maltose, and low or undetectable for glucose, isomaltose, cellobiose, or lactose. The reduced binding at the higher temperature was due to the rapid breakdown ...
Vol. 166, No. 1
JOURNAL OF BACTERIOLOGY, Apr. 1986, p. 95-99
0021-9193/86/040095-05$02.00/0 Copyright C 1986, American Society for Microbiology
Temperature-Sensitive Binding of oL-Glucans by Bacillus stearothermophilus T. FERENCI* AND K.-S. LEE Department of Microbiology, University of Sydney, Sydney, New South Wales 2006, Australia Received 14 August 1985/Accepted 16 January 1986
Bacillus stearothermophilus was found to bind strongly to starch and related a-glucans at 25°C but not at 55°C. The binding at the lower temperature could be assayed either by binding of fluorescein-labeled amylopectin to washed cell suspensions or through the reversible retention of bacteria by affinity chromatography in matrices containing immobilized starch. The bacteria exhibited amylopectin-dependent agglutination. The binding and agglutination were highest in bacteria grown on substrates containing a-1,4-glucosidic linkages such as maltose or dextrins. The binding affinity of cells was highest for maltohexaose, lower for maltose, and low or undetectable for glucose, isomaltose, cellobiose, or lactose. The reduced binding at the higher temperature was due to the rapid breakdown of the a-glucosides. The bacteria exhibited an extracellular a-amylase activity as well as a cell-associated a-glucosidase with high activity at 55°C but undetectable activity at 25°C. The inducibility, specificity, and protease sensitivity of the thermophilic a-glucosidase in whole cells were similar to those of the binding activity assayed at the lower temperature. Further evidence linking the binding and a-glucosidase activities came from a mutant, selected through affinity chromatography, which was reduced in starch binding at room temperature and also reduced in membraneassociated at-glucosidase activity at 55°C. These results suggest a novel survival mechanism whereby a bacterium attaches to a macromolecular substrate under nonoptimal growth conditions for possible utilization upon a shift to more favorable conditions.
cellular and surface-bound proteins in the metabolism of starch by this bacterium.
Bacteria are involved in a number of interactions with carbohydrates through surface-located proteins exhibiting sugar-specific binding sites. The properties of a number of such fimbrial adhesin-mediated interactions have been reviewed (7). Examples of specific nonfimbrial interactions have also been observed between bacteria and polysaccharides which are primarily sources of nutrients, such as the binding to cellulose of cellulolytic Clostridium thermocellum and Ruminococcus albus (8, 14) and the binding of starch by Escherichia coli (6). In addition to these defined examples, many members of mixed populations of rumen bacteria have also been shown to be able to adhere to starch or cellulose (12, 13). This study reports on a novel temperature-sensitive binding interaction between a starchdegrading Bacillus stearothermophilus and starch-related oligo- and polysaccharides. It is well known that members of Bacillus spp. produce both extracellular and surface-located enzymes involved in the breakdown of extracellular macromolecules (16). Some of these enzymes, in particular amylases and proteases, are of considerable current interest, not least because of their potential biotechnological significance (3). The soluble extracellular oa-amylase of B. stearothermophilus has been well studied enzymologically (15) and, more recently, at the genetic level (1, 10). A soluble cytoplasmic a-glucosidase of B. stearothermophilus has also been purified recently (17). Very little else is known about the role of these and other enzymes involved in the breakdown of starch by this organism, about how the breakdown products such as dextrins and maltose are utilized by this bacterium, or about how these processes are regulated. A study of the nature of the interaction between starch and B. stearothermophilus is therefore also of interest in understanding the role of extra*
MATERIALS AND METHODS Carbohydrates and affinity media. The a-glucosidase substrate p-nitrophenyl-a-maltoside and amylopectin were from Sigma Chemical Co., St. Louis, Mo., and maltohexaose was from Boehringer Mannheim, Mannheim, Federal Republic of Germany. Soluble starch was from Ajax Chemicals, Sydney, Australia. Fluorescein isothiocyanateconjugated amylopectin (FITC-amylopectin) was prepared as previously described (6). The oxirane activation of Sepharose and the covalent coupling of starch were also performed as described (5). The starch content of the affinity matrices used in this study ranged from 6.5 to 9 mg of immobilized starch per ml of packed gel. Other carbohydrates were from commercial sources. Bacterial strain and culture conditions. The bacterium used, B. stearothermophilus ATCC 7953, was a laboratory strain originating from the culture collection of the CSIRO Division of Food Research, Sydney. In routine experiments, the bacteria were grown at 55°C on nutrient broth supplemented with 0.2% (wt/vol) maltose and harvested in the late exponential phase of growth. To test the effects of carbon sources, the minimal medium of Manian and Ward (9) was used. Bacterial suspensions were washed twice in minimal medium A (11) at room temperature before binding or enzyme assays or before column chromatography. Binding studies. The binding of B. stearothermophilus to starch-Sepharose columns was exactly as performed for the affinity chromatographic studies using E. coli (5). For the binding studies involving FITC-amylopectin, the procedures previously described were used (6). Bacterial numbers in fractions were obtained from optical density measurements at 580 nm which were calibrated using a standard curve of optical density at 580 nm against total cell counts. Total cell
Corresponding author. 95
FERENCI AND LEE
96
S20-
I: C-
C-
o
~30O
Cc)(d
LiJ
10
0
13 1 9 13 5 Fraction Number FIG. 1. Affinity chromatography of B. stearothermop/hilus on starch-Sepharose. Bacteria grown on the given carbon source were washed in minimal medium A and suspended to a density of 2 x 101i to 4 x 1010 per ml in the same buffer. Columns containing 9 mg of 1
