Fibrillogenesis in gelsolin-related familial amyloidosis.

To clarify the mechanisms involved in amyloid formation in Finnish-type familial amyloidosis (FAF), we have tested the in vitro fibrillogenicity of synthetic wild-type and mutated gelsolin peptide analogs and studied the fragmentation patterns of gelsolin in the circulation of FAF patients with the Asn-187 or Tyr-187 gelsolin mutation. Fibril formation of synthetic peptides having sequence homology with wild-type or mutant gelsolins was monitored by Congo-red staining and polarization microscopy, negative staining electron microscopy and quantitative thioflavine-T fluorometry. Immunoblotting with anti-gelsolin and amyloid-specific antibodies and sequence analyses were used to study the fragmentation pattern of gelsolin. Ultrastructurally amyloid-like fibrils were formed from mutant Asn-187 and Tyr-187 gelsolin peptides. Fluorometric analysis revealed highly accelerated fibril formation from the mutant peptides as compared with the corresponding wild-type peptides. Addition of mercaptoethanol alone or in combination with dithiotreitol tended to enhance fibril formation of the 9-mer and 11-mer Asn peptides. Blocking of the C-terminal carboxyl of the mutant Asn-187 gelsolin182-192 peptide by amidation increased amyloidogenicity. The Tyr-187 gelsolin mutation, corresponding to the naturally occurring mutation in the Danish subtype of FAF, required acidic conditions to form fibrils meeting the criteria of amyloid. In FAF patients, in addition to the full-sized gelsolin, a series of lower-molecular mass C-terminal fragments of gelsolin (70,000-45,000 Da) was found in the circulation. In homozygous FAF(Asn-187) the 65-kDa fragment containing the amyloid forming region and the 55-kDa fragment, devoid of that region, was the major gelsolin species in the plasma. The results indicate that the 65-kDa gelsolin fragment derived by alpha-gelsolinase cleavage at the mutation-induced novel proteolysis site Arg172-Ala173 represents the putative circulating precursor protein of tissue amyloid in FAF and that the Asp187Asn/Tyr substitution in gelsolin creates a conformation that is highly fibrillogenic.

Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria.

The mode of antimicrobial action of chitosan (polymeric beta-1,4-N-acetylglucosamine) on gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM). Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced (E. coli, salmonellae) or abolished (P. aeruginosa) by MgCl2. No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl2 and was reversible by washing chitosan-treated cells prior to SDS exposure. Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection.

Colonization strategies and conjugal gene transfer of inoculated Pseudomonas syringae on the leaf surface.

Survival, colonization and activity of Pseudomonas syringae bacteria inoculated onto the leaf surface of the common bean (Phaseolus vulgaris) was studied. Inoculated Ps. syringae cells shortened by half their size in 100% humidity and by an average of one fifth in 40-60% humidity. The respiring portion of the population, measured by the formation of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-formazan crystals, decreased more in 40-60% humidity than in 100% humidity. In scanning electron micrographs, the bacterial cells on leaf surfaces were seen embedded in a mucoid matrix. Intraspecies conjugation of plasmid RP1 also occurred in 40-60% humidity conditions. The portion of transconjugants temporally rose higher than the same portion in 100% humidity conditions. Therefore, although only a small proportion of the inoculated cells remained active on the leaf surface in 40-60% humidity, a relatively high rate of conjugation was still seen. Gene spreading was thus efficient on the leaf surface also when conditions did not allow bacterial population growth.

Frigoribacterium faeni gen. nov., sp. nov., a novel psychrophilic genus of the family Microbacteriaceae.

The taxonomic position of five actinobacterial strains isolated from dust, an animal shed, the air inside a museum and soil was investigated using a polyphasic approach. The growth characteristics were unusual for actinomycetes. Optimal growth was at temperatures ranging from 2 to 10 degrees C. After small-step adaptation (5 degrees C steps) to higher temperatures, the strains were also able to grow at 20 degrees C. Cell wall analyses revealed that the organisms showed a hitherto undescribed, new group B-type peptidoglycan [type B2beta according to Schleifer & Kandler (1972), but with lysine instead of ornithine]. All strains contained menaquinone MK-9. Mycolic acids were not detected. Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were detected in the polar lipid extracts. The main fatty acids were 12-methyl-tetradecanoic acid (15:0 anteiso), 12-methyl-tetradecenoic acid (15:1 anteiso), 14-methyl-pentadecanoic acid (16:0 iso) and 14-methyl-hexadecanoic acid (17:0 iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso-pentadecane (15:0 anteiso-DMA). The G+C content of DNA was approximately 71 mol%. The results of 16S rRNA gene sequence comparisons revealed that the strains represent a new lineage in the suborder Micrococcineae and the family Microbacteriaceae of the order Actinomycetales. On the basis of these results the new genus Frigoribacterium gen. nov. is proposed, harbouring the new species Frigoribacterium faeni sp. nov. (type strain = 801T = DSM 10309T).

