Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

Influence of iron restriction on growth and the expression of outer membrane proteins by Haemophilus influenzae and H. parainfluenzae

Abstract The growth of both Haemophilus influenzae and H. parainfluenzae was progressively inhibited in media containing increasing concentrations of the iron chelator desferrioxamine. Iron restriction had little effect on the outer-membrane (OM) protein profiles of type b or non-typable H. influenzae although replacement of haemin with protoporphyrin IX resulted in the induction of an M _r 73 000 protein in the type b strains. H. parainfluenzae , however, responded to iron restriction by inducing several new proteins in the range of M _r 70 000 to 86 000.     

Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions

Abstract The outer membrane protein composition of mucoid Pseudomonas aeruginosa recovered without subculture from the sputum of a cystic fibrosis patient was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicated that three outer membrane proteins in the range of M _r 80 000–90 000 were induced. The induction of these proteins can be simulated by growing the same isolate under iron-restricted conditions in laboratory media. This initial study gives the first direct biochemical evidence that mucoid P. aeruginosa grows under iron restricted conditions in the lungs of the cystic fibrosis patient.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Dextran sulphate enhancement of lipopolysaccharide-induced tumour necrosis factor-α production by murine peritoneal macrophages: Correlation with macrophage blockade

Abstract Proteose peptone-induced murine peritoneal macrophages (Mø) were preincubated with 100–800 μg/ml of dextran sulphate (DS) 500 ( M _r 500 000) or DS1000 ( M _r 1 000 000). After 2–24 h of the preincubation, the Mø were stimulated with 1 μg/ml of lipopolysaccharide (LPS) in vitro for 18 h in DS-free culture medium. The culture supernatants were then collected for TNF assay. The LPS-induced TNF activity of Mø supernatant preincubated with DS500 or DS1000 for 6 h was enhanced by up to about ten-fold compared with those preincubated without DS. This enhancing effect was not observed when Mø were preincubated with 100–800 μg/ml of low molecular weight DS5 ( M _r 5000) or neutral dextran (Dex) 500 ( M _r 500 000). The enhancement of LPS-induced TNF-α production from Mø was observed after 2 or 4 h of incubation with DS1000 or DS500, respectively. The phagocytic activity of Mø was determined in vitro by the ingestion index and phagocytic capacity using Saccharomyces cerevisiae . Treatment with DS500 or DS1000 significantly suppressed the phagocytic activity from 2 h after the incubation, but this suppression was not observed in Mø incubated with DS5 or Dex500. Our experiments indicate that DS500 and DS1000 act directly on Mø and enhance LPS-induced TNF-α production from Mø, and that the enhancement is closely related to the suppression of Mø phagocytic function.     

Intergeneric protoplast fusion in lactic acid bacteria

Abstract Crystals from Bacillus thuringiensis var. israelensis appeared to contain three major proteins of M _r 230 000, 130 000 and 28 000. These proteins were solubilized from the crystals by incubation in 10 mM DTT, pH 9.5, and purified by sucrose gradient centrifugation. The M _r 230 000 and 130 000 crystal proteins showed mosquitocidal properties, whereas the M _r 28 000 crystal protein contained haemolytic activity. Immobilization of these proteins on latex beads did not alter these properties. Partial proteolytic degradation showed that the M _r 130 000 and 28 000 proteins are structurally different.     

Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis

Abstract Highly purified preparations of inner, i.e. cytoplasmic and intracytoplasmic, membranes and outer membranes were isolated from Nitrobacter hamburgensis strain X14 by sucrose density-gradient centrifugation of cell-free extracts. The two membrane fractions differed markedly in morphology, density, and protein composition as determined by polyacrylamide gel electrophoresis. The inner membrane fraction was enriched in NADH oxidase and nitrite oxidase activity. It contained four major protein bands of apparent M _rs of 28 000, 32 000, 70 000, and 116000. The outer membrane fraction was characterized by the presence of 2-keto-3-deoxyoctonate and contained two major proteins of apparent M _rs of 13 000 and 50 000. There was no evidence for differences between cytoplasmic and intracytoplasmic membranes.     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

Molecular masses of cAMP-binding proteins in yeasts

Abstract The cAMP-binding proteins of different yeasts were photoaffinity labeled using 8- N ₃-[³²P]cAMP, and the M _r values of the labeled proteins estimated by SDS-polyacrylamide gel electrophoresis. The M _r values of the cAMP-binding proteins may be grouped into two size classes: (A) M _r of about 50 000 represented by Saccharomyces cerevisiae and S. uvarum , and (B) M _r of about 60 000 represented by Kluyveromyces fragilis, K. lactis, K. marxianus, S. globosus and S. rouxii .     

