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DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli
FEMS Microbiology Letters 125 (1995) 71-76
DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli Ronit Bar Ness-Greenstein a, Mel Rosenberg atbY *, R.J. Doyle ‘, Nachum Kaplan d a The Maurice and Gabriela Gokischleger School of Dental Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel b Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel ’ Department of Microbiology/Immunology, University of Louisville, Louisville, Kv, USA ’ Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, 69978 Tel-Aviv, Israel Received 16 September 1994; revised 1 November 1994; accepted 2 November 1994
Abstract In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli, E. coli DHk cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized component(s) on the transformant outer surface. Keywords: Serratia marcescens; Hydrophobin; Escherichia coli
Cell surface hydrophobicity;
1. Introduction
The physicochemical outer surface properties of Escherichia coli have importance both from basic and applied viewpoints. Surface hydrophobicity and retention of E. coli cells on solid surfaces are a function of cell cycle as well as of the substratum surface energy [l]. In biotechnology, hydrophobized E. coli cells may be more readily immobilized than normal cells. Cell surface hydrophobicity may also play a role in adhesion of E. coli to host surfaces. Compared to many other microorganisms, most
* Corresponding author.
Hexadecane;
Ammonium
sulfate aggregation;
strains of E. coli do not adhere readily to hydrophobic surfaces, such as hydrocarbons and plastics [7,8,14,17]. In the present study, we tested the feasibility of increasing cell surface hydrophobicity of a commonly used E. coli strain, following transformation with DNA from Serratia marcescens, a microorganism with pronounced adhesion to hydrophobic substrata [16]. S. murcescens was reported to possess hydrophobic surface properties by Mudd and Mudd 70 years ago [11,12]. Since then, cell surface hydrophobicity has been linked to partitioning of S. marcescens at air/water and oil/water interfaces, as well as adhesion to solid surfaces including catheters and other plastics [2]. Hydrophobic surface properties of S. marcescens appear to be mediated by
037%1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378-1097(94)00475-7
72
R. Bar Ness-Greenstein et at. / FEMS Microbiology Letters 125 (1995) 71-76
several hydrophobins, including prodigiosin, a lipophibc tripyrrole pigment [3,5,6,15], and a 70-kDa surface protein, serraphobin [4].
2. Materials and methods
S. marcescens RZ was originally obtained from R. Zack, Tel Aviv University. Mutant 3164, a nonhydrophobic mutant, is derived from strain RZ 1131. Bacteria were maintained on brain heart infusion (BHI) agar (Difco Laboratories, Detroit, MI) at 4°C and transferred monthly. E. coli DH5a competent cells (library efficiency), were obtained from Gibco (BRL, Middlesex, UK) and were maintained at - 70°C. All molecular biology and cloning procedures used in this study were essentially as described by Maniatis et al. [lo]. Chromosomal DNA was extracted from S. marcescens RZ and dissolved to yield a concentration of 0.25 mg ml- ‘. DNA was digested with either BumHI (New England Biolabs, Beverley, MA) or Sau3A (Biolabs). The BamHI digest was selected for further treatments because it yielded a larger amount of DNA fragments of the desired size (5-10 kb) as compared to the Sau3A digest. The BamHI digest was fractionated by sedimentation through a sucrose gradient (5-20%), and the appropriate DNA fractions (i.e. size of 5-10 kb as determined by agarose gel electrophoresis), were pooled for ligation. The DNA fractions were ligated with T4 DNA ligase (Biolabs) to BumHI-cleaved pUC19 DNA (BioRad, Richmond, CA). Following CaCl, transformation of the ligation mixture into E. coli DHSa competent cells, transformants were selected on Luria Bertani (LB) plates (1% Tryptone, 0.5% yeast extract, 2.0% agar (Difco) 1% (w/v) NaCl), containing 50 pg per ml ampicillin (Sigma, St. Louis, MO) and 50 pg per ml X-Gal (5-bromo4-chloro-3-indolyl-P-D-galactopyranoside (Sigma, St. Louis, MO)). White colonies which were ampicillin resistant were transferred again to the same plates and screened for hydrophobicity by testing the adhesion of the bacteria to liquid hexadecane as follows: to a 0.5-ml colony suspension in phosphate buffer (PB) contain-