Danish type gelsolin related amyloidosis: 654G-T mutation is associated with a disease pathogenetically and clinically similar to that caused by the 654G-A mutation (familial amyloidosis of the Finnish type).

BACKGROUND: Familial amyloidosis of the Finnish type (FAF, Finnish hereditary amyloidosis) is caused by a 654G-A mutation in the gelsolin gene on chromosome 9 resulting in the expression of mutant Asn-187 gelsolin which is abnormally proteolytically processed generating amyloidogenic fragments that polymerize into amyloid fibrils. We have recently shown that in a Danish and a Czech family with a clinical syndrome similar to FAF, including corneal lattice dystrophy, cranial neuropathy and skin changes, the disease is caused by another mutation at the same position, namely 654G-T predicting a Try-for-Asp substitution at 187 in secreted gelsolin. AIM: To undertake a closer examination of the Danish subtype of FAF and report immunohistochemical and biochemical findings. RESULTS: Immunostaining of plasma gelsolin isolated from heterozygous FAF of the Danish subtype revealed a pattern similar to that found in FAF-Asn 187. The > 60 kDa gelsolin species contain an epitope characteristic of the amyloid forming region as revealed by an amyloid specific antibody, whereas the approximately 50 kDa fragments are devoid of it. Compared with the wild-type gelsolin peptide (Asp-187), the corresponding mutant peptide (Tyr-187) showed dramatically increased fibrillogenicity as revealed by quantitative thioflavine-T based fluorimetry; ultrastructurally, amyloid-like fibrils were formed by the mutant peptide. Immunohistochemistry showed that antibodies directed against residues 231-242 of secreted gelsolin, representing the carboxy terminus of the sequence forming the amyloid protein (residues 173-243) laid down in the tissues in a fibrillar form in FAF, specifically labelled the amyloid deposited in rectum and skin in the Danish (654G-T) subtype. CONCLUSIONS: The 654G-T mutation in the gelsolin gene gives rise to an amyloid disease clinically and pathogenetically similar to that caused by the 654G-A mutation.

Mutational analysis of the Pseudomonas syringae pv. tomato hrpA gene encoding Hrp pilus subunit.

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus-dependent autoagglutination of the bacteria. The majority of insertions in the amino-terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy-terminus appeared to be needed for pilus assembly. Insertions in the 5' non-translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

Alzheimer's disease-associated presenilins 1 and 2: accelerated amyloid fibril formation of mutant 410 Cys-->Tyr and 141 Asn-->Ile peptides.

Mutations in the presenilin 1 gene on chromosome 14 and in the related gene on chromosome 1 have been identified in individuals with early-onset familial Alzheimer's disease. The functions of the presenilin gene products as well as the relation of the presenilin mutations to amyloidogenesis are unclear. Here we show that peptides homologous to two disease-associated mutant forms of presenilin 1 and 2, respectively, show highly increased amyloid fibril formation as compared with the wild-type peptide homologues. The 410 Cys -->Tyr (S-182) 14-residue peptide and the 141 Asn-->Ile (E5-1) 15-residue peptide spontaneously assemble to fibrils of 7 to 9 nm width with strong Congophilic characteristics. Thioflavine-T fluorometry reveals a 7- to 18-fold higher rate of amyloid fibril formation of the mutant peptides as compared with the corresponding wild-type homologues. The results provide new insights into the mechanisms by which presenilin mutations lead to Alzheimer-type neuropathology: missense mutations in the presenilin genes may create products that are intrinsically highly amyloidogenic and directly involved in pathogenesis.

Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000.

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.

Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria.

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.

Description of chlorophenol-degrading Pseudomonas sp. strains KF1T, KF3, and NKF1 as a new species of the genus Sphingomonas, Sphingomonas subarctica sp. nov.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine.

The gafD gene encoding the N-acetyl-D-glucosamine-specific fimbrial lectin (adhesin) protein GafD of uropathogenic Escherichia coli was cloned and subjected to genetic analysis. The corresponding gene product was isolated as a MalE fusion protein. The lectin gene was identified with the aid of deletion mutagenesis; mutations in gafD impaired either receptor binding or both receptor binding and fimbria production, depending on the mutation created. All mutants converted to wild-type expressors when complemented in trans with the cloned intact gafD gene. The predicted 354-amino-acid sequence of GafD, deduced from the nucleotide sequence, is closely related to those of the fimbria-associated F17-G and F17b-G proteins coded for by enterotoxigenic and invasive E. coli strains. Isolated GafD was shown to recognize N-acetyl-D-glucosamine by virtue of specific binding to an immobilized receptor, thus proving directly that GafD is a sugar-binding protein. Our results indicate that GafD as such is sufficient for receptor recognition and that the protein also participates in fimbrial biogenesis.