S
9
immobilized starch per ml of matrix were prepared as described (5) and were loaded with 0.1 ml of bacterial suspension and eluted for seven 0.3-ml fractions with minimal medium A. Subsequent 0.3-ml fractions were eluted with 0.1 M maltose in the same medium. Elution of glucose-grown wild-type bacteria at 25°C is shown in panel a. Wild-type bacteria grown on maltose were eluted at 25C (b) and 55°C (c). The mutant JK3 at 25°C is shown in panel d. counts were performed by microscopic enumeration in a counting chamber. Assay and fractionation of a-glucosidase activity. aGlucosidase was assayed spectrophotometrically at 55°C by following the release of p-nitrophenol at 420 nm from the chromogenic substrates. Washed, intact cell suspensions in minimal medium A as buffer were routinely mixed with substrate at 0.2 mM concentration in 1 ml of total volume. For fractionation of cell components, washed cells were suspended in 10 mM phosphate buffer (pH 7.2) and broken by two passages through a French pressure cell at 12,000 lb/in2. Intact cells were removed by low-speed centrifugation (6,000 rpm for 5 min in a Sorvall SS34 centrifuge rotor). The broken-cell fraction was then subjected to centrifugation at 40,000 x g for 1 h to separate cytoplasmic and particulate components. Assay of a-amylase was by published procedures (2). RESULTS In screening strains of Bacillus, it was found that washed cell suspensions of B. stearothermophilus ATCC 7953 showed a strong, reversible interaction with starch. This was initially demonstrated through chromatography of bacterial populations on starch-Sepharose columns (Fig. 1). Maltosegrown bacteria showed high retention by these columns, and the binding was reversed by competing ca-glucosides such as maltose, maltodextrins, or soluble starch. Bacteria grown on other carbon sources showed lower retention by these columns, as shown with glucose-grown bacteria in Fig. 1.
J. BACTERIOL.
Even starch-grown bacteria were retained to a lesser extent than maltose-grown bacteria. The columns used in Fig. 1 contained 9 mg of immobilized starch per ml of matrix. The binding to these columns was indeed dependent on the starch content of the columns, as were the low recoveries shown in Fig. 1. Half-maximal starch-dependent retention occurred at about 2 mg of starch immobilized per ml of column matrix (Fig. 2). Over 95% retention of bacteria required over 8 mg of starch immobilized per ml of matrix. Some retention was also observed in the absence of starch; the residual binding to the agarose matrix was also partially reversed by eluting with 0.1 M maltose, suggesting an affinity of the bacteria to the carbohydrate component of the matrix. As was to be expected, the ability of 0.1 M maltose to elute bacteria decreased with increasing amounts of starch immobilized. The above results suggested that the B. stearothermophilus cell surface possessed a suitable binding site for at-glucans. This property was also reflected in the ability of amylopectin to agglutinate B. stearothermophilus suspensions. This agglutination was also maltose inducible and inhibited by the presence of 0.1 M maltose (results not shown). To obtain a more quantitative estimate of the affinity of the binding site for sugars, assays involving the binding of fluorescein-labeled amylopectin, as developed for starch binding by E. coli (6), were adopted. Washed suspensions of bacteria bound fluorescent amylopectin, and the binding of this ligand could be used to assay the competition of various sugars for the binding site. The best inhibitor of FITCamylopectin binding was maltohexaose (Table 1). Maltose and macromolecules containing a-1,4-glucosidic linkages were also effective inhibitors. In contrast, a-glucosides such as isomaltose and sucrose were decreasingly effective as competitors, and p-glycosides such as cellobiose or lactose were ineffective. Glucose was also ineffective, suggesting the binding site was relatively specific for compounds containing linear a-1,4-linked glucosides. These results correlated very well with the efficiency with which these sugars
0 O
._
4-I
10 Ias
I:
co
Al
._
'a
2
4 6 8 Starch Immobilized (mg/rmi) FIG. 2. Effects of starch concentration on retention and elution from starch-Sepharose columns. Columns containing different concentrations of immobilized starch were prepared as previously described (5). Bacterial suspensions (2 x 109) were applied to each column and eluted with 2 ml of minimal medium A. Elution was then changed to the same medium containing 0.1 M maltose, and the proportion of bacteria in each fraction was determined from absorbance measurements. Symbols: 0, proportion of applied bacteria retained (i.e., not eluted by medium); 0, proportion of applied bacteria eluted by 0.1 M maltose.
VOL. 166, 1986
STARCH BINDING BY B. STEAROTHERMOPHILUS
97
could elute bacteria from starch-Sepharose columns (results not shown), suggesting that both assays reflect similar binding activity. The binding affinities of washed bacteria for maltooligosaccharides were determined (Fig. 3); the results suggested that the binding sites were half-saturated at 1.8 x 10-3 and 3.4 x 10' M for maltose and maltohexaose, respectively. Hence the affinity of binding is greatest for longer
maltooligosaccharides. The above experiments were conducted at 20 to 24°C, far below the growth optimum of the thermophilic Bacillus strain. When binding experiments were conducted at 55°C, a surprisingly different pattern emerged. The majority of bacteria did not bind starch-Sepharose but were eluted in a diffuse peak starting close to the void volume of the columns (Fig. 1). A similarly reduced binding was found in FITCamylopectin binding assays. To test whether the reduced binding was due to possible breakdown of the ligands, 160 ,ug of FITC-amylopectin was mixed with 1.1 x 109 bacteria in washed suspension for 15 min at 55°C, and the products of the incubation were analyzed for breakdown. Gel-exclusion chromatography of the products on Bio-Gel P2 is shown in Fig. 4. Since this matrix excludes molecules of
