Makoto Kuroda

Structural basis for multimeric heme complexation through a specific protein-heme interaction: the case of the third neat domain of IsdH from Staphylococcus aureus.

To elucidate the heme acquisition system in pathogenic bacteria, we investigated the heme-binding properties of the third NEAT domain of IsdH (IsdH-NEAT3), a receptor for heme located on the surfaces of pathogenic bacterial cells, by using x-ray crystallography, isothermal titration calorimetry, examination of absorbance spectra, mutation analysis, size-exclusion chromatography, and analytical ultracentrifugation. We found the following: 1) IsdH-NEAT3 can bind with multiple heme molecules by two modes; 2) heme was bound at the surface of IsdH-NEAT3; 3) candidate residues proposed from the crystal structure were not essential for binding with heme; and 4) IsdH-NEAT3 was associated into a multimeric heme complex by the addition of excess heme. From these observations, we propose a heme-binding mechanism for IsdH-NEAT3 that involves multimerization and discuss the biological importance of this mechanism.

Chromosome-Determined Zinc-Responsible Operon czr in Staphylococcus aureus Strain 912

A novel operon, czrAB (zinc‐responsible genes), was identified in the chromosome of Staphylococcus aureus. The operon consists of two genes, czrA and czrB. The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S. aureus plasmid pI258 and smtB of Synechococcus PCC7942. The czrB, coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium‐resistant factor of Bacillus subtilis and Alcaligenes eutrophus. In the presence of zinc (0.1‐10 mM), the transcription of czrAB was enhanced in a concentration‐dependent manner. Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression. The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM). The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight. The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.

Staphylococcus aureus giant protein Ebh is involved in tolerance to transient hyperosmotic pressure

Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.

A Helical String of Alternately Connected Three-Helix Bundles for the Cell Wall-Associated Adhesion Protein Ebh from Staphylococcus aureus

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.

The hsp Operons Are Repressed by the hrc37 of the hsp70 Operon in Staphylococcus aureus

The heat‐shock proteins are coded for the polycistronic operons hsp70 and hsp60 in Staphylococcus aureus. The hsp70 operon is comprised of five genes, hrc37, hsp20, hsp70, hsp40 and orf35, and the hsp60 is comprised of two genes, hsp10 and hsp60. The hsp70 operon transcribed five different sizes of mRNA from three promoters: P1, the most active promoter, transcribed 6.0 and 3.6 kb mRNAs; P2 transcribed a single 1.8 kb mRNA; and P3 transcribed 4.2 and 2.4 kb mRNAs. The hsp60 operon transcribed a single 2.1 kb transcript from only one promoter, P1. Both operons had a common structure of inverted repeat element (CIRCE, Controlling Inverted Repeat of Chaperon Expression) at the promoter region. All of the transcripts were heat (46 C) inducible. One of the unidentified genes, hrc37, was characterized. The disruptant of the hrc37 in the hsp70 operon enhanced the transcription of both operons at 37 C (derepression). Complementation of the disruptant with the cloned hrc37 plasmid recovered the repression of the transcription of both operons at 37 C. The product of hrc37, Hrc37, was found to bind to the CIRCE element. These findings indicated that Hrc37 from the hsp70 operon repressed the transcription of both the hsp70 and hsp60 operons by binding to the CIRCE element located at the promoter region.

Crystal structure analysis reveals a novel forkhead-associated domain of ESAT-6 secretion system C protein in Staphylococcus aureus.

Pathogenic bacteria secrete many types of virulence factor or exotoxin to infect host cells and cause disease. They are usually secreted by the Sec translocon across the membrane by a mechanism that requires N-terminal signal peptides for recognition. However, Sec-independent systems for secretion of virulence factors, including Gram-negative type III and IV protein secretion systems, have been identified in Group A streptococci and other Gram-positive species.1–4 The Esx secretion system in Staphylococcus aureus has recently been proposed to be one such Sec-independent secretion system.3,5 Esx is a small secreted protein belonging to the early secreted antigen target 6 kDa (ESAT-6)-like proteins, which have a common WXG motif at the center of the sequence.6 Esx homologs have also been identified in other bacteria, including Mycobacterium tuberculosis, Clostridium acetobutylicum, Bacillus subtilis, and Listeria monocytogenes.5,6 The present study was performed in the pathogenic bacterium S. aureus, which has two Esx proteins, EsxA and EsxB, the molecular masses of which are 6 and 10 kDa, respectively, within a gene cluster comprised of eight ORFs [Fig. 1(A)].7 Based on the deletion of the esxa gene in all versions of the attenuated Mycobacterium bovis Bacille Calmette–Guérin vaccine strain,8 the observation that EsxA induces a strong T-cell response during infection,9 and that mutants that fail to secrete EsxA and EsxB show defects in the pathogenesis of S. aureus murine abscesses,5 these proteins in pathogenic bacteria are thought to be among the most important virulence factors in pathogenesis.5 Interestingly, in every species in which esx genes are found, there is an ORF with two or three FtsK/SpoIIIE domains, which is ESAT-6 secretion system C protein (EssC) in the case of S. aureus, in the gene cluster that includes the esx genes [Fig. 1(A,B)].6 The FtsK/SpoIIIE domain—PFAM entry PF01580—was found in the FtsK cell division protein from Escherichia coli and in the SpoIIIE sporulation protein that regulates prespore-specific gene expression in B. subtilis.10,11 This domain contains a putative ATP-binding P-loop motif, and is categorized as an AAAþ domain observed in

Expression, purification, crystallization and preliminary diffraction analysis of CapF, a capsular polysaccharide-synthesis enzyme from Staphylococcus aureus

Capsular polysaccharides (CPs) are important virulence factors of Staphylococcus aureus. The biosynthesis of type 5 and type 8 CPs (CP5 and CP8), which are produced by most clinical isolates of S. aureus, is catalyzed by 16 CP-assembling proteins. One of these proteins is the enzyme CapF, which catalyzes the synthesis of UDP-N-acetyl-L-fucosamine, a component of both CP5 and CP8. Here, the cloning, expression, purification, crystallization and diffraction analysis of CapF are reported. Optimization of the crystallization conditions by differential scanning calorimetry afforded a crystal of selenomethionine-substituted CapF that diffracted to a resolution of 2.80 A. The crystal belongs to space group P3(2)21, with unit-cell parameters a = b = 119.6, c = 129.5 A.

Over-expression of the peptide containing the ATP-binding site in Na/K-ATPase α-subunit

The deduced primary structure of Na/K-ATPase from the cloned nucleotide sequence revealed that the structure of ATP-hydrolysing active site was highly conserved among P-type ATPases (1,2,4). However, the minute conformation and molecular mechanism of ion transport have not been made clear yet. X-ray crystallography may be an efficient method to reveal the conformation of the protein, but such a hydrophobic membrane-protein has been unable to be purifed without using detergents. We made an assumption that out-of-membrane domain of Na/K-ATPase α-subunit facing to hydrophilic side with catalytic activity could be crystallized like regular soluble proteins (3). We report here that the hydrophilic, catalytic domain (50kDa) of α-subunit has been over-expressed in E. coli and purified without using any detergent, and discuss on the characterization for the peptide.