Transfer of polychlorophenol-degrading Rhodococcus chlorophenolicus (Apajalahti et al. 1986) to the genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov.

Three independently isolated polychlorophenol-degrading strains of bacteria were characterized on the basis of chemotaxonomic and nutritional characteristics. Previously, these strains were assigned to the species Rhodococcus chlorophenolicus, which was described on the basis of the properties of one of the strains, strain PCP-I(T) (T = type strain) (J. H. A. Apajalahti, P. Kärpänoja, and M. S. Salkinoja-Salonen, Int. J. Syst. Bacteriol 36:246-251, 1986). However, the results of analyses of mycolic acids suggested that these organisms should be transferred to the genus Mycobacterium as Mycobacterium chlorophenolicum. These bacteria have meso-diaminopimelic acid, arabinose, and galactose as cell wall constituents, mycolic acids containing 75 to 80 carbon atoms, and a predominant menaquinone with nine isoprenoid units and one hydrogenated double bond. The fatty acids include mainly straight-chain saturated and monounsaturated fatty acids with 10 to 18 carbon atoms and a large proportion of 10-methyloctadecanoic acid (tuberculostearic acid). The G+C contents of the DNAs of the three strains range from 67 to 69 mol%.

Amyloid fibril formation in gelsolin-derived amyloidosis. Definition of the amyloidogenic region and evidence of accelerated amyloid formation of mutant Asn-187 and Tyr-187 gelsolin peptides.

BACKGROUND: We have recently shown that the actin-modulating cytoskeletal and plasma protein gelsolin is involved in the pathogenesis of familial amyloidosis of Finnish type. To define the amyloidogenic region(s) in gelsolin and clarify the mechanisms involved in amyloid formation, we tested the amyloidogenicity of synthetic gelsolin peptide analogues. EXPERIMENTAL DESIGN: The in vitro amyloid fibril formation was studied using 22 synthetic peptides 7 to 30 residues long having sequence homology with wild-type or mutant gelsolins. Amyloid formation was monitored by Congo-red staining and polarization microscopy of the peptide aggregates, by negative staining electron microscopy, and by quantitative fluorometry with thioflavine T. RESULTS: Ultrastructurally, amyloid-like fibrils were formed from the mutant Asn-187 and Tyr-187 gelsolin peptides corresponding to the naturally occurring missense mutations found in familial gelsolin amyloidosis syndromes, as well as from a gelsolin peptide having a Val-187 substitution. The shortest peptide tested that was capable of forming amyloid-like fibrils was 9-residue mutant Asn-187 peptide. The corresponding wild-type peptide did not form amyloid. Quantitative fluorometry at the emission maximum 482 nm revealed highly accelerated amyloid fibril formation of the mutant Asn-187, Tyr-187 and Val-187 peptides as compared with the corresponding wild-type peptides. CONCLUSIONS: We have defined the amyloidogenic region of gelsolin to a 9-residue sequence in the highly conserved repetitive motif B and showed that residue 187 represents a critical site where a substitution of an amino acid with a charged side chain (Asp) with an amino acid with an uncharged (Asn) or hydrophobic side chain (Tyr, Val) creates a conformation that is highly amyloidogenic thus providing an explanation for the amyloidogenicity of the Asn-187 and Tyr-187 gelsolin variants.

Intermediate filament-like network formed in vitro by a bacterial coiled coil protein.

The TlpA protein encoded by the virulence plasmid of Salmonella enterica is an alpha-helical 371-amino acid protein possessing characteristics similar to eukaryotic coiled coil proteins (Koski, P., Saarilahti, H., Sukupolvi, S., Taira, S., Rikkonen, P., Osterlund, K., Hurme, R., and Rhen, M. (1992) J. Biol. Chem. 267, 12258-12265). In this paper we have investigated inter- and intramolecular associations and the morphology of structures formed by TlpA. Dynamics and temperature stability of TlpA dimers were studied by examining the feasibility and conditions in which TlpA would form an artificial heterodimer with its truncated derivative. Formation of heterodimers, bridged by Cu(2+)-catalyzed air oxidation of adjacent Cys residues, showed that TlpA dimers are dynamic chain exchanging structures at 37 degrees C, whereas they were nonexchanging at room temperature or on ice. Chemical cross-linking suggested higher order interaction between TlpA dimers. Electron microscopy studies revealed two levels of TlpA organization in vitro: thin filaments and rods, 2-5 nm in diameter, and a higher ordered filament network consisting of tonofilament-like formations with a diameter of 8-15 nm. Electron microscopy of thin-sectioned Escherichia coli over-producing TlpA showed an extraordinary intracellular assembly of proteinacious lamellae with a striated appearance and a 38-nm periodicity. This study describes for the first time a bacterial protein capable of organizing itself into an ordered and suspectedly dynamic intermediate filament-like architecture.

