- Volume 132, Issue 7, 1986

Summary: Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 × 104mouse LD50 (mg protein)−1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 × 105LD50 (mg protein)−1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of M r 144 000 was demonstrated before trypsin treatment and two bands of M r 100 000 and 55 000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.

Summary: Glutamine synthetase (GS) (EC 6.3.1.2) was purified from an Escherichia coli glnA deletion strain containing the Thiobacillus ferrooxidans GS structural gene. The apparent Mr of the cloned T. ferrooxidans GS subunit was approximately 60000. This indicates a particle Mr for the undissociated enzyme of 720000, assuming the enzyme is the typical dodecamer. Electron microscopy of purified GS revealed characteristic disc shaped molecules with central holes. The cloned T. ferrooxidans GS was regulatedby Mg2+ or Mn2+,adenylylation, and nitrogen source but was not affected by feedback modifiers. The GS has a γ-glutamyltransferase isoactivity point of pH 7.71.

Summary: NAD(P)H nitrate reductase (EC 1.6.6.2) from the yeast Candida nitratophila has been purified to electrophoretic homogeneity. The concentrated enzyme has a molecular mass of 365 kDa and consists of four subunits each of 95 kDa, but appears to dissociate into dimers after dilution. The absorption spectrum of the homogeneous protein is typical of a b-type cytochrome and its isoelectric point is pH 5.4. The enzyme utilizes both NADH and NADPH but is more active with NADH. Preincubation of pure enzyme with NAD(P)H and cyanide, but not NAD(P)H and ADP, leads to a reversible redox inactivation of the enzyme. Preincubation with NAD(P)H alone activates the enzyme.

Summary: Immunoelectrophoresis, using antibodies raised against homogeneously pure nitrate reductase (NR; EC 1.6.6.2), was used to study the regulation of NR synthesis in Candida nitratophila. Extracts from ammonium-grown cultures did not contain cross-reacting material, but cross-reacting material was detected in cultures transferred to nitrogen-free and nitrate medium. The appearance of NR activity in these extracts appeared to be the result of de novo protein synthesis. The possible role of nitrate in the stimulation of active NR synthesis is discussed. Losses of NR activity that occurred in nitrate-grown cultures transferred to ammonium medium correlated with decreases in cross-reacting material.

Summary: Stationary phase cells of Candida albicans can form either a bud or a hypha, depending upon the pH of the medium into which they are released. At low pH, cells form an ellipsoidal bud and at high pH, cells form an elongated hypha. By staining cells with rhodamine-conjugated phalloidin, we have compared the dynamics of actin localization during the formation of buds and hyphae. Before evagination, actin granules were distributed throughout the cytoplasmic cortex in both budding and hypha-forming cells. Just before evagination, actin granules clustered at the site of evagination, then filled the early evagination in both budding and hypha-forming cells. With continued bud growth, the actin granules then redistributed throughout the cytoplasmic cortex. In marked contrast, with continued hyphal growth, the majority of actin granules clustered at the hyphal apex. This distinct difference in actin granule localization may be related to the distinct differences in the expansion zones of the cell wall recently demonstrated between growing buds and hyphae. The spatial and temporal dynamics of the large neck actin granules and of actin fibres are also described.

Summary: The Bacillus subtilis spoVE locus was isolated from a λ clone bank and a 4.7 kbp EcoRV fragment subcloned into the shuttle vector pHV33. The resulting plasmid complemented chromosomal spoVE mutations. Its structure was stable in recE4 strains, but plasmid and chromosomal rearrangements occurred in rec +strains. New spoVE mutations were obtained by mutagenesis of the plasmid; all the mutations tested mapped within three adjacent HindIII fragments of total length 1140 bp.

Summary: We have determined the nucleotide sequence of a 1159 bp DNA fragment containing the spoVE locus of Bacillus subtilis. The locus contained a single open reading frame of 293 codons. On the basis of the predicted amino acid sequence, the product of the spoVE gene is believed to be a protein with an M r of 31539. The amino-terminal portion of the spoVE gene was used to construct a translational fusion with the lacZ’ gene. The hybrid spoVE-lacZ’ gene was shown to be expressed in Escherichia coli and, therefore, it seems reasonable to conclude that the proposed open reading frame for the spoVE gene does indeed function in vivo.

Summary: DNA-dependent RNA polymerase has been isolated from Rhodomicrobium vannielii. Like those from other eubacteria, the enzyme contained four subunits: beta and beta prime (M r 155000), alpha (M r 38000), and sigma (M r 98000). Analysis by isoelectric focussing showed that both alpha and sigma had several forms with different isoelectric pH values. The enzyme was sensitive to rifampicin (5 ng rifampicin ml−1gave 50% inhibition) and capable of specific promoter selection with DNA from R. vannielii, calf thymus and phage T7D111.