Nicotine dehydrogenase from Arthrobacter oxidans: A molybdenum-containing hydroxylase

Abstract The nicotine dehydrogenase from Arthrobacter oxidans was purified 40-fold to homogeneity with 26% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to M _r of 82 000, 30 000 and 15 000. The M _r of the native enzyme was calculated to be 12 0000 by gel chromatography. The enzyme contained about 1 FAD, 1 molybdenum, 4 iron and 2 labile sulfur.     

Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D

Abstract Poly(3-hydroxybutyric acid) granules, which harbored only four major granule-associated proteins as revealed by SDS polyacrylamide gel electrophoresis, were isolated from crude cellular extracts of Chromatium vinosum D by centrifugation in a linear sucrose gradient. N-Terminal amino acid sequence determination identified two proteins of M _r 41 000 and M _{r 40 000} as the phaE _Cv and phaC _Cv translational products, respectively, of C. vinosum D. In a previous study it was shown that both proteins are required for the expression opf poly(3-hydroxyalkanoic acid) synthase activity. The N-terminus of the third protein ( M _r 17 000) exhibited no homology to other proteins. Lysozyme, which was during purification of the granules, exhibited a strong affinity to PHB granules and was identified as the fourth protein enriched with the granules.     

Purification and properties of l-lactate dehydrogenase from Acinetobacter calcoaceticus

Abstract Membrane-bound l -lactate dehydrogenase has been purified almost to homogeneity from Acinetobacter calcoaceticus . The enzyme is an oligomeric protein of sub-unit M _r 40 000 containing non-covalently bound FMN as a prosthetic group. Purified l -lactate dehydrogenase has an apparent K _m of 83 μM for l -lactate but has no activity with, and is not inhibited by, d -lactate. The enzyme is strongly inhibited by HgCl₂, but other thiol reagents and metal-chelating compounds have little or no effect upon its activity.     

Bacteria recovered without subculture from infected human urines expressed iron-regulated outer membrane proteins

Abstract Twelve enteric bacterial strains were recovered by differential centrifugation of urines which were collected from clinically diagnosed and microbiologically confirmed cases of urinary tract infection. The outer membrane protein (OMP) profiles of the clinical isolates were then analysed by sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that 5 of the 12 isolates (3 Escherichia coli strains, 1 Klebsiella pneumoniae and 1 Proteus mirabilis strain) expressed 2 or more high M _r proteins in the range of 66000 to 85000. These high M _r proteins were expressed by the same organisms during growth in vitro in iron-restricted conditions but not in iron-sufficient media.
In addition, it was found that the major outer membrane proteins expressed by the clinical isolates varied considerably and that, in many cases, fresh isolates expressed fewer porin proteins than the same bacterial strains after growth in vitro in trypticase soy broth. This is thus the first evidence the E. coli, K. pneumoniae and P. mirabilis grow under iron-restricted conditions in the urinary tract of humans and that the outer membrane protein profile of clinical isolates differ from in vitro grown bacteria.     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Plasmid-associated bacteriocin production in a JK-type coryneform bacterium

Abstract The outer membrane of Escherichia coli K-12 has a variety of proteolytic activities. We were able to label several outer membrane proteins with [³H]diisopropylfluorophosphate (DFP). This suggests that they are serine proteases. The number of labelled proteins detected varied with the E. coli K-12 strain used. Strains bearing a tolC mutation, in addition, gave better labelling and/or had more labelled proteins. A previously described [³H]DFP-labelled outer membrane protein was shown not to be the TolC protein since it has a slightly lower M _r, it is not labelled more intensely in a TolC-overproducing strain, and it is still labelled in tolC mutant strains.     

Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.     

Cloning and expression in Escherichia coli of a species-specific Chlamydia trachomatis outer membrane antigen

Abstract A gene library of Chlamydia trachomatis serovar L₂ (strain 434) was constructed in Escherichia coli using plasmid pBR322. Amongst 200 recombinants we have identified and characterized a recombinant E. coli that expresses a protein antigen of M _r 74 000 similar in size to an outer membrane antigen produced by elementary bodies of C. trachomatis . Immunologically, the molecule synthesised by E. coli has the same specificity as the protein encoded by serovar L₂. A 1.8 kb DNA fragment from the recombinant insert, used as a hybridization probe, confirmed the species specificity of this clone at the gene level.