ing 22.2 g K,HPO,. 3H,O, 7.26 g KH,PO, per litre, pH 7.1, 0.1 ml hexadecane (Sigma) was added and the mixture was mixed vigorously for 1 min. Following phase separation, a sample (10 /.& of the upper hexadecane phase was taken for microscopic observation of bacteria adhering to the hexadecane droplets. Measurement of the percent adhesion of the bacteria to hexadecane was performed as described previously [17]. Briefly, to 1.0 ml bacteria suspended to 1.0 OD in PB in a polystyrene cuvette, 0.1 ml hexadecane (Sigma) was added. The mixture was vortexed vigorously for 1 min. Following phase separation, the decrease in OD of the lower aqueous phase was measured in a spectrophotometer and the percentage of cells adhering to the hydrocarbon upper phase was determined. Antiserum against whole cells of S. marcescens RZ was raised in rabbits as follows: 2 X lo9 cells were injected i.v. three times with 3 days intervals between injections. 10 days following the last injection, blood was collected and antiserum was precipitated in the presence of 40% ammonium sulfate and was stored at -20°C. Additional blood collections were performed 3 days following a booster injection of lo9 cells; blood samples were treated similarly. The kinetics of ammonium sulfate-mediated aggregation was measured as follows: cells were inoculated 1:200 and grown overnight at 37°C in 50 ml of brain heart infusion broth (Difco). Bacteria were harvested by centrifugation and washed twice in PBS and suspended in 0.1 M ammonium sulfate to yield initial optical densities between 1 and 4.5 (400 nm, 1 cm light path). Aggregation over time was monitored by the decrease in optical density. Anti-RZ antibodies, precipitated in 40% ammonium sulfate, were adsorbed onto non-hydrophobic mutant 3164 cells, as well as onto E. coli DHSa recipient cells harboring pUC19. For adsorption, cultures of S. marcescens 3164 were grown in 100 ml BHI for 24 h at 30°C. Following growth, cells were washed three times in 0.15 M NaCl by 20 min centrifugation at 10000 X g. The washed pellet was resuspended in 2 ml of the above antiserum preparation and the suspension was incubated with gentle shaking for 3 h at 4°C. The cells were then centrifuged as above and the supematant was used to suspend a fresh washed cell pellet of strain 3164.
73
R. Bar Ness-Greenstein et al. / FEMS Microbiology Letters 125 (I 995) 71-76
The procedure was repeated 3-6 times until sufficient specificity was obtained. Western colony blotting was performed as described by Maniatis et al. [lo]. Blots were incubated with the adsorbed anti-RZ antiserum.
A
E
c 8 d Y
3. Results and discussion
ti
6
Among about 1500 clones possessing recombinant pUC19 plasmids with inserts of serratial DNA, one transformant, DH5a (pRB2551, was found to exhibit adhesion to hexadecane (31 f 23%, mean f SD) which was significantly higher (P < 0.005, rtest) than that of control cells with the plasmid alone (1.3 &-3.26%); wild-type hydrophobic donor strain S. marcescens RZ showed high adhesion to hexadecane (78 f 5%, mean f SD). The transformant was maintained on LB-amp plates (LB plates, containing 50 pg ml-’ ampicillin) at 4°C. Restriction mapping of recombinant plasmid pRB255 revealed that it contained an insert of 5.4 kb (Fig. 1). In addition to the restriction sites depicted in Fig. 1, the insert contained four unresolved sites for SphI, three for MluI, three for MscI, three for BstEII, and two for PstI. The insert contained no sites for the enzymes HindIII, EcoRI, ScaI, XhoI, and KpnI. Ammonium sulfate has been proposed to aggregate E. coli cells, based on enhancement of hydrophobic interactions [9,14]. The kinetics of ammonium sulfate-mediated aggregation of adherent transformant DH5a (pRB255) and non-adherent recipient strain DH5a were compared at two initial cell densities (Fig. 2). At an initial optical density of 1, kinetics of aggregation were similar. However, when the initial cell density was raised to an OD of 5, different kinetics were observed. As compared to a constant decrease in optical density of the recipient
NsiI Sac1
I I
I
I
0
100
200
“I
.
0
Ii0
Minutes
2tJo
1 400
8
.
Minutes
300
360
.