Summary: A Transformant of Bacillus stearothermophilus carrying a recombinant plasmid, pLP11 (9.5 MDa), on which the penicillinase gene (penP) and kanamycin resistance gene (kan) were located was subjected to mutagenesis, and a mutant plasmid (9.5 MDa; penP kan), designated pTRA 117, obtained. A transformant of B. stearothermophilus carrying pTRA117 could grow at 63 °C in medium containing kanamycin, whereas a transformant carrying pLP11 could not. Although pTRA117 was deteeted as covalently closed circular (ccc) DNA when it was extracted from transformants cultured at 48 °C, it was integrated into the host chromosome when the culture temperature was shifted up to 63 °C. If the culture temperature was lowered to 48 °C from 63 °C, a new plasmid (10.7 MDa; penP kan), designated pTRZ117, could be detected as ccc DNA, the size of this plasmid suggested that it was pTRA117 plus a 1.2 MDa DNA fragment of the host chromosome, and this was confirmed by Southern hybridization. pTRZ90 (7.9 MDa; kan) was constructed from pTRZ117 by the deletion of a 2.8 MDa DNA fragment that contained penP. Fresh transformants of B. stearothermophilus that carried either pTRZ117 or pTRZ90 could grow at 65 °C.

Summary: To determine whether enzyme electrophoretic polymorphism in Escherichia coli populations was influenced by environmental background, the mobilities of four electrophoretically variable esterases (A, B, C and I) were examined. The distinction between isolates was established by significant differences in the electrophoretic distribution and the genetic diversity coefficient of individual esterases. Principal components analysis on each population and on all strains revealed three groups of allozymes. The first, characterized by slow electrophoretic mobilities of esterase B, was frequently observed in strains obtained from human extra-intestinal infections and rarely in commensal organisms. The second, characterized by fast mobilities of esterases A and B, was frequently found in animal isolates. The third, characterized by prominence of the most common mobilities of esterases B and A, was recovered in all populations. These results were confirmed by discriminant analysis. Among the 610 strains investigated, 316 electrophoretic types (distinctive combinations of allozymes of the four varieties of esterases) were distinguished, illustrating high esterase polymorphism.

Summary: One hundred and ninety one strains of Escherichia coli isolated from extra-intestinal infections and 85 strains isolated from the stools of healthy human beings were compared for electrophoretic mobility and isoelectric point of carboxylesterase B, and for production of α-haemolysin and the presence of mannose resistant haemagglutinin. Fast and slow electrophoretic mobilities were distinguished among the strains. The frequency of strains showing slow mobilities was considerably higher when they originated from extra-intestinal infections (40%) than when they were obtained from the stools of healthy individuals (7%). In a two-dimensional electrophoretic profile, the fast and slow mobility variants of carboxylesterase B were resolved into two patterns, B1 and B2, respectively. The frequency of pathogenic strains that concomitantly produced α-haemolysin and mannose resistant haemagglutinin was 48.7% for strains of pattern B2 but only 2.8% for strains of pattern B1. Thus, the electrophoretic pattern B2 of carboxylesterase B appears to be a molecular marker for a group of highly pathogenic E. coli strains which are frequently implicated in extra-intestinal infections.

Summary: We have tested the ability of pairs of colicin E plasmids to replicate stably in the same cell line. Although many of the pairs of E colicin plasmids were compatible, plasmids ColE3-CA38, ColE7-K317 and ColE8-J were mutually incompatible, as were ColE5-099, ColE6-CT14 and ColE9-J. Incompatibility between ColE6-CT14 and ColE5-099 or ColE9-J was asymmetrical, whereas incompatibility between the other plasmid pairs was symmetrical.

Summary: Monoclonal antibodies (McAb) that react with components of the bacteroid cell wall were isolated after immunization of rats with sonicated Rhizobium bacteroids from pea root nodules. More than 90% of the McAb reacted with lipopolysaccharides (LPS) from bacteroids and all of these cross-reacted with LPS from free-living cultures of the corresponding Rhizobium strains. Although LPS derived from free-living cultures of Rhizobium is very heterogeneous, detected as a ladder of immuno-staining bands after SDS–PAGE and electroblotting, the LPS from bacteroids is predominantly of the fast-migrating form. This observation suggests that LPS from bacteroids carries the core polysaccharide but lacks the repeating O-antigen oligosaccharide substituents that are usually found in free-living cultures. Three classes of McAb differed in their specificity for different Rhizobium strains, and differences in antigenicity were also observed between bacteroid and free-living cultures of the same strain.

Summary: Western blot analysis was used to identify antigenic components of Gardnerella vaginalis.Polypeptides bound to nitrocellulose membranes were probed with murine antisera raised to two strains of G. vaginalis. and antibody-antigen complexes were detected with 125I-labelled antimouse immunoglobulin followed by autoradiography. Although there was inter-strain variation in immunogenic polypeptide profiles, all 23 strains of G. vaginalis examined contained a common antigen of molecular mass 41 kDa. This antigen was not found in any of six other bacterial genera.