Search

It was confirmed that U. urealyticum produces an IgAl protease, which cleaves human IgAl only into intact Fab and Fcalpha fragments. By N-terminal amino acid sequencing of Fcalpha fragments, the site of digestion was identified as a Pro235-Thr236 peptide bond within the a chain hinge-region of IgAl. A number of assay systems were examined for their ability to detect and estimate IgAl protease activity. A reliable and reproducible immunoblotting method was developed, in conjunction with a quantifiable assay utilising [125I] IgAl. By these methods, IgAl protease activity was identified in fourteen serotypes of U. urealyticum, all of which appeared to digest IgAl at the same Pro235-Thr236 peptide bond. The enzyme was active over a broad range of pH (pH 3-10) and was inhibited by the serine-protease inhibitors 3,4-DCI and DFP. The IgAl protease was not located in 'spent' ureaplasma cultivation medium but appeared to be cell-associated. The activity was solubilised by a number of non-ionic detergents which were required in purification buffers to maintain enzyme stability, further suggesting a membrane-bound location. Although the enzyme was not purified to homogeneity, a number of protocols were established which provide a basis for future work. A genomic library of U. urealyticum DNA was produced and a variety of strategies adopted for identification of the iga gene. Radiolabelled DNA probes were generated from a plasmid containing the iga gene for N. gonorrhoeae (pIP503). By Southern blot hybridisation, no significant homology was identified between the heterologous probes and ureaplasma genomic DNA. Based on regions of high nucleotide conservation between the iga genes from N. gonorrhoeae and H. influenzae, degenerate PCR primers were designed. While amplification products did not appear to contain regions of the iga, such an approach may be adapted and extended for use in future studies.

Monoclonal antibodies (Mabs) raised against Ureaplasma urealyticum (serotype 8) revealed the presence of three membrane antigens. One major surface antigen of apparent molecular mass 96 kDa, shown to express four distinct epitopes, was found to be serotype-8 specific. Thus, Mabs raised against this polypeptide will unequivocally differentiate serotype 8 from the other serotypes of human origin. The binding of antibodies to this polypeptide partially suppressed the growth of the organisms. Membrane expressed antigenic polypeptides of apparent molecular masses 16 kDa and 17 kDa were expressed by those serotypes belonging to the large serocluster (A), whereas the 17 kDa polypeptide only was expressed in the smaller serocluster (B). Using this Mab probe serotype 13 was placed in the larger serocluster. Thus Mabs, which recognise one or both of these polypeptides, will unequivocally differentiate the two seroclusters of this organism. The cytosolic urease from U. urealyticum, serotype 8, was purified by immuno-affinity chromatography. Two active forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single peak with urease activity of apparent molecular mass 190 KDa was shown by FPLC. Freezing and thawing of the purified enzyme caused a partial breakdown to inactive sub-units whereas total inactivation of the enzyme and denaturation, achieved by boiling for two minutes in the absence of any added denaturing agents, revealed three subunit polypeptides of apparent molecular masses 72, 14 and 11 KDa. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The active enzyme displayed two pH optima of 6.9 and 6.15. Mabs raised against purified urease bound to both the active enzyme and to the inactive 72 kDa subunit. No evidence of antigenicity was found for the 14 and 11 KDa sub-units. These Mabs cross-reacted with ureases from all the other human serotypes. Competition assays revealed a minimum of four and possibly five distinct epitopes on the enzyme, all distinct from its active site. Ureaplasmas from 5 animal hosts were studied using the various Mabs. The 96 kDa antigen was not found in any of the non-human strains. Variations in the available epitopes on the ureases and the presence or absence of the 16/17 KDa antigens in the non-human strains allowed a putative identification of the source of the non-human ureaplasmas. Such investigations also showed that with the exception of the 96 KDa serotype 8-specific antigen, chimpanzee isolates could not be differentiated from the human ureaplasma serotypes belonging to the large serocluster. These Mabs were also used to develop fluorescent probes and other diagnostic assays which included a slide agglutination system and a sensitive urease catch assay which was also converted into a 'dip-stick' assay.