‘
0
Fig. 2. Kinetics of ammonium sulfate-mediated aggregation of recipient non-adherent strain DH5a (a) and adherent transformant DHSa (pRB255) (b). Cells were washed and suspended in 0.1 M ammonium sulfate as described in Materials and methods and the decrease in optical density at 400 nm (1 cm light path) was measured over time at two initial optical densities (approx. 1 (+I and approx. 4-5 ( 0 )).
strain, an S-type curve was observed for the transformant, with no aggregation during the first 100 min, followed by an extremely rapid decrease to low levels. In contrast, the recipient exhibited gradual
SmaI Sal1 BstXI
NsiI Ba@I
Fig. 1. Restriction map of recombinant plasmid pRB255. pRD255 DNA was cleaved with appropriate combinations of the depicted restriction enzymes in order to construct the restriction map. Restriction fragments were separated on 0.6% agarose gels and visualized following ethidium bromide staining. pUC19 sequences are represented by shaded boxes.
R. Bar Ness-Greenstein
et al. /FEMS
Microbiology
Letters 125 11995) 71-74
Fig. 3. Western colony blotting of the adherent transformant DH5a (pRB255). A colony of the adherent transformant DHSLU (pRB255) as well as control reference colonies of RZ, non-hydrophobic strain 3164, and DH5a with or without pUC19 plasmid, were blotted as described in Materials and methods. Blots were reacted with anti-RZ antiserum which was pre-adsorbed on 3164 and DH5cx cells. Right to left: 1, adherent transformant DH5cr (pRB255); 2, RZ; 3, 3164; 4, DHScx; 5, DH5o (pUC19).
aggregation throughout the experiment (Fig. 2). The density-dependent aggregation in the transformant suggests a positive cooperative interaction between the cells. Furthermore, aggregation in both strains was more pronounced at the higher optical density. The dramatic difference in the aggregation kinetics of the transformant provides further evidence of its altered physicochemical properties. Adherent transformant DHSa (pRB255) was examined by Western colony blotting (Fig. 31. A clear reaction with the anti-RZ antiserum was obtained with transformant cells and RZ cells, whereas the controls (DHSa with or without plasmid pUC19, and the non-hydrophobic mutant 3164) showed only a weak background reaction. Hence, we infer that the increased adhesion of the transformant to hexadecane, as well as the altered kinetics of aggregation in the presence of ammonium sulfate, are due to appearance of a S. marcescens antigen(s) on the surface of the transformed E. coli cells. The present study supports the rationale of rendering E. coli cells more hydrophobic by transformation with DNA from other, more hydrophobic species. The results presented here extend previous data showing that E. coli cells can be hydrophobized by chemical surface modification [7] as well as by
addition of organic cations [8]. Since these other treatments may affect viability, hydrophobization by genetic transformation may prove advantageous (e.g. in immobilization) to other methods which have an adverse affect on cell viability. This prospect is currently under investigation.
Acknowledgements We are grateful to Yardena Mazor and Sonia Rosenheck for excellent technical assistance. The work described here was carried out in the Alpha Omega Laboratories of the Maurice and Gabriela Goldschleger School of Dental Medicine, with the support of the United States-Israel Binational Science Foundation, Jerusalem, Israel, Grant 86-0023, and NIH-NIDR Grant DE-07199.
References [ll Rittle, K.H., Helmstetter, C.E., Meyer, A.E. and Baier, R.E. (1990) Escherkhin coli retention on solid surfaces as functions of substratum surface energy and cell growth phase. Biofouling 2, 121-130.
R. Bar Ness-Greenstein
121Ashkenazi,
et al. /FEMS Microbiology Letters 125 (1995) 71-76
S., Weiss, E. and Drucker, M.M. (1986) Bacterial adherence to intravenous catheters and needles and its influence by cannula type and bacterial surface hydrophobicity. J. Lab. Clin. Med. 107, 136-140. w Bar-Ness, R., Avrahamy, N., Matsuyama, T. and Rosenberg, M. (1988) Increased cell surface hydrophobicity of a Serratia marcescens NS 38 mutant lacking wetting activity. J. Bacterial. 170, 4361-4364. [41 Bar-Ness, R. and Rosenberg, M. (1989) Putative role of a 70 kD outer-surface protein in promoting cell surface hydrophobicity of Serratia marcescens RZ. J. Gen. Microbial. 135, 2277-2281. El Blanchard, D.C. and Syzdek, L.D. (1978) Seven problems in jet bubble and jet drop researches. Limnol. Ocean. 23, 389400. [61Hermansson, M., Kjelleberg, S. and Norkans, B. (19791 Interaction of pigmented wild-type and pigment-less mutant of Serratia marcescens with lipid surface film. FEMS Microbiol. Lett. 6, 129-132. [71Goldberg, S., Doyle, R.J. and Rosenberg, M. (1990) Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers. J. Bacterial. 172, 5650-5654. 181Goldberg, S., Konis, Y. and Rosenberg, M. (1990) Effect of cetylpyridinium chloride on microbial adhesion to hexadecane. Appl. Environ. Microbial. 56, 1678-1682. [91 Lindahl, M., Faris, A., Wadstrom, T. and Hijerten, S. (1981) A new test based on ‘salting out’ to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta 677, 471-476.