Structure and composition of biological slimes on paper and board machines.

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 mum). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-beta-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 x 10 to 2.6 x 10 (average, 7 x 10) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 mumol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 x 10 cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 x 10 to 1.5 x 10 (average, 4 x 10) cells per g (dry weight) of slime was obtained.

Creation of amyloid fibrils from mutant Asn187 gelsolin peptides.

The amyloid protein in familial amyloidosis, Finnish type, is a 71 amino acid long fragment of the inner region of mutant Asp187----Asn gelsolin. The mechanism of gelsolin amyloid formation was tested with synthetic 11 and 30 residue peptides corresponding to the normal and mutant sequence of gelsolin. Fibrils meeting the morphologic criteria of amyloid were formed from the mutant Asn187 peptides. Substitution of the normal Asp187 residue with the mutant Asn residue resulted in a 9-fold increase in fibrillogenicity as determined by quantitative fluorometry. The present study demonstrates the first successful in vitro creation of amyloid-like fibrils from Asn187 gelsolin peptides and provides evidence that amyloid formation in Finnish amyloidosis is a direct consequence of the Asp187----Asn substitution in gelsolin.

Ultrastructure of a Bacillus sp. strain KL8 isolated from indoor dust.

A motile Gram-positive bacterial strain (KL8) was isolated from indoor dust. It was identified by API-test50 CHB as a species of Bacillus. This Bacillus sp. strain KL8 was described using different electron microscopic techniques: negative staining, thin sectioning, metal shadowing and freeze-etching. An additional surface layer (S-layer) was the outermost layer of the cell wall of this flagellated bacterium. The hexagonally arranged protein lattice covering the cells had a lattice constant about 9-10 nm, which falls in the same range as that of Bacillus anthracis.

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk "Viili".

"Viili," a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.

Morphological and physiological characteristics and lipopolysaccharide composition of N2-fixing (C2H2-reducing) root-associated Pseudomonas sp.

A dinitrogen-fixing Pseudomonas sp. was isolated from the roots of the grass Deschampsia caespitosa. The motile organism, which had 4 to 10 polar flagella, was gram negative, obligately aerobic, oxidase positive, arginine dihydrolase positive, and fluorescent. To verify API20B, API20E, and Oxi-Ferm identifications, as well as results from standard microbiological tests and electron microscopic examinations, which all indicated the organism to be a Pseudomonas, we analyzed its lipopolysaccharide. The lipopolysaccharide contained neutral sugars, phosphorus, heptose, hexosamine, 2-keto-3-deoxyoctonate, and fatty acids, which were dodecanoic, 3-hydroxydecanoic, 2-hydroxydodecanoic, and 3-hydroxydodecanoic acids. Both the qualitative and quantitative compositions resembled known data of the genus Pseudomonas. Dinitrogen fixation, determined as C2H2 reduction in semisolid medium, was supported by several carbon sources including malate and glucose. The N2 fixation activity was decreased if the oxygen concentration of the gas phase was lowered to one-tenth of atmospheric concentration. The highest specific nitrogenase activity recorded was 954 nmol C2H4/mg bacterial protein per hour, which is about 30% of that noted for Azospirillum lipoferum used as reference.

The role of pili in the adhesion of Escherichia coli to human urinary tract epithelial cells.

Pili or fimbriae were purified by a new technique involving solubilization from the bacterial outer membrane by deoxycholate and separation from flagella by 6M urea. This technique was employed to clarify the role of pili for the adherence of urinary tract pathogenic E. coli; a virulence factor in urinary tract infection. The isolated pili formed single bands in SDS gels and were pure by serologic criteria. They retained the binding properties of the whole piliated bacteria, since they bound to uroepithelial cells and agglutinated erythrocytes. Antibodies to purified pili blocked adhesion. The adhesion and hemagglutination reactions by the strains used for pilus purification were mannose-resistant but globotetraos-sensitive, i.e. the strains recognized globoseries glycolipid receptors in the target cells. The occurrence of this property in a freshly collected material of strains was tested using erythrocytes of blood groups P1, P2k and p.