- Volume 132, Issue 6, 1986

Incubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: (a) being degraded when cells were incubated with protease; (b) being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; (c) reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.

The protein content of EDTA extracts from 76 strains of Gram-positive anaerobic cocci was examined using SDS-PAGE. Strains of Peptostreptococcus anaerobius produced almost identical profiles; greater heterogeneity was observed within the species Peptococcus magnus, Peptococcus prevotii and Peptococcus asaccharolyticus, but several strains within each biotype produced similar patterns. Serological investigation of these extracts by ELISA revealed numerous cross-reactions among the different biotypes. Immunoblot transfers from polyacrylamide gels demonstrated two common antigens within strains of the species Ps. anaerobius, but these were not species-specific.

An understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci.

Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 M r surface exposed region of the molecule.

Several monoclonal antibodies directed against gonococcal outer membrane protein IB have been used in in vitro assays to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, virulence of the variant P9-17 for epithelial cells in tissue culture was reduced in the presence of three of the four antibodies which recognized type-specific epitopes. Similarly, virulence of P9-17 as well as a recent isolate was reduced in the presence of the one antibody, SM24, which reacted with a conserved epitope. This antibody was also bactericidal in the presence of complement, and in addition was opsonic for several protein IB-expressing strains as determined by polymorphonuclear leucocyte chemiluminescence measurements. Similarly, all the type-specific antibodies were opsonic for P9 variants. However, only two of these antibodies mediated complement-dependent killing although those which were ineffective were nevertheless complement-fixing antibodies. These results indicate that antibodies to closely positioned epitopes on protein I vary in their biological activities and that the conserved epitope recognized by the antibody SM24 is potentially an effective target on the gonococcal surface for immunoprophylaxis.

The adherence of Chlamydia trachomatis LGV440(L1) to human HeLa 229 and mouse McCoy cells was stimulated by the lectin wheat germ agglutinin (WGA) and inhibited by the sugars N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and chitobiose, but only when the chlamydiae had been passaged several times in HeLa cells. After passage in McCoy cells, the lectin and the sugars elicited little response. The non-LGV serovar UW-31(K), however, differed from LGV440(L1) in that, regardless of passage, the lectin and sugar effects were observed only in HeLa cells. Affinity chromatography on WGA-agarose confirmed that HeLa-grown LGV-440(L1) bound to a significantly greater extent relative to McCoy-grown chlamydiae. In addition, participation of heterogeneous chlamydial ligands was suggested by the observation that the adherence of heated (60°C, 5 min) UW-31(K) to HeLa cells at 37°C was not inhibited at all, but at 5°C, the adherence rate was greatly reduced, indicating the participation of heat-stable as well as heat-labile ligands. These data are interpreted to indicate that the passage history of C. trachomatis results in the acquisition of altered surface components that participate in the initial interaction of the bacterium with the host.

Cadmium-adapted Pseudomonas putida exhibited a long lag phase (6 h) on incubation in a defined medium containing 3 mM-Cd2+. During this time extensive blebbing of the outer membrane was observed by electron microscopy and polyphosphate granules containing Cd2+ were present in the cells. Cells from exponential-phase cultures of cadmium-adapted P. putida were found in clusters. They were much smaller than control cells grown without cadmium, and contained electron-dense aggregates. Cadmium-adapted cells released more lipopolysaccharide and protein into the external medium than did control cells; the addition of Ca2+, but not Mg2+, to the medium prevented this increased release. Cadmium-adapted cells also showed greatly increased sensitivity towards certain antibiotics, including the aminoglycosides, cyclic polypeptides and doxycycline. It is suggested that this is related to changes in membrane structure.

A streptomycin-resistant variant of Xanthomonas campestris was grown in defined nutrient-deficient media in both batch and continuous culture. The production, composition and viscosity of the extracellular polysaccharide (xanthan) synthesized by this strain were influenced by the fermentation time and nutrient exhaustion in batch culture and by the dilution rate in continuous culture. The specific rate of exopolysaccharide synthesis was maximal during exponential growth in all of the nutrient-deficient media studied although some xanthan was formed during stationary phase. The concentration of exopolysaccharide decreased at later stages of stationary phase in some cultures. Both the extent of acylation and the consistency index of xanthan isolates were low or minimal during exponential growth, maximal in polysaccharide isolated as the growth rate fell and usually lower after this time. Between dilution rates of 0·03 and 0·06 h−1 in chemostat culture, the cell and exopolysaccharide dry weights were independent of dilution rate, the specific rate of xanthan synthesis decreasing at lower growth rates. Although the variation in the acyl content and consistency index was less than that observed in batch culture, exopolysaccharide isolated at higher dilution rates tended to have a higher acetyl content, lower pyruvyl content and lower consistency index.

