Amyloids are ordered protein aggregates, found in all kingdoms of life, and are involved in aggregation diseases as well as in physiological activities. In microbes, functional amyloids are often key virulence determinants, yet the structural basis for their activity remains elusive. We determined the fibril structure and function of the highly toxic, 22-residue phenol-soluble modulin α3 (PSMα3) peptide secreted by PSMα3 formed elongated fibrils that shared the morphological and tinctorial characteristics of canonical cross-β eukaryotic amyloids. However, the crystal structure of full-length PSMα3, solved de novo at 1.45 angstrom resolution, revealed a distinctive "cross-α" amyloid-like architecture, in which amphipathic α helices stacked perpendicular to the fibril axis into tight self-associating sheets. The cross-α fibrillation of PSMα3 facilitated cytotoxicity, suggesting that this assembly mode underlies function in.

The phenol-soluble modulin (PSM) peptide family, secreted by Staphylococcus aureus, performs various virulence activities, some mediated by the formation of amyloid fibrils of diverse architectures. Specifically, PSMα1 and PSMα4 structure the S. aureus biofilm by assembling into robust cross-β amyloid fibrils. PSMα3, the most cytotoxic member of the family, assembles into cross-α fibrils in which α-helices stack into tightly mated sheets, mimicking the cross-β architecture. Here we demonstrated that massive T-cell deformation and death is linked with PSMα3 aggregation and co-localization with cell membranes. Our extensive mutagenesis analyses supported the role of positive charges, and especially Lys17, in interactions with the membrane, and suggested their regulation by inter-and intra-helical electrostatic interactions within the cross-α fibril. We hypothesize that PSMα3 cytotoxicity is governed by the ability to form cross-α fibrils and involves a dynamic process of co-aggregation with cell membrane, rupturing it.

Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that has a 38-kb gene cluster for the utilization of arabinan, a branched polysaccharide that is part of the plant cell wall. The bacterium encodes a unique three-component regulatory system (araPST) that includes a sugar-binding lipoprotein (AraP), a histidine sensor kinase (AraS), and a response regulator (AraT) and lies adjacent to an ATP-binding cassette (ABC) arabinose transport system (araEGH). The lipoprotein (AraP) specifically bound arabinose, and gel mobility shift experiments showed that the response regulator, AraT, binds to a 139-bp fragment corresponding to the araE promoter region. Taken together, the results showed that the araPST system appeared to sense extracellular arabinose and to activate a specific ABC transporter for arabinose (AraEGH). The promoter regions of the arabinan utilization genes contain a 14-bp inverted repeat motif resembling an operator site for the arabinose repressor, AraR. AraR was found to bind specifically to these sequences, and binding was efficiently prevented in the presence of arabinose, suggesting that arabinose is the molecular inducer of the arabinan utilization system. The expression of the arabinan utilization genes was reduced in the presence of glucose, indicating that regulation is also mediated via a catabolic repression mechanism. The cluster also encodes a second putative ABC sugar transporter (AbnEFJ) whose sugar-binding lipoprotein (AbnE) was shown to interact specifically with linear and branched arabino-oligosaccharides. The final degradation of the arabino-oligosaccharides is likely carried out by intracellular enzymes, including two ␣-L-arabinofuranosidases (AbfA and AbfB), a ␤-L-arabinopyranosidase (Abp), and an arabinanase (AbnB), all of which are encoded in the 38-kb cluster.The natural degradation of biomass from plants is a key step in the carbon cycle (53,69,79). This process is carried out mainly by microorganisms that can be found either free or as part of the digestive system in higher animals (76). The three main polysaccharides in the plant cell wall are cellulose, hemicellulose, and pectin, which are rigidified by lignin, a heterogeneous aromatic polymer (28, 60). Pectin is a complex polysaccharide and may account for up to 30% of the dry weight of the plant cell wall (46). Arabinan is a pectic polysaccharide consisting of a backbone of ␣-1,5-linked L-arabinofuranosyl units, which are further decorated mainly with ␣-1,2-and ␣-1,3-linked arabinofuranosides (46).Three general strategies are taken by the microbial world for plant cell wall degradation and can be described as follows. Anaerobic bacteria, such as Clostridium spp., have evolved unique multienzyme complexes, named cellulosomes, that integrate many cellulolytic and hemicellulolytic enzymes and mediate both the attachment of the cell to the crystalline polymer and its controlled hydrolysis (9,16,23,65). Aerobic fungi, such as Trichoderma and Aspergillus, secrete a large variety of free cellulases, hemicellulases...

Type I galactan is a pectic polysaccharide composed of b-1,4 linked units of D-galactose and is part of the main plant cell wall polysaccharides, which are the most abundant sources of renewable carbon in the biosphere. The thermophilic bacterium Geobacillus stearothermophilus T-6 possesses an extensive system for the utilization of plant cell wall polysaccharides, including a 9.4-kb gene cluster, ganREFGBA, which encodes galactanutilization elements. Based on enzyme activity assays, the ganEFGBA genes, which probably constitute an operon, are induced by short galactosaccharides but not by galactose. GanA is a glycoside hydrolase family 53 b-1,4-galactanase, active on high molecular weight galactan, producing galactotetraose as the main product. Homology modelling of the active site residues suggests that the enzyme can accommodate at least eight galactose molecules (at subsites À4 to +4) in the active site. GanB is a glycoside hydrolase family 42 b-galactosidase capable of hydrolyzing short b-1,4 galactosaccharides into galactose. Applying both GanA and GanB on galactan resulted in the full degradation of the polymer into galactose. The ganEFG genes encode an ATP-binding cassette sugar transport system whose sugar-binding lipoprotein, GanE, was shown to bind galactooligosaccharides. The utilization of galactan by G. stearothermophilus involves the extracellular galactanase GanA cleaving galactan into galactooligosaccharides that enter the cell via a specific transport system GanEFG. The galacto-oligosaccharides are further degraded by the intracellular b-galactosidase GanB into galactose, which is then metabolized into UDP-glucose via the Leloir pathway by the galKET gene products.

Arabinanases are glycosidases that hydrolyse alpha-(1-->5)- arabinofuranosidic linkages found in the backbone of the pectic polysaccharide arabinan. Here we describe the biochemical characterization and the enzyme-substrate crystal structure of an inverting family 43 arabinanase from Geobacillus stearothermophilus T-6 (AbnB). Based on viscosity and reducing power measurements, and based on product analysis for the hydrolysis of linear arabinan by AbnB, the enzyme works in an endo mode of action. Isothermal titration calorimetry studies of a catalytic mutant with various arabino-oligosaccharides suggested that the enzyme active site can accommodate at least five arabinose units. The crystal structure of AbnB was determined at 1.06 A (1 A=0.1 nm) resolution, revealing a single five-bladed-beta-propeller fold domain. Co-crystallization of catalytic mutants of the enzyme with different substrates allowed us to obtain complex structures of AbnBE201A with arabinotriose and AbnBD147A with arabinobiose. Based on the crystal structures of AbnB together with its substrates, the position of the three catalytic carboxylates: Asp27, the general base; Glu201, the general acid; and Asp147, the pKa modulator, is in agreement with their putative catalytic roles. In the complex structure of AbnBE201A with arabinotriose, a single water molecule is located 2.8 A from Asp27 and 3.7 A from the anomeric carbon. The position of this water molecule is kept via hydrogen bonding with a conserved tyrosine (Tyr229) that is 2.6 A distant from it. The location of this molecule suggests that it can function as the catalytic water molecule in the hydrolysis reaction, resulting in the inversion of the anomeric configuration of the product.