Cell Wall Cross-linking Research Articles

Reactions of dehydrodiferulates with ammonia

Lignocellulosic materials derived from forages and agricultural residues are potential sustainable resources for production of bioethanol or other liquid biofuels. However, the natural recalcitrance of such materials to enzymatic hydrolysis is a major obstacle in their efficient utilization. In grasses, much of the recalcitrance is associated with ferulate cross-linking in the cell wall, i.e., with polysaccharide-polysaccharide cross-linking that results from ferulate dehydrodimerization or with lignin-polysaccharide cross-linking that results from the incorporation of (polysaccharide-bound) ferulates or diferulates into lignin, mainly via free-radical coupling reactions. Many pretreatment methods have been developed to address recalcitrance, with ammonia pretreatments in general, and the AFEX (Ammonia Fiber Expansion) process in particular, among the more promising methods. In order to understand the polysaccharide liberating reactions involved in the cleavage of diferulate cell wall cross-links during AFEX pretreatment, reaction products from five esters modeling the major diferulates in grass cell walls treated under AFEX-like conditions were separated and characterized by NMR and HR-MS. Results from this study indicate that, beyond the anticipated amide products, a range of degradation products derive from an array of cleavage and substitution reactions, and reveal various pathways for incorporating ammonia-based nitrogen into biomass.

Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Identification of ferulate oligomers from corn stover

Cross-links between plant cell wall polymers negatively impact forage digestibility. Hydroxycinnamates and their oligomers act as cross-links between polysaccharides and/or polysaccharides and lignin. Higher ferulate oligomers such as dehydrotrimers were identified in cereal grains but not in vegetative organs of grasses. The aim of this study was to characterize ester-linked hydroxycinnamate oligomers from corn stover with special emphasis on ferulate dehydrotrimers. With the exception of the 4-O-5-dehydrodiferulic acid all known ferulate dehydrodimers, including the recently described 8-8(tetrahydrofuran) dimer, were identified in the alkaline hydrolyzate of corn stover after chromatographic fractionation. Next to dehydrodimers, 18 cyclobutane dimers made up of ferulic acid and/or p-coumaric acid were identified by GC-MS of the dimeric size exclusion chromatography fraction. Ferulate dehydrotrimers were isolated by using multiple chromatographic procedures and identified by UV spectroscopy, MS and NMR. Four trimers were unambiguously identified as 5-5/8-O-4-, 8-O-4/8-O-4-, 8-8(aryltetralin)/8-O-4-, and 8-O-4/8-5-dehydrotriferulic acids, a fifth tentatively as 8-5/5-5-dehydrotriferulic acid. The formation of ferulate dehydrotrimers is not limited to reproductive organs of grasses but also contribute to network formation in the cell walls of vegetative organs. Although radically coupled hydroxycinnamate dimers and oligomers were in the focus of researchers over the last decade, the earlier described cyclobutane dimers significantly contribute to cell wall cross-linking.

Improving the hardness of thermally processed carrots by selective pretreatments

Abstract The aim of this study was to improve the texture of thermally processed carrots by selective pretreatments modifying plant-intrinsic properties. Pretreatments were a combination of a thermal or high-pressure (HP) treatment followed by a 1 h soak in a specific solution. Lowering the degree of methyl-esterification (DM) of the carrot pectin was confirmed to be one strategy to reduce texture degradation. The thermal or HP pretreatment resulted in pectin with a lower DM which is less susceptible to β-eliminative depolymerization. A subsequent Ca2+ soak resulted in an even better texture by enhancing the amount of pectin cross-links within the cell wall. Lowering the pH of the carrots was proven to be another strategy. A thermal or HP pretreatment followed by soaking carrots in solutions of low pH proved to be effective in lowering the internal carrot pH, hereby retarding β-elimination and consequently texture degradation. The composition of the low pH solution was shown to be of importance; soak solutions containing cations and/or Ca2+ complexing agents have to be avoided. Ferulic acid proved to be a good acidifying candidate. In conclusion, for texture improvement of thermally processed carrots, lowering the susceptibility for β-elimination and enhancing cell wall cross-links are two main targets which both can be reached by manipulating different plant-intrinsic properties.

