Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.     

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.     

Hydrophobic adsorptive and hemagglutinating properties ofEscherichia coli possessing colonization factor antigens (CFA/I or CFA/II), type 1 pili,or other pili

Hydrophobic and hemagglutinating activities of piliated enterotoxigenicEscherichia coli possessing colonization factor antigens (CFA)/I and putative CFA/II, strains with type 1 pili, and piliated strains of nonenterotoxigenicE. coli from urinary tract infections were compared. When passed through columns of hydrophobic Phenyl Sepharose in the presence of buffered ammonium sulfate, the strains with CFA adsorbed most strongly. Similarly, the CFA strains showed a tendency to autoagglutinate at a lower (NH₄)₂SO₄ concentration than the other strains studied. The degree of hydrophobicity of the strains tested is in the order CFA/I>CFA/II>type 1 pili>urinary tract strains. Rough variants ofE. coli strains were more hydrophobic than their smooth parents. Electron microscopy showed large numbers of pili on CFA strains, whereas type 1 piliated strains possessed fewer pili. CFA-negative clones possessed few or no, pili and did not adsorb to the gel. A highly piliated mutant strain (PAK/2PfS) ofPseudomonas aeruginosa bound to the Phenyl Sepharose while the poorly piliated wild-type strains did not. Strains, lost their adsorptive capacity after blending, sonication, heating, or trypsin treatment. It is concluded that the hydrophobicity of enteric organisms, as measured by hydrophobic interaction chromatography, is a function of the type and number of pili on the cell surface.     

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM₁) and, to a lesser extend, ll³N-acetylneuraminosylgangliotetraosyl ceramide (GM₁) in solid-phase binding assays. Asialo-GM₁, but not GM₁, inhibited both PAK and PAK pili binding to immobilized asialo-GM₁ on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM₁, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM₁ and pili occurs at the pilus tip as asialo-GM₁ coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)^ox-OH and PAO(128–144)^ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM₁ and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM₁ Monoclonal antibody PK3B had no effect on PAK pili binding to asialo-GM₁ Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip-associated phenomenon Involving a tip-exposed C-terminal region of the pilin structural subunit.     

α<Superscript>5</Superscript>β<Subscript>1</Subscript> integrins and fibronectin are involved in adherence of the<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ER97314 clinical strain to A549 cells

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.     

Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin

We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches. Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin. We conclude that flagellin is an adhesin of P. aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

Pseudomonas aeruginosa Type IV pilus expression in Neisseria gonorrhoeae: effects of pilin subunit composition on function and organelle dynamics

Type IV pili (TFP) play central roles in the expression of many phenotypes including motility, multicellular behavior, sensitivity to bacteriophages, natural genetic transformation, and adherence. In Neisseria gonorrhoeae, these properties require ancillary proteins that act in conjunction with TFP expression and influence organelle dynamics. Here, the intrinsic contributions of the pilin protein itself to TFP dynamics and associated phenotypes were examined by expressing the Pseudomonas aeruginosa PilA(PAK) pilin subunit in N. gonorrhoeae. We show here that, although PilA(PAK) pilin can be readily assembled into TFP in this background, steady-state levels of purifiable fibers are dramatically reduced relative those of endogenous pili. This defect is due to aberrant TFP dynamics as it is suppressed in the absence of the PilT pilus retraction ATPase. Functionally, PilA(PAK) pilin complements gonococcal adherence for human epithelial cells but only in a pilT background, and this property remains dependent on the coexpression of both the PilC adhesin and the PilV pilin-like protein. Since P. aeruginosa pilin only moderately supports neisserial sequence-specific transformation despite its assembly proficiency, these results together suggest that PilA(PAK) pilin functions suboptimally in this environment. This appears to be due to diminished compatibility with resident proteins essential for TFP function and dynamics. Despite this, PilA(PAK) pili support retractile force generation in this background equivalent to that reported for endogenous pili. Furthermore, PilA(PAK) pili are both necessary and sufficient for bacteriophage PO4 binding, although the strain remains phage resistant. Together, these findings have significant implications for TFP biology in both N. gonorrhoeae and P. aeruginosa.     