75
[lo] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 316-319. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [ll] Mudd, S. and Mudd, E.B.H. (1924) The penetration of bacteria through capillary spaces. IV. A kinetic mechanism in interfaces. J. Exp. Med. 40, 633-645. [12] Mudd, S. and Mudd, E.B.H. (1924) Certain interfacial tension relations and the behaviour of bacteria in films. J. Exp. Med. 40, 647-660. [13] Rosenberg, M. (1984) Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity. J. Bacterial. 160, 480-482. [14] Rosenberg, M. (1984) Ammonium sulphate enhances adherence of Escherichia coli J-5 to hydrocarbon and polystyrene. FEMS Microbial. Lett. 25, 41-45. [15] Rosenberg, M., Barki, M., Bar-Ness, R., Goldberg, S. and Doyle, R.J. (1991) Microbial adhesion to hydrocarbons (MATH). Biofouling 4, 121-128. [16] Rosenberg, M., Blumberger, Y., Judes, H., Bar-Ness, R., Rubinstein, E. and Mazor, Y. (1986) Cell surface hydrophobicity of pigmented and nonpigmented Serratia marcescens strains. Infect. Immun. 52, 932-935. 1171 Rosenberg, M., Gutnick, D. and Rosenberg, E. (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbial. Lett. 9, 29-33.
DNA from Serratia marcescens confers a hydrophobic character in Escherichia coli Ronit Bar Ness-Greenstein a, Mel Rosenberg atbY *, R.J. Doyle ‘, Nachum Kaplan d a The Maurice and Gabriela Gokischleger School of Dental Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel b Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel ’ Department of Microbiology/Immunology, University of Louisville, Louisville, Kv, USA ’ Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, 69978 Tel-Aviv, Israel Received 16 September 1994; revised 1 November 1994; accepted 2 November 1994
Abstract In order to determine whether hydrophobic surface properties of Serratia marcescens can be transferred to Escherichia coli, E. coli DHk cells were transformed by DNA fragments from S. marcescens RZ. Fifteen-hundred E. coli transformants were screened for adhesion to hexadecane and polystyrene. One transformant exhibited increased adhesion to hexadecane droplets, as well as altered kinetics of aggregation in the presence of ammonium sulfate. Western colony blotting revealed that antibodies raised against S. marcescens RZ recognized component(s) on the transformant outer surface. Keywords: Serratia marcescens; Hydrophobin; Escherichia coli
Cell surface hydrophobicity;
1. Introduction
The physicochemical outer surface properties of Escherichia coli have importance both from basic and applied viewpoints. Surface hydrophobicity and retention of E. coli cells on solid surfaces are a function of cell cycle as well as of the substratum surface energy [l]. In biotechnology, hydrophobized E. coli cells may be more readily immobilized than normal cells. Cell surface hydrophobicity may also play a role in adhesion of E. coli to host surfaces. Compared to many other microorganisms, most
* Corresponding author.
Hexadecane;
Ammonium
sulfate aggregation;
strains of E. coli do not adhere readily to hydrophobic surfaces, such as hydrocarbons and plastics [7,8,14,17]. In the present study, we tested the feasibility of increasing cell surface hydrophobicity of a commonly used E. coli strain, following transformation with DNA from Serratia marcescens, a microorganism with pronounced adhesion to hydrophobic substrata [16]. S. murcescens was reported to possess hydrophobic surface properties by Mudd and Mudd 70 years ago [11,12]. Since then, cell surface hydrophobicity has been linked to partitioning of S. marcescens at air/water and oil/water interfaces, as well as adhesion to solid surfaces including catheters and other plastics [2]. Hydrophobic surface properties of S. marcescens appear to be mediated by
037%1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378-1097(94)00475-7
72
R. Bar Ness-Greenstein et at. / FEMS Microbiology Letters 125 (1995) 71-76
several hydrophobins, including prodigiosin, a lipophibc tripyrrole pigment [3,5,6,15], and a 70-kDa surface protein, serraphobin [4].
