- Volume 139, Issue 7, 1993

Summary: Many strains of oral streptococci secrete glucosyltransferases (GTFs) that polymerize sucrose into glucans that form an integral part of the plaque matrix on the tooth surface. Recently, we reported the cloning of two closely linked GTF-encoding genes (gtfJ and gtfK) from Streptococcus salivarius ATCC 25975 as well as the sequence of gtfJ, which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtfK, which encodes a primer-dependent GTF that synthesizes a soluble product (a GTF-S), as well as the sequence of a small downstream open reading frame of unknown function. The deduced sequence of GtfK was compared with those of seven other streptococcal Gtfs and an unrooted phylogenetic tree constructed. This analysis suggested that Gtfs with similar product specificities do not form phylogenetic clusters and was consistent with currently accepted phylogenetic schemes. The tree was tested by constructing a series of “sub-trees” from different blocks of the alignment. Evidence was obtained for recombination events involving gtfB and gtfC from S. mutans GS-5, gtfJ and gtfK from S. salivarius, as well as the gtfI genes from S. downei and S. sobrinus. The recombination events between gtfB and gtfC, and between the two gtfI genes, were confirmed by examining divergences at silent sites.

Summary: Insertion element IS6110 occurs in multiple copies throughout the Mycobacterium tuberculosis genome, and the variability of its insertion sites is the basis for the IS6110 restriction fragment length polymorphism (RFLP) method for typing. We describe a novel gene amplification method to assess the variability of the location of IS6110. A unilateral-nested polymerase chain reaction and hybridization procedure was used to measure the variability in the distances between IS6110 elements and copies of a major polymorphic tandem repeat sequence of M. tuberculosis. The pattern of amplicons produced could be used to cluster epidemiologically related strains of M. tuberculosis into groups which correlated with the groups formed using IS6110-RFLP typing. Reliable patterns can be generated directly from sputum specimens as well as from M. tuberculosis cultures. We designated the novel method as IS6110-ampliprinting.

Summary: A B-cell epitope, AEFPLDIT, was located to the variable sequent 3 of the major outer-membrane protein (MOMP) using the monoclonal antibody L3-1, raised to the Chlamydia trachomatis serovar L3 MOMP. By Western blot and inclusion immunofluorescence assay the monoclonal antibody recognized all the C complex and C-related complex serovars of C. trachomatis, except serovar C. Dot-blot and ELISA data using native elementary bodies indicated that the epitope was surface exposed. The monoclonal antibody, at concentrations of 10 and 100 μg per 107 chlamydial inclusion-forming units, was able to neutralize the infectivity of chlamydia in an in vivo assay but did not neutralize chlamydia in vitro or in a mouse toxicity assay. A peptide corresponding to the variable sequent 3 has previously been shown to also elicit a T-cell response; thus, careful consideration should be given to inclusion of this region of the major outer-membrane protein in a subunit vaccine.

Summary: Several species of the opportunistic fungal pathogen Candida produce an extracellular aspartyl proteinase that may assist the organism to invade and colonize host tissues, evade the host immune response and assimilate nitrogen from proteinaceous sources. Although addition of exogenous proteins, such as bovine serum albumin (BSA), to cultures of C. albicans is known to elicit proteinase production, the precise molecular mechanisms controlling regulation of proteinase induction are unknown. We have examined the ability of a variety of macromolecules to induce proteinase production using a chemically-defined nitrogen-limited growth medium and a rapid, sensitive microtitre fluorescent assay for proteinase activity in culture supernatants. BSA and the extracellular matrix protein collagen induced proteinase production. Homopolymers of both poly-L- and poly-D-glutamate also induced proteinase activity, whereas polyglycine, heparin sulphate and dextran sulphate did not. Thus, molecular recognition of proteinase-inducing stimuli is not highly stereospecific, but apparently requires both main- and side-chain interactions. Peptides 8 or more residues in length generally induced proteinase production while most shorter peptides did not. These data reveal that internalization of small peptides with less than 7 residues by peptide transport was not the inducing signal for proteinase production, since Candida dipeptide and oligopeptide permeases do not efficiently transport peptides of more than 6-7 residues. In addition a tight-binding synthetic inhibitor of Candida proteinase (K i = 0.17 nM) prevented growth of C. albicans on BSA as a sole nitrogen source by blocking protein degradation. Immunodetection of proteinase in these culture supernatants suggests that fully intact proteins, in addition to peptide fragments of sufficient size, are capable of inducing proteinase production. A model involving stimulation of a plasma membrane signal transduction event by extracellular protein and/or polypeptide ligands of more than seven residues is compatible with these data.

Summary: The mycelial growth of Eurotium chevalieri was examined at different water activities (a w) using glycerol as the osmoticum. Growth was optimal at 0.90 a w and restricted at 0.995 a w highlighting the xerophilic behaviour of E. chevalieri. Decreased a w produced an increase in the proportion of oleic acid (C18:1) at the expense of the proportion of linoleic acid (C18:2) of cellular phospholipids. The degree of unsaturation of phospholipid fatty acids showed a 20 % decrease between 0.995 and 0.80 a w of growth. Steady-state fluorescence anisotropy (r s) and fluorescence lifetime (τ) measurements for liposomes prepared from cellular phospholipids of E. chevalieri and labelled with DPH (1,6-diphenyl-1,3,5-hexatriene) were made at 25 °C. The lipid order parameter (S, describing molecular order) and the rotational correlation time (ϕ, describing molecular dynamics) were calculated from r s and τ data. Except at 0.995 a w, a decrease in a w was accompanied by increasing r s and S values, indicating a rigidification of membranes, while ϕ values were not significantly different. Plots of order parameters and their first derivatives as a function of temperature exhibited break areas in the temperature range 20-48 °G. These large temperature ranges for lipid transitions could correspond to chain melting of complex lipid systems which made up the liposomes prepared from phospholipids of E. chevalieri. However, as a w decreased, the transition temperatures increased globally, between 0.97 and 0.90 a w.

Summary: The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Summary: Using pulsed-field gel electrophoresis (PFGE), we have demonstrated clonal variation in the size of chromosome XII in a diploid strain of Sacchavomyces cerevisiae X2180-2D. The sizes of the two chromosome XII homologues were very different: 2600 (L-type) and 1450 kb (S-type). The frequency with which we detected clonal size variation in the diploid, compared to that of the parental clones, was about 15-50% of the progeny clones and the range of the size variation of the homologues was 2580-2680 kb (L-type) and 1340-1500 kb (S-type), respectively. The homologue of the L-type appeared to be more frequently variable than that of the S-type. The size variation was shown to be derived from size changes in the rDNA cluster region, which is present in chromosome XII, by digesting the chromosome with XhoI, whose cutting site is not present in a rDNA repeat unit, and hybridizing to rDNA probes. The clonal size variation was also investigated in haploids from spores after meiosis. The L-type and S-type chromosomes segregated 2:2 in an ascus and the sizes of all the S-type chromosomes were shifted up, compared to the original diploid, though the L-type ones were stable. The S-type sizes of 1340, 1450 and 1780 kb in the original diploids changed into the ranges of 1475-1610 kb, 1520-1680 kb and 1820-2010 kb, respectively, in the segregants. Furthermore, we observed that the size of S-type chromosomes in haploid cells was gradually increasing in mitosis during successive subcultures. The rDNA units appeared to be amplified on the S-type chromosome.