Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes we have determined the structure of the widely-occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active site cofactor comprising two antiferromagnetically-coupled Fe 3+ ions, three Ca 2+ ions, and a μ 3 -bridging oxo group. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.Phosphate-containing macromolecules and metabolites are essential components of living cells. Under conditions of phosphate deficiency microorganisms obtain phosphate from biologically-derived organic compounds by producing extra-cytoplasmic alkaline phosphatases (1, 2). Prominent amongst these enzymes are phosphate monoesterases of the PhoA and PhoX families which are found in all three domains of life. The archetypal PhoA enzyme of Escherichia coli has been extensively studied (2) but PhoX alkaline phosphatases are minimally characterized and do not exhibit sequence similarity to other phosphotransfer enzymes. Genes encoding PhoX are abundant in ocean bacteria (3)(4)(5) and are also present in bloom-forming cyanobacteria (6), human pathogens (7,8), and eukaryotic green algae including the model organism Chlamydomonas reinhardtii (9).

Character count:Current: 171 words in first paragraph 1497 words in rest of text (excluding methods, references, legends).Permitted: 200 words (300 words max) for first paragraph 1500 words max for rest (excluding methods, references, legends). The crystal structure of the fimbrial usher protein FimD revealed an intricate, five-domain architecture: a β-barrel domain, incorporated into which is a β-sandwich plug domain, an Nterminal periplasmic domain (NTD) and two C-terminal periplasmic domains (CTD1 and CTD2) 6,7 ( Supplementary Fig. S1). The mechanism for folding and assembling such a large, multi-domains, protein into the outer membrane is unclear, and currently no assay system is available to study the process in vivo. A candidate to catalyze this assembly reaction was the β-barrel assembly machinery (BAM complex): composed of the essential proteins BamA and BamD, assisted by the proteins BamB, BamC and BamE; Fig. 1a. In addition, bacteria haveTamA and TamB (Fig. 1a) that constitute a translocation and assembly module (TAM) [10][11][12] . The

SummaryAdhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro.

Background: PhoD family enzymes liberate phosphate from organic compounds.Results: The structure of PhoD reveals an active site with one Fe3+ and two Ca2+ ions.Conclusion: PhoD represents a new class of phosphatase related to purple acid phosphatases.Significance: The requirement of PhoD for iron ions may limit bacterial phosphate acquisition in low iron environments.

We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.

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Shigella flexneri Spa15 is a chaperone of the type 3 secretion system, which binds a number of effectors to ensure their stabilization prior to secretion. One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG. In this study, Spa15 alone and in complex with IpgB1 has been studied by double electron electron resonance, an experiment that gives distance information showing the spacial separation of attached spin labels. This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets. The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.

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