2. Materials and methods
S. marcescens RZ was originally obtained from R. Zack, Tel Aviv University. Mutant 3164, a nonhydrophobic mutant, is derived from strain RZ 1131. Bacteria were maintained on brain heart infusion (BHI) agar (Difco Laboratories, Detroit, MI) at 4°C and transferred monthly. E. coli DH5a competent cells (library efficiency), were obtained from Gibco (BRL, Middlesex, UK) and were maintained at - 70°C. All molecular biology and cloning procedures used in this study were essentially as described by Maniatis et al. [lo]. Chromosomal DNA was extracted from S. marcescens RZ and dissolved to yield a concentration of 0.25 mg ml- ‘. DNA was digested with either BumHI (New England Biolabs, Beverley, MA) or Sau3A (Biolabs). The BamHI digest was selected for further treatments because it yielded a larger amount of DNA fragments of the desired size (5-10 kb) as compared to the Sau3A digest. The BamHI digest was fractionated by sedimentation through a sucrose gradient (5-20%), and the appropriate DNA fractions (i.e. size of 5-10 kb as determined by agarose gel electrophoresis), were pooled for ligation. The DNA fractions were ligated with T4 DNA ligase (Biolabs) to BumHI-cleaved pUC19 DNA (BioRad, Richmond, CA). Following CaCl, transformation of the ligation mixture into E. coli DHSa competent cells, transformants were selected on Luria Bertani (LB) plates (1% Tryptone, 0.5% yeast extract, 2.0% agar (Difco) 1% (w/v) NaCl), containing 50 pg per ml ampicillin (Sigma, St. Louis, MO) and 50 pg per ml X-Gal (5-bromo4-chloro-3-indolyl-P-D-galactopyranoside (Sigma, St. Louis, MO)). White colonies which were ampicillin resistant were transferred again to the same plates and screened for hydrophobicity by testing the adhesion of the bacteria to liquid hexadecane as follows: to a 0.5-ml colony suspension in phosphate buffer (PB) contain-
ing 22.2 g K,HPO,. 3H,O, 7.26 g KH,PO, per litre, pH 7.1, 0.1 ml hexadecane (Sigma) was added and the mixture was mixed vigorously for 1 min. Following phase separation, a sample (10 /.& of the upper hexadecane phase was taken for microscopic observation of bacteria adhering to the hexadecane droplets. Measurement of the percent adhesion of the bacteria to hexadecane was performed as described previously [17]. Briefly, to 1.0 ml bacteria suspended to 1.0 OD in PB in a polystyrene cuvette, 0.1 ml hexadecane (Sigma) was added. The mixture was vortexed vigorously for 1 min. Following phase separation, the decrease in OD of the lower aqueous phase was measured in a spectrophotometer and the percentage of cells adhering to the hydrocarbon upper phase was determined. Antiserum against whole cells of S. marcescens RZ was raised in rabbits as follows: 2 X lo9 cells were injected i.v. three times with 3 days intervals between injections. 10 days following the last injection, blood was collected and antiserum was precipitated in the presence of 40% ammonium sulfate and was stored at -20°C. Additional blood collections were performed 3 days following a booster injection of lo9 cells; blood samples were treated similarly. The kinetics of ammonium sulfate-mediated aggregation was measured as follows: cells were inoculated 1:200 and grown overnight at 37°C in 50 ml of brain heart infusion broth (Difco). Bacteria were harvested by centrifugation and washed twice in PBS and suspended in 0.1 M ammonium sulfate to yield initial optical densities between 1 and 4.5 (400 nm, 1 cm light path). Aggregation over time was monitored by the decrease in optical density. Anti-RZ antibodies, precipitated in 40% ammonium sulfate, were adsorbed onto non-hydrophobic mutant 3164 cells, as well as onto E. coli DHSa recipient cells harboring pUC19. For adsorption, cultures of S. marcescens 3164 were grown in 100 ml BHI for 24 h at 30°C. Following growth, cells were washed three times in 0.15 M NaCl by 20 min centrifugation at 10000 X g. The washed pellet was resuspended in 2 ml of the above antiserum preparation and the suspension was incubated with gentle shaking for 3 h at 4°C. The cells were then centrifuged as above and the supematant was used to suspend a fresh washed cell pellet of strain 3164.