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.

Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5

NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D (13)C-(1)H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4 : 1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d(6)-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

Infection induced oxidative cross-linking of hydroxyproline-rich glycoproteins (HRGPs) is associated with restriction of Colletotrichum sublineolum in sorghum

Hydroxyproline-rich glycoproteins (HRGPs) accumulation and oxidative cross-linking is one of the earliest defense responses in plants against pathogen infection. In the present study HRGP accumulation in three sorghum genotypes i.e. SC146 (resistant), cv. SC326 (intermediately resistant) and BTx623 (susceptible) as a response to Colletotrichum sublineolum isolate CP2126 infection is elucidated. HRGPs were monitored by hydroxyproline (Hyp) estimation. In genotypes SC146 and genotypes SC326 there was a significantly higher amounts of Hyp at 2 days after inoculation (dai) compared to genotype BTx623, indicating an infection induced accumulation of HRGPs. Western blot analysis of acid-ethanol extracted proteins with polyclonal antibody raised against pearl millet purified HRGPs identified four bands with molecular masses of ~65, 45, 17 and 14 kDa as HRGPs. Insolubilization of the 45 kDa protein in genotypes SC146 and SC326 upon infection with C. sublineolum indicates a role of this protein in cell wall cross-linking, coinciding with heavier accumulation of hydrogen peroxide. In addition, tissue print analysis using polyclonal antibody of pearl millet HRGPs recognized these cross-linked proteins to be HRGPs. These findings indicated that HRGPs in sorghum is a component of defense reaction against C. sublineolum infection.

Chemistry and occurrence of hydroxycinnamate oligomers

Hydroxycinnamates such as ferulic acid, sinapic acid and p-coumaric acid ester-linked to plant cell wall polymers may act as cross-links between polysaccharides to each other, but also to proteins and lignin. Although sinapates and p-coumarates also form cell wall cross-links by the formation of radically or photochemically formed dimers, ferulate derivatives are the quantitatively most important cross-links in the plant cell wall. While the first radically generated ferulate dimer was already identified almost 40 years ago, the spectrum of known ferulate dimers was considerably broadened within the last 15 years. Higher ferulate oligomers were generated in model systems, but also isolated from plant materials. Different model systems using either free hydroxycinnamic acids or their esters are reviewed, highlighting a discussion of the relevance of these models for the plant cell wall. The first ferulate trimer from plant material was discovered in 2003 and seven dehydrotrimers of ferulic acid were isolated from maize bran since. Some of these trimers were also identified in other plant materials such as wheat and rye grains, corn stover, sugar beet and asparagus. Formation mechanisms of ferulate trimers and implications for the plant cell wall are discussed. Ferulate tetramers are the highest oligomers isolated from plant materials so far. These compounds can theoretically cross-link up to four polysaccharide chains, assuming all cross-links are formed intermolecularly. Formation of intramolecular versus intermolecular polysaccharide cross-links is a key question to be answered in the future if we want to judge properly the importance of hydroxycinnamate cross-links in the plant cell wall.

Infection biology and defence responses in sorghum againstColletotrichum sublineolum

To investigate the infection biology of Colletotrichum sublineolum (isolate CP2126) and defence responses in leaves of resistant (SC146), intermediately resistant (SC326) and susceptible (BTx623) sorghum genotypes. Infection biology and defence responses were studied quantitatively by light microscopy, H(2)O(2) accumulation by DAB staining and HRGP accumulation by immunological methods. Inhibition of conidial germination and appressorium formation may represent prepenetration defence responses on the leaf surface. Inducible defence responses in the resistant genotypes included decreases in formation of appressoria as well as accumulation of H(2)O(2), HRGPs and phytoalexins. Concomitant with these inducible responses, fungal growth was stopped during or just after penetration in genotypes SC146 and SC326. High levels of H(2)O(2) accumulating at late infection stages (5 days after inoculation) in the susceptible genotype BTx623 correlated with necrosis and tissue degeneration. The early accumulation of H(2)O(2) and HRGPs indicates roles in defence whereas the late accumulation in genotype BTx623 correlated with successful pathogenesis. The fact that there is a significant correlation between induced accumulation of H(2)O(2), papilla formation and cell wall cross-linking, as evidenced by HRGP accumulation, and cessation of pathogen growth in resistant genotypes may help exploit host resistance in sorghum.

