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. 2000 Oct;182(19):5365-72.
doi: 10.1128/JB.182.19.5365-5372.2000.

Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron

J A Shipman  1 , J E Berleman, A A Salyers
Affiliations

Characterization of four outer membrane proteins involved in binding starch to the cell surface of Bacteroides thetaiotaomicron

J A Shipman et al. J Bacteriol. 2000 Oct.

Abstract

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of (14)C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.

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Figures

FIG. 1
FIG. 1
(A) Immunoblot showing SusD expression from a multicopy plasmid. Approximately 50 μg of protein was loaded in each lane. All membrane fractions were obtained from cells grown on defined medium with maltose as the sole carbohydrate source. Lanes: 1, membrane fraction from B. thetaiotaomicron 5482; 2, membrane fraction from B. thetaiotaomicron ΩsusC; 3, membrane fraction from B. thetaiotaomicron ΩsusC(pSDC27); 4, membrane fraction from B. thetaiotaomicron ΩsusD; 5, membrane fraction from B. thetaiotaomicron ΩsusD(pSDC27); 6, membrane fraction from B. thetaiotaomicron ΩsusE. This and all other immunoblots shown in this paper were scanned using an Epson Perfection 636U scanner and incorporated into a figure using both Adobe Photoshop 5.5 and Adobe Illustrator 8.0. (B) The Sus operon, showing polar insertional disruption mutations used in these studies.
FIG. 2
FIG. 2
Starch binding by B. thetaiotaomicron mutants compared to that of the wild type. For simplicity, error bars are shown for B. thetaiotaomicron 4007, ΩsusF, ΩsusE, and ΩsusD(pSDC27) only. Error bars for other cases are comparable in size.
FIG. 3
FIG. 3
(A) Immunoblots showing proteolytic sensitivity of SusC and SusD in wild-type intact cells expressing all the Sus proteins. Approximately 100 μg of protein from cell extracts was loaded in each lane. Cells were treated with proteinase K (final concentration, 2 mg/ml). Degradation products of SusC due to proteinase K are shown by the arrows under the original SusC. No degradation products were observed for SusD. The lanes are labeled according to the time that had elapsed after addition of proteinase K. In all cases, the amount of the periplasmic protein, SusA, was the same at all stages of digestion, and no breakdown products were detected (not shown). (B) Immunoblots showing changes in proteolytic sensitivities of SusC and SusD in various mutants. Approximately 100 μg of protein from cell extracts was loaded in each lane. The protein detected on the immunoblot is shown on the right, with the corresponding mutant strains expressing the protein labeled on the left-hand side. The lanes are labeled according to the time that had elapsed after addition of proteinase K.
FIG. 4
FIG. 4
Immunoblots showing proteolytic sensitivity and protection from proteolysis by amylopectin of SusE and SusF in wild-type cells. Approximately 100 μg of protein from cell extracts was loaded in each lane. The immunoblots are labeled above according to whether amylopectin was added to the proteinase K treatment. +AP, addition of amylopectin; −AP, no amylopectin was added. The lanes are labeled according to the time that had elapsed after addition of proteinase K. In all cases, the amount of the periplasmic protein SusA was the same at all stages of digestion, and no breakdown products were detected (not shown).
FIG. 5
FIG. 5
(A) SDS-PAGE gel showing selectivity of amylose-agarose affinity chromatography for Sus OMPs. Molecular markers are shown to the left of the blot. Each protein labeled on the right was confirmed by immunoblots using antisera directed against the corresponding protein. Lanes: 1, membrane proteins of B. thetaiotaomicron 5482; 2, n-octyl-β-d-glucopyranoside-solubilized membrane proteins; 3, wash fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins after loading onto an amylose-agarose column; 4, maltose eluant fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins. (B) Immunoblots showing changes between various mutants in amylose binding activity of SusC, SusD, and SusE. The protein is labeled on the right of the blot, with the mutant strains expressing the corresponding protein labeled to the left of the blot. Lanes: 1, membrane fraction of the mutant; 2, n-octyl-β-d-glucopyranoside-solubilized membrane fraction of the mutant; 3, wash fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins of the mutant after loading onto amylose-agarose column; 4, maltose eluant of n-octyl-β-d-glucopyranoside-solubilized membrane fraction of mutants. Although not evident in the figure, lane 4 of the immunoblot for mutant ΩsusE did show a low level of SusC. The designation ΩsusD + ΩsusC(pSDC27) indicates that solubilized membrane fractions from a strain producing only SusD and one producing only SusC were incubated together overnight before being loaded on the column.
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