73
R. Bar Ness-Greenstein et al. / FEMS Microbiology Letters 125 (I 995) 71-76
The procedure was repeated 3-6 times until sufficient specificity was obtained. Western colony blotting was performed as described by Maniatis et al. [lo]. Blots were incubated with the adsorbed anti-RZ antiserum.
A
E
c 8 d Y
3. Results and discussion
ti
6
Among about 1500 clones possessing recombinant pUC19 plasmids with inserts of serratial DNA, one transformant, DH5a (pRB2551, was found to exhibit adhesion to hexadecane (31 f 23%, mean f SD) which was significantly higher (P < 0.005, rtest) than that of control cells with the plasmid alone (1.3 &-3.26%); wild-type hydrophobic donor strain S. marcescens RZ showed high adhesion to hexadecane (78 f 5%, mean f SD). The transformant was maintained on LB-amp plates (LB plates, containing 50 pg ml-’ ampicillin) at 4°C. Restriction mapping of recombinant plasmid pRB255 revealed that it contained an insert of 5.4 kb (Fig. 1). In addition to the restriction sites depicted in Fig. 1, the insert contained four unresolved sites for SphI, three for MluI, three for MscI, three for BstEII, and two for PstI. The insert contained no sites for the enzymes HindIII, EcoRI, ScaI, XhoI, and KpnI. Ammonium sulfate has been proposed to aggregate E. coli cells, based on enhancement of hydrophobic interactions [9,14]. The kinetics of ammonium sulfate-mediated aggregation of adherent transformant DH5a (pRB255) and non-adherent recipient strain DH5a were compared at two initial cell densities (Fig. 2). At an initial optical density of 1, kinetics of aggregation were similar. However, when the initial cell density was raised to an OD of 5, different kinetics were observed. As compared to a constant decrease in optical density of the recipient
NsiI Sac1
I I
I
I
0
100
200
“I
.
0
Ii0
Minutes
2tJo
1 400
8
.
Minutes
300
360
.
‘
0
Fig. 2. Kinetics of ammonium sulfate-mediated aggregation of recipient non-adherent strain DH5a (a) and adherent transformant DHSa (pRB255) (b). Cells were washed and suspended in 0.1 M ammonium sulfate as described in Materials and methods and the decrease in optical density at 400 nm (1 cm light path) was measured over time at two initial optical densities (approx. 1 (+I and approx. 4-5 ( 0 )).
strain, an S-type curve was observed for the transformant, with no aggregation during the first 100 min, followed by an extremely rapid decrease to low levels. In contrast, the recipient exhibited gradual
SmaI Sal1 BstXI
NsiI Ba@I
Fig. 1. Restriction map of recombinant plasmid pRB255. pRD255 DNA was cleaved with appropriate combinations of the depicted restriction enzymes in order to construct the restriction map. Restriction fragments were separated on 0.6% agarose gels and visualized following ethidium bromide staining. pUC19 sequences are represented by shaded boxes.
R. Bar Ness-Greenstein
et al. /FEMS
Microbiology
Letters 125 11995) 71-74
Fig. 3. Western colony blotting of the adherent transformant DH5a (pRB255). A colony of the adherent transformant DHSLU (pRB255) as well as control reference colonies of RZ, non-hydrophobic strain 3164, and DH5a with or without pUC19 plasmid, were blotted as described in Materials and methods. Blots were reacted with anti-RZ antiserum which was pre-adsorbed on 3164 and DH5cx cells. Right to left: 1, adherent transformant DH5cr (pRB255); 2, RZ; 3, 3164; 4, DHScx; 5, DH5o (pUC19).