Seasonal patterns of leaf H2O2 content: reflections of leaf phenology, or environmental stress?

H2O2 is an ubiquitous compound involved in signalling, metabolic control, stress responses and development. The compatibility of leaf tissue levels with these functions has, however, often been questioned. The objective here is to document H2O2 levels and variability under natural conditions, and their underlying causes. Using the FOX method, bulk H2O2 concentrations were analysed in leaf samples from 18 species of herbs and trees throughout the 2006 growing season. Sampling addressing targeted predictions was emphasised in 2007 and 2008. H2O2 levels varied 100-fold through the year, with a main peak in spring. Two hypotheses were examined: (H1) that H2O2 reflects seasonally variable responses to environmental stresses, and (H2) that it reflects metabolism associated with leaf development. Based on poor or inappropriate correlations between H2O2 and indicators of light, temperature or drought stress, support for H1 was minimal. H2 was supported both by seasonal patterns and by targeted analyses of concentration changes throughout leaf development. This study concludes that bulk tissue H2O2 concentrations are poor indicators of stress, and are generally too high to reflect either signalling or metabolic control networks. Instead, the linkage of H2O2 and leaf phenology appears to reflect the roles of H2O2 in cell expansion, lignification and wall cross-linking.

Cross-linking of plant cell walls with dehydrated fructose by smoke-heat treatment

Cross-linking of plant cell walls with dehydrated fructose by smoke-heat treatment

Structural Insight into the Transglycosylation Step of Bacterial Cell-Wall Biosynthesis

Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.

Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by “rescuing” structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316–319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, l-galactono-γ-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.

N-thiolated β-lactams: Studies on the mode of action and identification of a primary cellular target in Staphylococcus aureus

This study focuses on the mechanism of action of N-alkylthio β-lactams, a new family of antibacterial compounds that show promising activity against Staphylococcus and Bacillus microbes. Previous investigations have determined that these compounds are highly selective towards these bacteria, and possess completely unprecedented structure–activity profiles for a β-lactam antibiotic. Unlike penicillin, which inhibits cell wall crosslinking proteins and affords a broad spectrum of bacteriocidal activity, these N-thiolated lactams are bacteriostatic in their behavior and act through a different mechanistic mode. Our current findings indicate that the compounds react rapidly within the bacterial cell with coenzyme A (CoA) through in vivo transfer of the N-thio group to produce an alkyl-CoA mixed disulfide species, which then interferes with fatty acid biosynthesis. Our studies on coenzyme A disulfide reductase show that the CoA thiol-redox buffer is not perturbed by these compounds; however, the lactams appear to act as prodrugs. The experimental evidence that these β-lactams inhibit fatty acid biosynthesis in bacteria, and the elucidation of coenzyme A as a primary cellular target, offers opportunities for the discovery of other small organic compounds that can be developed as therapeutics for MRSA and anthrax infections.

Crh1p and Crh2p are required for the cross‐linking of chitin to β(1‐6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.