The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces

Pseudomonas aeruginosa readily binds to stainless steel and other abiotic surfaces, causing major problems in both the medical and food industries. In this study, we show that P. aeruginosa binds to abiotic surfaces in a concentration-dependent, saturable manner during the initial stages of biofilm formation. P. aeruginosa type IV pili mediate binding to stainless steel as a pilus-deficient strain does not bind to steel, purified type IV pili bound in a concentration-dependent, saturable manner, and purified pili competitively inhibited whole cell binding. PAK pili can also bind polystyrene and polyvinylchloride in a concentration-dependant and saturable manner. As an antibody specific for the C-terminal pilin receptor binding domain inhibited adherence to abiotic surfaces, the role of the C-terminal receptor binding domain in mediating binding to steel surfaces was examined. A synthetic peptide of the PAK pilin epithelial cell receptor binding domain [PAK(128-144)ox] bound directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide with an apparent Ki of approximately 0.2 nM and effectively inhibited the binding of viable homologous and heterologous P. aeruginosa strains to steel with an apparent Ki of approximately 4 nM. A single point mutation (K130I) in the PAO receptor binding domain was observed to abolish binding to stainless steel while binding to human buccal epithelial cells was enhanced. Therefore, the C-terminal receptor binding domain appears to have evolved for binding a variety of surfaces.     

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

PAK is regulated by PI3K, PIX, CDC42, and PP2Calpha and mediates focal adhesion turnover in the hyperosmotic stress-induced p38 pathway

Fractionation of brain extracts and functional biochemical assays identified PP2Calpha, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2Calpha dephosphorylated PAK1 and p38, both of which were activated upon hyperosmotic shock with the same kinetics. In comparison to growth factors, hyperosmolality was a more potent activator of PAK1. Therefore we characterize the PAK signaling pathway in the hyperosmotic shock response. Endogenous PAKs were recruited to the p38 kinase complex in a phosphorylation-dependent manner. Overexpression of a PAK inhibitory peptide or dominant negative Cdc42 revealed that p38 activation was dependent on PAK and Cdc42 activities. PAK mutants deficient in binding to Cdc42 or PAK-interacting exchange factor were not activated. Using a panel of kinase inhibitors, we identified PI3K acting upstream of PAK, which correlated with PAK repression by pTEN overexpression. RNA interference knockdown of PAK expression reduced stress-induced p38 activation and conversely, PP2Calpha knockdown increased its activation. Hyperosmotic stress-induced PAK translocation away from focal adhesions to the perinuclear compartment and resulted in disassembly of focal adhesions, which are hallmarks of PAK activation. Inhibition of PAK by overexpression of PP2Calpha or the kinase inhibitory domain prevented sorbitol-induced focal adhesion dissolution. Inhibition of MAPK pathways showed that MEK-ERK signaling but not p38 is required for full PAK activation and focal adhesion turnover. We conclude that 1) PAK plays a required role in hyperosmotic signaling through the PI3K/pTEN/Cdc42/PP2Calpha/p38 pathway, and 2) PAK and PP2Calpha modulate the effects of this pathway on focal adhesion dynamics.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle.

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na+ + K+ + Mg2+)-ATPase was similar (1.40 and 1.65 micronmol Pi/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio (Na+/K+) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types. The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a Mr=28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca2+ and Mg2+) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor. These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

Purification and characterization of the CS2 pili of colonization factor antigen II produced by human enterotoxigenic Escherichia coli

A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied. Analysis revealed that CS2-possessing ETEC was predominant among isolates from traveller's diarrhea at Osaka, Japan. TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl-Sepharose CL-4B. It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili. This suggests that TH61 pili purified in this study are CS2 pili. Subunit (pilin) of the purified pili has a molecular weight of about 16,000. Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili. These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.