aggregation throughout the experiment (Fig. 2). The density-dependent aggregation in the transformant suggests a positive cooperative interaction between the cells. Furthermore, aggregation in both strains was more pronounced at the higher optical density. The dramatic difference in the aggregation kinetics of the transformant provides further evidence of its altered physicochemical properties. Adherent transformant DHSa (pRB255) was examined by Western colony blotting (Fig. 31. A clear reaction with the anti-RZ antiserum was obtained with transformant cells and RZ cells, whereas the controls (DHSa with or without plasmid pUC19, and the non-hydrophobic mutant 3164) showed only a weak background reaction. Hence, we infer that the increased adhesion of the transformant to hexadecane, as well as the altered kinetics of aggregation in the presence of ammonium sulfate, are due to appearance of a S. marcescens antigen(s) on the surface of the transformed E. coli cells. The present study supports the rationale of rendering E. coli cells more hydrophobic by transformation with DNA from other, more hydrophobic species. The results presented here extend previous data showing that E. coli cells can be hydrophobized by chemical surface modification [7] as well as by
addition of organic cations [8]. Since these other treatments may affect viability, hydrophobization by genetic transformation may prove advantageous (e.g. in immobilization) to other methods which have an adverse affect on cell viability. This prospect is currently under investigation.
Acknowledgements We are grateful to Yardena Mazor and Sonia Rosenheck for excellent technical assistance. The work described here was carried out in the Alpha Omega Laboratories of the Maurice and Gabriela Goldschleger School of Dental Medicine, with the support of the United States-Israel Binational Science Foundation, Jerusalem, Israel, Grant 86-0023, and NIH-NIDR Grant DE-07199.
References [ll Rittle, K.H., Helmstetter, C.E., Meyer, A.E. and Baier, R.E. (1990) Escherkhin coli retention on solid surfaces as functions of substratum surface energy and cell growth phase. Biofouling 2, 121-130.
R. Bar Ness-Greenstein
121Ashkenazi,
et al. /FEMS Microbiology Letters 125 (1995) 71-76
S., Weiss, E. and Drucker, M.M. (1986) Bacterial adherence to intravenous catheters and needles and its influence by cannula type and bacterial surface hydrophobicity. J. Lab. Clin. Med. 107, 136-140. w Bar-Ness, R., Avrahamy, N., Matsuyama, T. and Rosenberg, M. (1988) Increased cell surface hydrophobicity of a Serratia marcescens NS 38 mutant lacking wetting activity. J. Bacterial. 170, 4361-4364. [41 Bar-Ness, R. and Rosenberg, M. (1989) Putative role of a 70 kD outer-surface protein in promoting cell surface hydrophobicity of Serratia marcescens RZ. J. Gen. Microbial. 135, 2277-2281. El Blanchard, D.C. and Syzdek, L.D. (1978) Seven problems in jet bubble and jet drop researches. Limnol. Ocean. 23, 389400. [61Hermansson, M., Kjelleberg, S. and Norkans, B. (19791 Interaction of pigmented wild-type and pigment-less mutant of Serratia marcescens with lipid surface film. FEMS Microbiol. Lett. 6, 129-132. [71Goldberg, S., Doyle, R.J. and Rosenberg, M. (1990) Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers. J. Bacterial. 172, 5650-5654. 181Goldberg, S., Konis, Y. and Rosenberg, M. (1990) Effect of cetylpyridinium chloride on microbial adhesion to hexadecane. Appl. Environ. Microbial. 56, 1678-1682. [91 Lindahl, M., Faris, A., Wadstrom, T. and Hijerten, S. (1981) A new test based on ‘salting out’ to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta 677, 471-476.
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[lo] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 316-319. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [ll] Mudd, S. and Mudd, E.B.H. (1924) The penetration of bacteria through capillary spaces. IV. A kinetic mechanism in interfaces. J. Exp. Med. 40, 633-645. [12] Mudd, S. and Mudd, E.B.H. (1924) Certain interfacial tension relations and the behaviour of bacteria in films. J. Exp. Med. 40, 647-660. [13] Rosenberg, M. (1984) Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity. J. Bacterial. 160, 480-482. [14] Rosenberg, M. (1984) Ammonium sulphate enhances adherence of Escherichia coli J-5 to hydrocarbon and polystyrene. FEMS Microbial. Lett. 25, 41-45. [15] Rosenberg, M., Barki, M., Bar-Ness, R., Goldberg, S. and Doyle, R.J. (1991) Microbial adhesion to hydrocarbons (MATH). Biofouling 4, 121-128. [16] Rosenberg, M., Blumberger, Y., Judes, H., Bar-Ness, R., Rubinstein, E. and Mazor, Y. (1986) Cell surface hydrophobicity of pigmented and nonpigmented Serratia marcescens strains. Infect. Immun. 52, 932-935. 1171 Rosenberg, M., Gutnick, D. and Rosenberg, E. (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbial. Lett. 9, 29-33.