- Volume 141, Issue 4, 1995

Growth of gonococci in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) has previously been shown to induce resistance to the bactericidal effect of normal human serum and is accompanied by sialylation of the gonococcal lipopolysaccharide (LPS). We have used monoclonal antibodies (mAbs) to compare the effect of LPS sialylation on recognition of gonococci and complement-mediated killing by antibodies directed either against LPS or against defined epitopes on outer-membrane protein PI. Despite differences in binding to sialylated LPS on Western blots, all three mAbs directed against LPS showed considerably reduced binding to gonococci grown in the presence of CMP-NANA and a concomitant reduction in ability to promote complement-mediated killing. In contrast, mAbs directed against previously defined epitopes on a surface exposed loop of PI showed little difference in binding between sialylated and non-sialylated gonococci and promoted killing of the sialylated gonococci. Similarly a mAb directed against an epitope on a loop of the outer-membrane Rmp protein, which had previously been shown to block killing by antibodies directed against other surface antigens, also exerted a blocking effect with sialylated gonococci. Thus in the present study the continued biological effect of mAbs was correlated with the ability of the antibody to recognize surface-exposed epitopes on sialylated gonococci. Despite the presence of the sialylation which is likely to occur in vivo , it should be possible to induce complement-mediated killing by focusing the immune response to those surface-exposed epitopes which are least susceptible to the potential inhibitory effect of LPS sialylation.

Bacteroides forsythus, a bacterial species frequently associated with disease periodontal sites, is known to possess trypsin-like activity. The present study was undertaken to determine the major characteristics of this activity. The trypsin-like activity was mainly found on the surface of the bacteria and could be solubilized with a zwitterionic detergent (Zwittergent 3-14). Using N-a-benzoyl-DL-arginine-p-nitroanilide as substrate, the optimum pH was between 7·5 and 8·5 and the optimum temperature was 35°C. The evidence suggests that the enzyme is a serine protease since it was strongly inhibited by diisopropylfluorophosphate (DFP), N-a-p-tosyl-L-lysine chloromethyl ketone hydrochloride, leupeptin and antipain. The B. forsythus trypsin-like enzyme cleaved numerous chromogenic synthetic peptides containing either an arginine or lysine bond, but could not hydrolyse native proteins including casein, gelatin and BSA. Incubation of a cell envelope extract of B. forsythus the presence of [3H]DFP, which is known to bind irreversibly to serine proteases, labelled two bands at 70 and 81 kDa following SDS-PAGE (under reducing conditions) and fluorography. It is suggested that the B. forsythus trypsin-like enzyme may be mainly involved in the degradation of small peptides resulting from hydrolysis of larger proteins by other oral bacteria.

A toxin gene (bceT) on a 2·9 kb DNA fragment of Bacillus cereus B-4ac was cloned and expressed in Escherichia coli, and its nucleotide sequence determined. The DNA fragment contained an open reading frame capable of encoding a polypeptide of 336 amino acids with a molecular mass of 41039 Da. The translated product in E. coli exhibited Vero cell cytotoxicity, and was positive in a vascular permeability assay. It also caused fluid accumulation in a ligated mouse ileal loop and was lethal to mice upon injection. These biological activities are considered characteristic of diarrhoeal enterotoxins. We therefore conclude that this gene, designated bceT, encodes one of the enterotoxic proteins of B. cereus which cause food-borne diarrhoea.

An ultradian oscillation is described for Schizosaccharomyces pombe which meets the criteria for a cellular clock, i.e. timekeeping device. The rhythm can be induced by transfer from circadian conditions (stationary phase or very slow growth) to ultradian conditions (rapid growth). It can also be synchronized by ultradian temperature cycles of 6°C difference. Released to constant temperature, the rhythm persists for 20 h without damping. The period of the free-running rhythm is temperature-compensated and in no experiment did period length fall outside the narrow range between 40 and 44 min. The parameter observed is the septum index, i.e. the percentage of cells occupying the last stage of the cell cycle in wild-type cells before final division. The results suggest control of the cell division processes by the ultradian clock.

A temperature-sensitive, conditionally lethal actin mutant of Saccharomyces cerevisiae, DBY 1693, was used to study, using light and electron microscopy, dysfunction of the actin cytoskeleton in the morphogenesis of the cell wall. Cells of this mutant strain survived at least 24 h at the restrictive temperature (37°C). These cells showed isodiametric growth. Mutant cells accumulated vesicles, probably as a consequence of chaotic secretory transport caused by loss of polarity. A conspicuous morphological response to the dysfunction of actin was the formation of an aberrant wall over the whole surface of the isodiametrically-growing cell. This wall was of loose texture with protruding glucan microfibrils incompletely masked with amorphous matrix. It resembled the regenerating cell wall on the surfaces of yeast protoplasts. The localization of wall synthesis over the whole surface of temperature sensitive actin mutant cells was in accordance with an even distribution of submembranous actin in the form of patches (similarly to regenerating protoplasts). Delocalization of finger-like invaginations of the plasma membrane from the bud region to the whole surface of the growing cell was also found in mutant cells.

We investigated whether the concentration of H+ or metal ions such as Ca2+, or both, on the external side of the cytoplasmic membrane is involved in coupling of folding and secretion of Bacillus subtilis levansucrase by studying the modulation of each isolated event. In vitro at 30°C, in the absence of Ca2+, the equilibrium between the unfolded and the folded states of levansucrase was rapidly and totally displaced toward the folded state by a small pH shift from 7·4 to 6·0. Ca2+ (> 5 mM) acted as a catalyst of folding at pH ≥ 7. In vivo pulse-chase experiments at 30°C showed that, in the absence of Ca2+, the rate and the yield of the second step of levansucrase secretion were strongly dependent on the external pH. In acidic growth medium (pH 5·8), secretion was efficient. In contrast, at pH ≥ 7, the presence of Ca2+ was essential for secretion. In bacteria grown at high temperature (48°C), both external acidic pH and Ca2+ were required for efficient secretion. Moreover, a levansucrase variant altered in its calcium affinity was efficiently secreted only under acidic growth conditions. Depending on the culture conditions, the differences in H+ or Ca2+ concentrations which are maintained between the opposite sides of the energized cytoplasmic membrane could be adequate to catalyse conformational transition which could play a critical role in the second step of the protein release. These environmental parameters could also affect the yield of secretion of some other secretory proteins, leading to the hypothesis that several different secretion mechanisms could coexist in B. subtilis.

Streptomyces exfoliatus SMF13 produced leupeptin, chymotrypsin-like protease (CTP), metalloprotease, and trypsin-like protease (TLP) extracellularly. The activity of TLP was specifically inhibited by leupeptin. Production of leupeptin was closely associated with growth but leupeptin was inactivated by leupeptin-inactivating protein (LIP) when growth reached the stationary phase in submerged cultures, or when aerial mycelia started to form on surface cultures. Autolysis of mycelia after the stationary phase in submerged cultures was apparently retarded by the addition of leupeptin; on surface cultures, aerial mycelium formation was clearly retarded by the addition of leupeptin. We propose that CTP participates primarily in utilization of a proteinaceous nitrogen source, that TLP functions as an essential enzyme involved in the metabolism of mycelial protein, that leupeptin inhibits the activity of TLP and that LIP inactivates leupeptin. The cascade of regulatory actions of the compounds, which are produced sequentially during mycelium development, may provide selective advantages in adverse culture conditions.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7·5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli -pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31 % esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.