A circular dichroism study of sheath contraction in pyocin R1.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.

Spectral properties of a mixture of fluorescent pigments produced by Pseudomonas aeruginosa.

The major chromophore of a mixture of fluorescent pigments produced by Pseudomonas aeruginosa ATCC 9027 had pH-dependent absorption, excitation, and emission spectra, such that two ionic forms existed in the ground state and three in the excited states. The pigments could complex with several metal ions to change fluorescence and absorption spectra. Although the pigments were separable into several components, spectra indicated that the same fluorescent chromophore was present in each component. Hydrolysis of the mixture of pigments gave amino acids which did not include alanine or lysine. These pigments must therefore differ from those described by other workers, even though similarities of the chromophores were evident from comparisons with data in the literature, and from comparisons of a hydrolytic product of the mixture of pigments, termed compound F, with the chromophore of the fluorescent pigment of Azotobacter vinelandii. Drastic hydrolysis of the latter chromophore also yielded compound F.

1H-NMR studies of the coordination geometry at the heme iron and the electronic structure of the heme group in cytochrome c-552 from Euglena gracilis.

The 1H-NMR lines of heme c in reduced and oxidized cytochrome c-552 from Euglena gracilis were individually assigned and the coordination geometry of the axial ligands was investigated. The electronic structure of the heme and the chirality of the axially bound methionine were found to be of the same type as in mammalian cytochrome c, but different from cytochrome c-551 from Pseudomonas aeruginosa. These results provide additional support for a previously proposed correlation between the chirality of attachment of the axial methionine and the electronic wave functions in oxidized cytochromes of the c type. Comparison of mammalian cytochrome c, cytochrome c-551 and cytochrome c-552 indicates that the chirality of the axially bound methionine is not linked with the evolutionary increase of the polypeptide chain length.

Mössbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins.

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe Mössbauer spectroscopy. The Mössbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.

Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa.

The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single-channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented.

Influence of sodium dodecyl sulphate quality on the electrophoretic mobility of the outer membrane proteins of mucoid and non-mucoid Pseudomonas aeruginosa.

The electrophoretic mobilities of proteins F and H1 from the outer membrane of Pseudomonas aeruginosa (mucoid and non-mucoid) in polyacrylamide gel electrophoresis were affected by the quality of sodium dodecyl sulphate (SDS) used. In particular, the sodium tetradecyl sulphate impurity present in crude SDS influenced the mobilities of F and H1. These observations explain conflicting reports on changes in outer membrane proteins with strain and growth conditions. Synthesis of H1 was induced by growth in magnesium depleted medium but repressed when calcium or manganese were added to magnesium depleted medium.

Specificity of the glucose channel formed by protein D1 of Pseudomonas aeruginosa.

Protein D1 from the outer membrane of Pseudomonas aeruginosa was purified and reconstituted into proteoliposomes. Many small molecules were shown to diffuse through the D1 channel in the proteoliposomes, and the permeation rates of D-glucose, L-glucose, D-xylose, and L-xylose were much higher than expected for their size. This finding and the permeation rates of various glucose analogs suggest that, although the channel has a specific recognition site for glucose, it functions in a manner very different from that of the glucose carrier of erythrocytes.

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution.

The crystal structure of cytochrome c5 from Azotobacter vinelandii has been solved and refined to an R value of 0.29 at 2.5 A resolution. The structure of the oxidized protein was solved using a monoclinic crystal form. The structure was solved by multiple isomorphous replacements, re-fit to a solvent-leveled multiple isomorphous replacement map, and refined by restrained least squares. The structure reveals monomers associated about the crystallographic 2-fold axis by hydrophobic contacts at the "exposed heme edge". The overall conformation for the monomer is similar to that of Pseudomonas aeruginosa cytochrome c551. However, relative to a common heme conformation, c5 and c551 differ by an average of 6.8 A over 82 alpha-carbon positions and the propionates of c5 are much more exposed to solvent. The shortest heme--heme contact at the "dimer" interface is 6.3 A (Fe to Fe 16.4 A). Alignment of c5 and c551 shows that the two cytochromes, in spite of sequence differences, have remarkably similar charge distributions. A disulfide stacks on a tyrosine between the N- and C-terminal helices.

Reconstituted and native iron-cores of bacterioferritin and ferritin.

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells.

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

Resistance to ciprofloxacin appearing during therapy.