Cell wall construction in Saccharomyces cerevisiae

In this review, we discuss new insights in cell wall architecture and cell wall construction in the ascomycetous yeast Saccharomyces cerevisiae. Transcriptional profiling studies combined with biochemical work have provided ample evidence that the cell wall is a highly adaptable organelle. In particular, the protein population that is anchored to the stress-bearing polysaccharides of the cell wall, and forms the interface with the outside world, is highly diverse. This diversity is believed to play an important role in adaptation of the cell to environmental conditions, in growth mode and in survival. Cell wall construction is tightly controlled and strictly coordinated with progression of the cell cycle. This is reflected in the usage of specific cell wall proteins during consecutive phases of the cell cycle and in the recent discovery of a cell wall integrity checkpoint. When the cell is challenged with stress conditions that affect the cell wall, a specific transcriptional response is observed that includes the general stress response, the cell wall integrity pathway and the calcineurin pathway. This salvage mechanism includes increased expression of putative cell wall assemblases and some potential cross-linking cell wall proteins, and crucial changes in cell wall architecture. We discuss some more enzymes involved in cell wall construction and also potential inhibitors of these enzymes. Finally, we use both biochemical and genomic data to infer that the architectural principles used by S. cerevisiae to build its cell wall are also used by many other ascomycetous yeasts and also by some mycelial ascomycetous fungi.

Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.

Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8–8-coupling product acylating cereal plant cell walls

A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically derives from 8-8-coupling of ferulate followed by water addition to one of the incipient quinone methide moieties and internal trapping of the other. The finding adds complexity to the analysis of plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways occurring in planta.

Ascochyta blight of chickpea: infection and host resistance mechanisms

Ascochyta rabiei (teleomorph Didymella rabiei) is a directly penetrating, necrotrophic fungus that infects all aboveground parts of chickpea (Cicer arietinum). During spore germination and infection, germ tubes secrete a mucilaginous substance to facilitate attachment to the host surface, and the invading fungus produces cell-wall-lytic enzymes to penetrate the host. The pathogen produces several phytotoxins (solanapyrones A, B, and C, cytochalasin D, and a proteinaceous toxin) that seem to be responsible for necrosis and cell death. The pathogen can degrade antimicrobial compounds and suppress their production in chickpea. On the basis of aggressiveness, the population of A. rabiei can be classified into two broad pathotypes: pathotype I (less aggressive) and pathotype II (aggressive). Complete resistance to A. rabiei has not been found in chickpea; the resistance present in superior cultivars used in chickpea production is partial or incomplete. There is a high degree of variation in resistance among chickpea cultivars, and the resistance declines as the plant matures. The symptoms of infection and disease severity follow a quantitative continuum based on aggressiveness of the pathogen, genetic resistance present in the cultivar, and age of the plant. The well-established defense responses in chickpea are cross-linking of cell walls mediated by hydrogen peroxide, production of pathogenesis-related (PR) proteins (chitinase, β-1,3-glucanase, and thaumatin-like proteins), and accumulation of phytoalexins. However, expression of these induced defense responses does not correlate with pathotype-specific resistance, indicating that other constitutive or unknown components may be involved in providing resistance to aggressive pathotypes. Lack of information about the attribute that makes the pathogen aggressive, as well as inadequate knowledge of pathotype-specific defense mechanisms and the causes for decline in resistance, are major constraints in developing cultivars with durable resistance.

Anchor Structure of Staphylococcal Surface Proteins: V. ANCHOR STRUCTURE OF THE SORTASE B SUBSTRATE IsdC

Staphylococcus aureus sortase A cleaves surface protein precursors bearing C-terminal LPXTG motif sorting signals between the threonine and glycine residues. Using lipid II precursor as cosubstrate, sortase A catalyzes the amide linkage between the carboxyl group of threonine and the amino group of pentaglycine cross-bridges, thereby tethering C-terminal ends of surface proteins to the bacterial cell wall envelope. Staphylococcal sortase B also anchors its only known substrate, the IsdC precursor with a C-terminal NPQTN motif sorting signal, to the cell wall envelope. Herein, we determined the cell wall anchor structure of IsdC. The sorting signal of IsdC is cleaved between threonine and asparagine of the NPQTN motif, and the carboxyl group of threonine is amide-linked to the amino group of pentaglycine crossbridges. In contrast to sortase A substrates, the anchor structure of IsdC displays shorter glycan strands and significantly less cell wall cross-linking. A model is proposed whereby sortases A and B recognize unique features of sorting signals and peptidoglycan substrates to deposit proteins with distinct topologies in the cell wall envelope.