was 0.052 per well. The amount of EPShs then decreased in proportion to the decrease in the amount of colonized cells and reached 20 μg cellulose equivalent per well at A492 = 0.015 per well, which suggested that the production of EPShs was dependent on the amount of colonized cells. In evaluating the amount of EPShs using our newly developed GFPCBM3 protein, the fluorescence intensity (F538) indicated that the EPSh content of the microcolonies corresponded to F538 = 40 per well at A492 = 0.052 per well. The fluorescence intensity also decreased in a linear manner as the amount of microcolonies decreased. These results show that both methods provided similar measurements for the amount of EPShs against the colonized cells, which verifies that the GFP-CBM3 protein could therefore be used for the quantitative evaluation of the EPShs present in E. coli microcolonies. A GFP-CBM3 fused protein was designed in the current study bearing a linker between the two individual domains that could be readily cleaved by a site-specific protease to allow for the quantitative and non-destructive analysis of the EPShs in E. coli microcolonies. The fluorescence intensity of the released GFP can be measured without destroying the structure of the microcolonies. Burton et al. used WGA conjugated to Alexa-fluor 488 to probe the PGA present in biofilms (Burton et al., 2007). In their case, the biofilms were subjected to sonication to allow for the release of the fluorescent material and the measure of the fluorescence intensity. This also led to the destruction of the structure of biofilm. Our GFP-CBM3 method could be used to measure the EPShs present in biofilm in a non-destructive manner, which would allow the biofilms to be subjected to further analysis. However, there is a limitation in the extent to which the GFP-CBM3 fused protein
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