A previous survey of enterococci had identified five strains of Streptococcus faecalis (K55 and SB94) - two subspecies liquefaciens (K60 and K88) and one zymogenes (K87) - and two S. faecium strains (K46 and SB69) which were resistant to tetracycline and streptomycin but susceptible to gentamicin. All the S. faecalis strains and K46 were in addition resistant to erythromycin but only the S. faecium strains were penicillin and ampicillin resistant. The minimal inhibitory concentrations of a further six antibiotics were determined. These values confirmed that in S. faecalis strains, erythromycin resistance was accompanied by resistance to lincomycin and pristinamycin IA, a phenotype typical of macrolide - lincosamide - streptogramin B - type (MLS) antibiotics resistance. The erythromycin resistant K46 however, although resistant to lincomycin, was pristinamycin susceptible and so the basis of resistance is unknown. S. faecalis K60, K87 and SB94 were resistant to kanamycin and neomycin as was S. faecium K46 but all strains were susceptible to spectinomycin. The phenotypes were consistent with resistance mediated by enzymic modification of streptomycin with adenyltransferase (6) and of kanamycin and neomycin with phosphotransferase (3') (5")-III. Erythromycin and tetracycline resistances were expressed constitutively in all strains. Only one S. faecalis (K88) was found to be chloramphenicol resistant and as is typical of Gram-positive bacteria, resistance was inducible. The ability to produce bacteriocin was restricted to beta-haemolytic strain K87 and to strain SB94. Subsequent results indicated that strain K87 probably produced more than one bacteriocin, the activity of which was repressed in the parental strain but which, in derivatives, could be enhanced by the presence of streptomycin. Evidence for the location of resistance, haemolysin and bacteriocin genes was sought from study of the transfer characteristics and stability of markers and from examination of the plasmid content of parental strains and their derivatives. The well characterised S. faecalis subspecies zymogenes strain DS5 (Clewell et al., 1982b) was included for comparison in transfer and curing experiments. All the S. faecalis strains aggregated in response to a cell free filtrate of a plasmid free recipient strain JH2-1, indicating the presence of at least one conjugative plasmid although the low transfer frequencies of most resistance genes in broth matings suggested that response was not necessarily encoded by antibiotic resistance plasmids. Transfer of beta-haemolytic activity and all resistance markers was observed after broth matings but the range of transfer frequencies between strains was wide. Furthermore, the incidence of transfer could be variable particularly in the transfer of DS5 erythromycin resistance and all K87 antibiotic resistances which seemed to be dependent on the production of active donor bacteriocin. Matings of S. faecalis strains carried out on membrane filters were only marginally more efficient in terms of transfer frequencies but were superior with regard to reproducibility of transfer. No antibiotic resistance transfer from S. faecium donors was observed after broth matings and only SB69 tetracycline resistance transferred after filter mating at very low frequency. Several resistance determinants and those encoding β-haemolysin were found to be capable of retransfer indicative of association with genes specifying conjugative ability. Analysis of transconjugant phenotypes revealed that the tetracycline resistance gene of K55, the streptomycin resistance gene of K88 and β-haemolytic activities were always transferred alone but some resistance markers were usually co-transferred with other donor markers.

Planococcus C.C.M. 316, a gram-positive facultative marine halophile, was studied with respect to growth and membrane composition of cells grown in media containing 0.5%, 3% and 10% sea salt. Membranes were prepared from cells grown in the three sea salt concentrations and analysed to determine any changes which may have been caused by the increasing concentrations of salt in the growth media. The three membrane preparations were found to have similar compositions to those reported for other gram-positive cocci. Cells grown in the 3% sea salt concentration contained membranes with a higher protein:lipid ratio and RNA content than the membranes from cells grown in the 0.5% and 10% concentrations. Amino acid analysis of the membrane proteins showed that the composition remained virtually unchanged in the three membrane preparations. The ratio of acidic: basic amino acid residues was nearer to the figures reported for non-halophiles than for those of the extreme halophiles. Examination of the lipids showed that phospholipids predominated to the extent of about 70% of the total lipids. Cardiolipin and lysocardiolipin were the major phospholipids, with phosphatidyl ethanolamine, phosphatidyl glycerol and phosphatidyl serine present as minor components. Glyco-lipids were found to decrease with increasing sea salt concentration in the medium, and in all three membrane preparations constituted only a very small proportion of the total lipids. Neutral lipids contained long chain alcohols, mono-, di- and tri-glycerides, as well as relatively large amounts of the isoprenoid compound squalene. The major fatty acid associated with the lipids was a branched saturated C15 acid which constituted 50 - 7% of the total fatty acids in most fractions. Although increasing salt in the medium produced changes within the proteins and lipids in the membranes, these changes were not such that they could be interpreted as an increase in the halophilic nature of the membrane. The carotenoids were shown to be derived from beta carotene and to consist mainly of 3'hydroxy 4' oxo compounds, although the extent of polar substitutions was dependent on both culture age and the concentration of salt in the medium.

