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. 2008 Feb;190(4):1350-8.
doi: 10.1128/JB.01149-07. Epub 2007 Dec 14.

Xylanase attachment to the cell wall of the hyperthermophilic bacterium Thermotoga maritima

Wolfgang Liebl  1 , Christoph Winterhalter, Wolfgang Baumeister, Martin Armbrecht, Michael Valdez
Affiliations

Xylanase attachment to the cell wall of the hyperthermophilic bacterium Thermotoga maritima

Wolfgang Liebl et al. J Bacteriol. 2008 Feb.

Abstract

The cellular localization and processing of the endo-xylanases (1,4-beta-D-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane ("toga") of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

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Figures

FIG. 1.
FIG. 1.
Xylanase zymogram staining of SDS-polyacrylamide gel of fractions of T. maritima cells grown on soluble starch (A), oat spelt xylan (B), or xylose (C). Lanes 1, molecular mass marker proteins; lanes 2, total crude extract; lanes 3, soluble fraction after spheroplast preparation (cleared supernatant; periplasmic fraction); lanes 4, residual fraction after spheroplast preparation (nonperiplasmic cell-bound proteins). In each gel, the samples applied to lanes 2, 3, and 4 were derived from identical amounts of cells. Dark bands are Coomassie blue-stained protein bands, and white bands against the gray background correspond to proteins with xylanase activity. The mobilities of full-length XynA (120 kDa) and XynB (40 kDa) are marked at the right.
FIG. 2.
FIG. 2.
Zymogram of SDS-polyacrylamide gel with xylanases from the supernatant of a T. maritima MSB8 culture and their separation via cellulose affinity chromatography. Lane 1, concentrated culture supernatant (10 μg protein) before cellulose affinity chromatography; lane 2, material with no affinity to microcrystalline cellulose (100 μg protein); lane 3, XynA eluted from the cellulose affinity column with 0.1 M cellobiose. Note that it is not possible to accurately estimate the relative amounts of XynA and XynB on the basis of the intensity of the active bands due to the fact that the two enzymes differ significantly in specific activity, pH and temperature optima, and molecular mass (43). The numbers at the right indicate the positions of molecular mass markers.
FIG. 3.
FIG. 3.
SDS-PAGE analysis of crude extract and membrane proteins of T. maritima. See footnote a of Table 1 for details of sample preparation. Lanes 1 and 5, crude extract; lanes 2 and 6, wash supernatant; lanes 3 and 7, washed membrane fraction; lane 4, molecular mass standard proteins. The sizes of the markers are indicated at the right. Ten micrograms of protein was applied to each lane. The left half of the gel was stained with Coomassie brilliant blue, while the right half of the gel was stained for xylanase activity.
FIG. 4.
FIG. 4.
Electron micrograph of immunogold-labeled thin sections of fixed, Lowicryl K4M-embedded Thermotoga maritima cells. The primary antibody used for labeling of XynA was raised against the recombinantly synthesized, purified catalytic domain of XynA. The secondary antibody was an anti-rabbit IgG gold (10 nm) conjugate.
FIG. 5.
FIG. 5.
Epifluorescence micrographs of cells of T. maritima strain MSB8 grown in medium containing 0.25% xylose after immunolabeling with anti-XynA antiserum and anti-rabbit IgG fluorescein isothiocyanate conjugate. (A) Formaldehyde-fixed cells labeled without ethanol pretreatment. This micrograph represents a double exposure under phase-contrast and epifluorescence microscope conditions in order to visualize both fluorescent (arrowheads) and nonfluorescent cells. (B) Epifluorescence micrograph of cells labeled after pretreatment with ethanol as described in Materials and Methods. In this case, all cells displayed green fluorescence.
FIG. 6.
FIG. 6.
Summary of processing events observed with toga-associated and secreted XynA derivatives produced by T. maritima strain MSB8. The hydrophobic core of the XynA signal peptide as well as charged residues and the predicted standard signal peptidase cleavage site (which apparently is not utilized in T. maritima) are also indicated.
FIG. 7.
FIG. 7.
Alignment of typical type IV prepilin signal peptides with the N-terminal sequence of T. maritima XynA. The highly conserved glycine and glutamic acid residues are boxed. An aspartic acid residue near the processing site is boxed in the case of the T. maritima enzyme, which lacks the glutamic acid at position +5. The arrow indicates the processing site.
FIG. 8.
FIG. 8.
(A) Schematic model of the T. maritima cell envelope, showing the postulated mode of anchoring of XynA in the toga via a hydrophobic N-terminal insertion signal. The five-domain modular structure of XynA consists of the central catalytic domain (B) flanked by repeated N-terminal domains (A1 and A2) and repeated C-terminal domains (C1 and C2), which represent CBMs. The most abundant proteins of the T. maritima cell envelope, i.e., Ompα (a dimeric coiled-coil protein apparently spanning the periplasm [14]) and Ompβ (a porin [13, 34]), are also indicated. The lipid content and composition of the T. maritima outer membrane and the mode of association of Ompα with murein or the cytoplasmic membrane are not clear. (B) Helical wheel representation of the hydrophobic cores of the postulated outer membrane anchors of XynA and Ompα of T. maritima. Hydrophobic residues are shaded in light gray. In both cases the glycine residues (marked with asterisks) occupy similar positions in the structure.
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