Geoffrey Coleman

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; α-l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.

The characteristics of extracellular protein secretion by Staphylococcus aureus (Wood 46) and their relationship to the regulation of alpha-toxin formation.

The progress of secretion of alpha-toxin and total extracellular protein by Staphylococcus aureus (Wood 46), grown aerobically at 37 degrees C, in a 3% (s/v) tryptone soya broth medium supplemented with vitamins was followed. Exoprotein was secreted at a high rate by intact bacteria during the exponential phase (to 9 h) and into the post-exponential phase. After 18 h, when exoprotein accounted for 33% of the total protein in the culture, no further exoprotein was secreted although the bacterial density continued to increase at a low rate beyond this time. During the phase of active secretion, alpha-toxin represented a constant proportion of total exoprotein, the differential rate of synthesis of which increased by a factor of four after the end of exponential growth. Concomitant with the increase in the differential rate of exoprotein formation there was a fourfold increase in the intracellular concentration of RNA precursor material.

The Metabolism of Cellulose, Glucose and Starch by the Rumen Ciliate Protozoon Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii incubated anaerobically in the presence of ampicillin utilized cellulose and to a lesser extent starch, but not soluble sugars. The protozoon incorporated 14C from 14C-labelled cellulose, glucose and starch; it synthesized protein from these compounds but only at a rate that would allow for the protozoa to divide every 8300, 1200 and 580 h, respectively, if this were the sole source of protein. These substrates were also metabolized with the production of acetic, propionic and butyric acids. Evidence is presented that 70 % of the cellulase inside the protozoa is soluble and not of bacterial origin. It had an activity of 11 to 28 pg cellulose digested h-1 protozoon-1 [0.55 to 3.3 μg cellulose digested h-1 (mg protein)-1].

Subcellular distribution of polysaccharide depolymerase and glycoside hydrolase enzymes in rumen ciliate protozoa

The distribution of polysaccharide depolymerase and glycoside (acid) hydrolase activity in nine genera of rumen entodiniomorphid and holotrich ciliate protozoa was examined by differential centrifugation. Sedimentable activity was detected in all of the protozoa examined and occurred principally in fractions that were prepared by centrifugation at 1000g for 10 min, 10,000g for 10 min, and 20,000g for 20 min (fractions F1, F2, and F3). Acid phosphatase was present in these subcellular fractions which contained membrane-bound vesicles 0.1–0.8 μm in size. The enzyme location profile of the subcellular fractions differed within the genera examined. The distribution of the enzyme activity in the subcellular fractions indicated the presence of distinct populations of hydrolase-containing organelles and other functional vesicles in the rumen ciliates.

A Rapid and Convenient Method for Determining the Dry Weight Content of Bacteria Which Show a Tendency to Form Large Aggregates

Bacillus amyloliquefaciens readily forms large aggregates (Welker & Campbell, I 967) and dry weights determined indirectly by bacterial counts, turbidimetry or nephelometry (Mallette, 1969) are not satisfactory and consequently the direct determination of dry weight has always been used (Coleman & Elliott, 1962). We have now developed a rapid turbidimetric technique for this determination which gives comparable results to those obtained by the direct method. METHOD Samples of bacterial culture, usually in the range 2 to 5 m1, were centrifuged for 2 min at 2500 g and resuspended to the original volume in distilled water. The resulting suspension was then treated with an ultrasonic disintegrator (100 W, 20 kHz; M.S.E. Ltd, Crawley, Sussex) for I min. A sample, usually I ml, of the disrupted preparation was then diluted with distilled water to 2 ml and z ml of 10 % (w/v) trichloroacetic acid was added. After thorough mixing the extinction of the resulting turbid suspension was measured at 600 nm in a cuvette of I cm light path by means of a Unicam SP 500 spectrophotometer.

Pleiotropic compensation in the regulation of extracellular protein formation by a low alpha-toxin-producing variant of Staphylococcus aureus (Wood 46).

The changes in bacterial density, total extracellular protein and haemolysin produced by bacteria from overnight cultures of Staphylococcus aureus (Wood 46) and a low alpha-toxin-producing variant suspended in fresh medium were followed at 37 degrees C. Although five extracellular proteins were produced at a reduced level by the variant (alpha-toxin formation was reduced more than tenfold), the differential rates of total extracellular protein formation by the two organisms were identical. The results are consistent with a common regulatory mechanism for extracellular protein formation in which a pleiotropic compensation may occur in order to saturate the extracellular protein-producing capability.

The effect of glucose on the differential rates of extracellular protein and α-toxin formation by Staphylococcus aureus (Wood 46)

The differential rates of formation of total extracellular protein and α-toxin by Staphylococcus aureus (Wood 46) were determined during aerobic growth, at 37°C, in a complex medium containing 0.0, 0.25 or 1.0% (wt/vol) glucose. Different inocula were employed from 1% (vol/vol) of an overnight culture to 100% where bacterial cells were washed and resuspended in fresh medium without change in density. It was shown that under all conditions examined the differential rates of total extracellular protein formation exhibited a biphasic pattern characteristics of regulation based on ‘competition’. This biphasic pattern was maintained even in the presence of a large inoculum and a high glucose concentration, conditions considered to favour the onset of catabolite repression. However, a lowering of the initial rate was observed with increasing glucose suggesting the superimposition of catabolite repression as a modulating effect under extreme conditions.In the case of the specific extracellular protein component, α-toxin, its differential rate of formation paralleled total exoprotein in all except the condition most favourable for catabolite accumulation when a deviation consistent with a pronounced catabolite repression of this component was demonstrated which was not pH-dependent.

Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria

The complete nucleotide sequence of the rpoB gene which encodes the beta subunit of S. aureus RNA polymerase has been determined. The RpoB protein, consists of 1182 amino acids and has a novel initiation codon UUG which initiates protein synthesis with methionine. There is a very strong Shine-Dalgarno complementarity and the -10 and -35 promoter hexameric sequences are TAATAT and CCGTTT, respectively. A rho-dependent termination site, CAATCAA, is present which overlaps the -35 promoter sequence of the adjacent rpoC gene a feature which may have a role in the co-ordinate expression of the two genes. A strong homology and conserved regions were found to exist over the predicted amino acid sequences coding for S. aureus rpoB and the equivalent proteins in Escherichia coli, Pseudomonas putida, Salmonella typhimurium, Chlamydia trachomatis, cyanobacterium Anabaena sp. strain PCC 7120.

A Comparison of the Patterns of Extracellular Proteins Produced by the High α-Toxin-secreting Organism Staphylococcus aureus (Wood 46) During Aerobic and Anaerobic Growth

Staphylococcus aureus (Wood 46) was grown aerobically and anaerobically in supplemented 3% (w/v) Tryptone Soya Broth medium for 24 h at 37 degrees C. Although the bacterial density achieved was 9 times higher in the aerobic culture, the exoprotein produced per unit of bacterial dry weight was only 1.4 times higher than in the anaerobic culture. However, the SDS-PAGE patterns of extracellular proteins were quite different: the aerobic products occurred almost exclusively in the mol. wt range 15-30000 compared with 30-60000 for those produced anaerobically. The only major component common to both preparations was alpha-toxin which accounted for 2.4 times more of the total exoprotein under aerobic than under anaerobic conditions.