The numbers and species of coliforms and group D streptococci isolated from water samples taken from above and below a sewer outfall by the membrane filtration method were compared. In addition, group D streptococci obtained from a variety of clinical sources were also speciated. The numbers of coliforms below the outfall were shown to increase by a factor of 36, whereas group D streptococci below increased by a factor of 150, as compared with the counts above. Speciation of the isolates indicated that Escherichia coli and Enterobacter species were the most common Coliform species from both sampling sites. Streptococcus faecalis strains were the most prevalent group D streptococci from below the outfall and from clinical sources whereas Streptococcus faecium var casseliflavus strains predominated above. The determination of the Faecal Coliform (FC) to Faecal Streptococci (FS) ratios indicated that pollution in both sites was mainly from human origin. The "in vitro" susceptibility of all isolates to antibacterial agents was tested by the agar dilution method. Chloramphenicol and trimethoprim were most active against all coliform isolates, followed by tetracycline, cephalexin, sulphamethoxazole, ampicillin and streptomycin in that order. Drug resistant coliforms were encountered from both sampling sites, and a significant number of multiple drug-resistant coliforms, particularly E. coli were detected. Both E. coli and Enterobacter species from below the outfall showed a statistically significant increase in resistance to ampicillin, and E. coli strains from below the outfall also showed a statistically significant increase in resistance to stilphamethoxazole as compared with isolates from above. Ampicillin and penicillin were the most active drugs against all species of group D streptococci. Of the two aminoglycosides tested gentamicin was more active than streptomycin. Erythromycin was highly active against more than half of the strains. Tetracycline resistance was most frequent followed by Streptomycin. Streptococcus bovis and Streptococcus equinus strains were, in general, more susceptible to the drugs tested than were the other species. Streptococcus faecium strains displayed the widest range of resistance to the drugs tested. No multiple-drug-resistant group D streptococci species were encountered above the outfall, but a few isolates from below and from clinical sources were multiple-drug-resistant. Statistical analysis showed no significant increase in drug resistance between isolates from above as compared with isolates from below. Conjugation studies indicated that for both groups of organisms, the drug resistance markers were transferable. Curing experiments with acridine orange showed a very low capacity of the agent to eliminate the R-factors from these bacteria.

The optimum temperature for both growth and. pigmentation of S. aurantiaca was found to be 28°. Growth of S. aurantiaca was limited above 40°. Pigmentation was maximal 65 hours after maximum bacterial numbers were obtained, but began to decline rapidly. Growth only occurred to any substantiated degree within narrow pH limits. The total membrane fraction from S. aurantiaca cells harvested at 17 hours (exponential phase), 27 hours (early stationary phase) and 57 hours (late stationary phase) were analysed to determine if any change occurred in the chemical components of the membranes from the exponential to the late stationary phases of growth. The percentage composition of the membrane components in all three membrane fractions were within the range of values reported for membranes isolated from other Gram-positive bacteria. Membranes from the two stationary phase cultures had a similar quantitative chemical composition but both differed in several respects from the membranes isolated from the exponential phase culture. The protein content remained approximately constant throughout the growth phases but the lipid content decreased and the carbohydrate content increased, with age. Since the overall recoveries of organic material from the total membranes decreased with age, it is thought that this could be explained by increased binding of the lipid to protein which would mean decreased lipid extraction and anomalously low lipid contents. This is substantiated by the observed increase in the relative amounts of the bound lipid and phospholipid fractions with age. Analysis of the lipid showed the presence of an unusually high amount of neutral lipid and a lo\v quantity of phospholipid in all three membrane prep0,rations, as compared with those found in other membranes. The fatty acids found in all the membrane hydrolysates were generally typical for a Gram-positive bacterium but there were considerable variations in the relative quantities of the individual fatty acids with age. Amino acid analysis indicated an amino acid content similar to membranes isolated from other sources, and there were again variations in the molar ratios of the amino acids with age. The monosaccharides detected in all the membrane preparations were galactose, glucose, mannose and ribose. The hexoses were shown to be constituents of the glycolipid and oarotenoid glycopeptide fractions. Only trace amounts of galacatosamine and glucosamine were found. All the carotenoids detected in a 27 hour culture of S. aurantiaca were shown to be derived from carotene. The free pigment consisting of carotene, zeinoxanthin and a dihydroxy carotene formed approximately 29% of the total pigment. The remaining 71% were carotenoid glycopeptides where the carotene most probably linked to galactose, glucose, mannose and to amino acids/peptides. The hexose moieties were linked to carotenoid and/or peptides or other hexose moieties by glycosidic bonds involving their reducing groups. Amino acid analysis of a water soluble carotenoid glyopep tide indicated an amino acid composition similar to the total membrane protein. This carotenoid glycopeptide appeared to be homogeneous despite the inclusion of three different hexoses. S°20 values of the carotfenoid glyopeptide indicated a molecular weight of the order of 20,000.