The development of resistance to ciprofloxacin in nine clinical isolates of Pseudomonas aeruginosa was investigated. Isolates had increases in minimal inhibitory concentrations (MICs) from 0.25 to 16 micrograms/ml. The isolates also became resistant to ofloxacin and norfloxacin, but did not show increases in MICs to aminoglycosides, antipseudomonas penicillins, or cephalosporins. One isolate from a patient with endocarditis showed a reduction in a 43-kD outer membrane protein and simultaneous increase in the imipenem MIC. This isolate also showed impaired uptake of ciprofloxacin. Respiratory isolates from cystic fibrosis patients did not show loss of outer membrane protein. MICs were lowered by ethylene diaminetetra-acetic acid, suggesting changes in lipopolysaccharide. Resistant isolates were synergistically inhibited by combinations of ciprofloxacin plus tobramycin or ceftazidime, but MICs remained beyond the achievable serum level.

Structure of the O-polysaccharide chain of lipopolysaccharide from Pseudomonas aeruginosa IID 1001 (ATCC 27577).

Structural studies were carried out on an acidic O-polysaccharide released by mild acid treatment from the lipopolysaccharide of Pseudomonas aeruginosa IID 1001 (ATCC 27577), which is serologically related to the serotypes Habs O3, Lanyi O1, and Wokatsch O13 in other serological classifications of Pseudomonas aeruginosa. The composition and data from structural analyses including H-NMR and C-NMR measurements, methylation, and Smith degradation showed that the structure of the IID 1001 O-polysaccharide was coincident with that of the Habs O3 and Lanyi O1 antigens (or Wokatsch O13). However, whereas solvolysis of the O-antigen of Habs O3 as well as that of Lanyi O1 by hydrogen fluoride has been reported to yield a reducing trisaccharide, GlcNAc(alpha 1----4)GalNAcA(alpha 1----3)Bac2NAc4Nacyl (acyl represents a 3-hydroxybutanoyl group), hydrogen fluoride hydrolysis of IID 1001 O-polysaccharide yielded a nonreducing trisaccharide with the reducing terminal bacillosamine residue linked at C-1 to the hydroxyl group of its N-acyl substituent, 3-hydroxybutanoic acid. These results, in combination with mass spectral data, led to the most likely structure for the tetrasaccharide repeating unit of the IID 1001 O-polysaccharide, (formula; see text) in which the location of N-acyl groups on bacillosamine residues differs from that in the O-antigens of Habs O3 and Lanyi O1 (or Wokatsch O13).

Subunit arrangement in the extended sheath of pyocin R.

An optical diffraction study has been made of electron micrographs of extended sheaths of pyocin R. It has been demonstrated that the extended sheath consists of annuli of six subunits, which are arranged in a helix of 3.57 annuli per one turn. The annulus repeat in the helix direction is 35A. The dimensions and probably the helical parameters of pyocin R sheath are different from those of T4 phage tail, but a structural correlation appears to exist between the extended sheath of pyocin R and that of the phage tail.

The effect of defocusing on the electron microscopic image of the extended particle of pyocin R1.

The effect of defocusing on the electron microscopic image of the extended particle of pyocin R1 was examined with the aid of optical diffraction. The results show that the through-focus image of the cross-striated particle changed to a fishbone-like image with a little under-focusing, which had been considered to be a transitional state during the sheath contraction of phage G. In the optical diffraction pattern of the defocused image, a strong meridional reflection corresponding to the distance of cross-striations in the through-focus image almost disappeared, supporting the change to the fishbone-like image on defocusing.

Comparative study on fibers isolated from four R-type pyocins, phage-tail-like bacteriocins of Pseudomonas aeruginosa.

Five R-type pyocins have been reported which are almost identical with one another in their morphology and subunit composition, though distinct in receptor-binding specificity. We isolated fibers from pyocin R2, R3, and R4 by essentially the same procedure as used in our previous isolation of pyocin R1 fiber with unimpaired receptor-binding ability. All the isolated fibers including R1 fiber were indistinguishable from one another, in terms of electron microscopic observation and subunit composition analysis by SDS gel electrophoresis. They consisted mainly of Subunit No. 2 (Mw 71,000) and No. 9 (31,000) proteins. Although Subunit No. 9 protein in every fiber was susceptible to trypsin and afforded a fragment with the same molecular weight (about 19,000) detectable in the SDS gel, Subunit No. 2 protein was cleaved with trypsin only after the fiber had been treated with an organomercurial, 4-(p-sulfophenylazo)-2-mercuriphenol. The cleavage of Subunit No. 2 protein proceeded to give several fragments with molecular weights ranging from 64,000 to 34,000, and the fragmentation patterns were electrophoretically distinct at least among R1 fiber, R3 fiber, and others (R2 and R4 fibers). The results indicate that Subunit No. 2 proteins of these fibers are different from one another in the structure surrounding trypsin-susceptible peptide bonds. Immunological investigations with anti-R1 fiber antibodies provided some additional information on the difference among R-type pyocins at the fiber level.