A wide range of nitriles and amides act as carbon and/or nitrogen sources for Nocardia rhodochrous LL100-21. Growth on a number of aliphatic nitriles or their corresponding amides resulted in the coinduction of both acetonitrile hydratase and acetamidase activities. In each case the specific activity of acetonitrile hydratase was higher than that of acetamidase. The substrate and inducer specificities were determined for the two enzymes and found to be quite distinct. Propionitrile, although an excellent inducer of both enzymes, was only slowly hydrolysed by acetonitrile hydratase. Conversely acrylonitrile was a good enzyme substrate but had a complete lack of effect as an inducer. Butyramide and methacrylamide acted as poor substrates for acetamidase but induced high levels of both acetonitrile hydratase and acetamidase. Dinitriles of long chain length, lactonitrile and iodoacetamide were inhibitory to growth and enzyme activity.

Neutral L-amino acids were added to liquid cultures of Pseudomonas syringae pv. atropurpurea that were entering the exponential phase of growth. For each amino acid addition experiment the production of the usual coronafacoyl compounds, coronatine and N-coronafacoyl-L-valine, was diminished and a new product was detected. This was isolated and purified, and established by mass spectrometry and GC analysis of the amino acid released by acid hydrolysis to be the N-coronafacoyl amide of each L-amino acid added to the culture. Coupling was established to occur between coronafacic acid and the L-amino acids alanine, ?-aminobutyric acid, norvaline, isoleucine, alloisoleucine, leucine and norleucine. These results suggest that an intracellular enzyme system is operating which lacks an overall rigid specificity in the coupling (amide bond formation) between coronafacic acid and aliphatic neutral amino acids.

Synthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 μg ml−1 h−1) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87% while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase (N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.

Analysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0·1-24·4 g (kg contents)−1, 7·7-66·0 mmol (kg contents)−1 and 1·5-11·1 mmol (kg contents)−1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3·5 to 19·8 mg azocasein hydrolysed h−1 (g faecal material)−1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1·0 × 1011-1·3 × 1012 (g dry wt faeces)−1] and Propionibacterium spp. [1·2 × 108-1·0 × 1010 (g dry wt faeces)−1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.

Forty-eight strains of Thermus isolated from hot springs in Yellowstone National Park, Wyomming, USA, and eight reference strains were subjected to a numerical taxonomic analysis using Gower's coefficient (SG ) with single and average linkage clustering. Two major groups were distringuished, which could be differentiated by colony morphology, ability to reduce nitrate and proteolytic activity. Cluster 1 contained Thermus aquaticus YT-1, the type strain of the species, and cluster 2 contained authentic strains of ‘T. flavus’ and ‘T. thermophilus’. T. ruber was recovered as a single member cluster. The mol% G + C of DNA from representative strains from each cluster was 64·4 to 66·8 for cluster 1, 62·2 to 67·1 for cluster 2 and 62·5 for T. ruber.

A method for the introduction of a bacteriophage DNA into Brevibacterium lactofermenturn protoplasts is described. Frequencies of 105 infective centres per μg DNA were easily achieved, the relationship between the number of infective centres and the amount of DNA being linear up to 5 μg DNA per assay. This method can be used to introduce foreign DNA into these bacteria.

Atomic absorption spectrophotometry was used to characterize aluminium uptake by the cyanobacterium Anabaena cylindrica. An EDTA-washing procedure was used to distinguish between adsorbed and intracellular aluminium. The intracellular aluminium content increased with increasing external concentration and time. The phosphorus concentration in the growth medium did not affect the rate of aluminium uptake nor did dark treatment or addition of CCCP, an uncoupler of phosphorylation. We therefore conclude that aluminium toxicity is due to intracellular aluminium rather than to interactions with nutrients in the growth medium and that aluminium uptake is independent of phosphorus uptake. The accumulation of aluminium in polyphosphate granules and cell walls of phosphorus-rich cells noted earlier is rather due to an increased binding capacity in these cellular compartments. Also, the rapid uptake of aluminium by A. cylindrica mainly occurs via passive diffusion.

Specific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponerna pallidurn among organisms cultivated in oitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).