1. The preparation, purification and properties of a water-soluble membrane component from S. flava using the synthetic detergent Lubrol L has been described. This fraction contained carotenoid, glucose and peptide, was highly stable to heat and pH extremes, and release of free carotenoid from it proved extremely difficult. The possible effect of the binding of the membrane components within detergent micelles is discussed and the dangers inherent, in the determination of molecular weights, by osmometry or ultracentrifugation, in the presence of detergent have been indicated. 2. The polar carotenoid subfractions from S. flava have been characterised and found to consist of carotenoid, glucose and peptide. The linkage between carotonoid and glucose is presumed to be glycosidic, and a model for the in vivo orientation of the carotenoid complex in the bacterial membrane has been proposed. A possible correlation between membrane stability and carotenoid content has been found, and this is discussed in relation to the model. 3. After complete removal of the free pigments from S. morrhuae by solvent extraction, a water soluble carotenoid fraction was isolated and characterised. The material, whose molecular weight is approximately 9,000 contains carotenoid, glucose and peptide. The bond between glucose and carotenoid is again presumed to be glycosidlc, and the peptide moiety contains high proportions of the acidic amino acids, the significance of which is discussed. This bound pigment is also thought to represent one form in which: carotenoid is bound in the bacterial membrane. 4. The effect of the age of the culture on the chemical composition of the total membrane fraction from S. flava has been investigated. Both protein and lipid contents decrease with age although there is little variation in carbohydrate content. It is suggested that the decreased lipid content is a reflection of the increased binding of lipid to protein with age. Considerable variation in the fatty acid composition with age was observed, which makes the use of the fatty acid profile as a taxonomic criterion for this species of doubtful value. The presence of a sterol in S. flava membrane lipids is indicated although from GLC data, it seems unlikely that this is cholesterol. Mono-saccharides detected in membrane hydrolysates were ribose, rhamnose, glucose, and mannose. The presence of glucosamine and galactosamine was also indicated. 5, The general ultrastructural features of whole cells of both S. flava and S. morrhuae have been described. S. flava exhibits many of the fine structural features common to Gram-positive organisms and was seen to form the packets of cells typical of the Sarcinae. Cell division in S. flava was shown to be of the cell membrane septation type, and a mechanism for this mode of division has been proposed. Preparation of protoplasts from S. flava using the method of Baird-Parker and Woodroffe (1967) was unsuccessful, but the treatment with lysozyme, revealed a layered appearance of the cell wall. Several intracytoplasmic membranous inclusions were seen in these cells and their relationship to mesosomes is discussed. Mesosomes as such were also present, but never in association with developing septa. The effect of varying conditions of fixation on the fine structure of S. flava was also studied. Evidence has been presented that, under certain conditions, sporulation may occur in S. flava. Good fixation of S. morrhuae cells proved difficult to achieve, and this may be due to insufficient concentration of salts in the fixation medium. Cells of S. morrhuae are approximately twice the size of S. flava cells, and division was seen to occur in a much more random fashion, producing irregular clumps of cells with common cell walls. Spherical or ovoid bodies of unknown composition were seen in association with the cytoplasmic membrane.

1. Studies on the carotenoid pigments of: S. flava indicated the presence of four main fractions, the amount of each being in direct proportion to its polarity. Little could be discovered about the least polar fraction, containing carotenes, and probably the colourless precursors, except that none of the normal carotene precursors appeared to be present. The three xanthophylls were found to be C50 carotenoids with a chromophore of nine conjugated double bonds. All contained hydroxyl groups. 2. Comparison by spectroscopy and chromatography of the carotenoids of S. flava with those of some other bacteria, indicated that the occurrence of these C50 carotenoids might be quite widespread. S. lutea was found not only to produce the same carotenoids, but also to have the same level of pigmentation as S. flava, when the bacteria were grown under the same conditions. 3. Investigations into the protoplast membrane of S. flava proved conclusively that all the carotenoid was contained within the membrane. A water-soluble fraction was obtained from the membrane, and studies showed that it was a lipoprotein complex, in which the bulk of the carotenoid was tightly bound.