- Volume 133, Issue 4, 1987

SUMMARY: The incorporation of specifically labelled sugars into alginate by mucoid strains of both Pseudomonas mendocina and Azotobacter vinelandii resulted in substantially different labelling patterns for fructose and glucose. Alginate was synthesized principally from degradation products of glucose, whereas the majority of the polymer produced from fructose was assembled from intact hexose units. A possible explanation for the limited synthesis of alginate from undegraded glucose molecules is that one of the necessary enzymes, glucose-phosphate isomerase, is subject to inhibition by 6-phosphogluconate, a metabolite involved in the catabolism of sugars via the Entner-Doudoroff pathway in both of the organisms.

Ethanol induced small amounts of cytochrome P-450 in Saccharomyces cerevisiae NCYC 754 under conditions in which it is not normally detectable. Moreover, in non-growing yeast the existing cytochrome P-450 content was increased by 50 % at a limited range of glucose concentrations (8–12 % in 0·1 m-potassium phosphate buffer, pH 7·0), in which ethanol is produced by fermentation, possibly at an optimum concentration for induction of cytochrome P-450. Added alkanols, other than ethanol, caused rapid degradation of cytochrome P-450 in non-growing yeast; the rate of loss was directly related to the lipid solubility of the alkanol. Ethanol therefore favoured the accumulation of cytochrome P-450 in yeast; this may be related to an important putative role of one of the isoenzymes in ethanol-tolerance of the yeast, by the oxidative removal of ethanol from the endoplasmic reticulum of the cell. It is the accumulation of dissolved oxygen, rather than ethanol, that occurs on cessation of yeast growth that is likely to trigger the rapid disappearance of cytochrome P-450 observed at this time.

The LPD1 gene of S. cerevisiae, which encodes lipoamide dehydrogenase (EC 1.8.1.4), has been cloned and characterized. The LPD1 gene is present as a single copy in the yeast genome and is transcribed to give a polyadenylated mRNA species of approximately 2·0 kb. The synthesis of lipoamide dehydrogenase in yeast is subject to carbon catabolite repression since both the level of the LPD1 transcript and the accumulation of the lipoamide dehydrogenase subunit polypeptide were greatly reduced in wild-type cells grown on glucose compared to those grown on a variety of non-fermentable carbon sources. Strains defective in LPD1 but transformed with the LPD1 gene on a high copy number vector exhibited elevated levels of the LPD1 transcript as well as increased lipoamide dehydrogenase activity when grown on glycerol. Immunoblotting experiments confirmed that such transformants over-expressed lipoamide dehydrogenase protein. Transcription from the LPD1 sequence on plasmid pGP1 still appeared to be subject to some catabolite repression despite the presence of multiple copies of the plasmid in the cell.

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage λ vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-α-d-glycosyl residues (~90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.

The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages SPR, ø3T, SPβ and ρ11 can be transferred by transfection and recombination to the genome of the related nonmodifying phage Z. Integration of the Mtase genes occurs in phage Z DNA at a unique location which is homologous with the flanking regions of the Mtase genes of the related phages. In lysogenic cells carrying recombinant phages, expression of the Mtase genes is repressed, irrespective of whether the Mtase genes were derived from phage donors which were homo- or heteroimmune to phage Z.

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 ° It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30° and also to the short form of these pili synthesized at 37 ° The reason for the failure to form plaques at 37 ° is not known. The adsorption site is a short length of the pilus shaft extending 100–200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.

UV irradiation and mitomycin C exposure trigger a protease-activity-dependent inhibition of cell division in Rhodobacter capsulatus, which begins about 2 h after the treatment is applied. UV irradiation also induces a dose-dependent mutagenesis with a maximal rate between 5 and 10 J m−2, with increased synthesis of a protein of Mr approximately 30000 between 2 and 3 h after UV irradiation. In addition, R. capsulatus has an efficient photoreactivation system that reverses the lethal effects of UV irradiation in the presence of intense visible light.

A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2·2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76·2 % in nucleotides and 85·4 % in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.

Mutants of mucoid Pseudomonas aeruginosa defective in fructose-bisphosphate aldolase (FBA), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAP) or 3-phosphoglycerate kinase (PGK) were unable to grow on gluconeogenic precursors like glutamate, succinate or lactate. The gap and pgk mutants could grow on glucose, gluconate or glycerol, but fba mutants could not. This suggests that the metabolism of glucose or gluconate does not require either PGK or NADP-linked GAP but does require the operation of the aldolase-catalysed step. For gluconeogenesis, however, all three steps are essential. Recombinant plasmids carrying genes for FBA, PGK, GAP or phospho-2-keto-3-deoxygluconate aldolase (EDA) activities were constructed from a genomic library of mucoid P. aeruginosa selecting for complementation of deficiency mutations. Analysis of their complementation profile indicated that one group of plasmids carried fba and pgk genes, while another group carried eda, 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate dehydrogenase (zwf) genes. The gap gene was not linked to any of these markers. Partial restoration of FBA activity in spontaneous revertants of Fba− mutants was accompanied by a concomitant loss of PGK activity. These experiments indicate a linkage between the fba and pgk genes on the P. aeruginosa chromosome.

SUMMARY: To assess the importance of aerobactin-mediated iron uptake as a bacterial virulence determinant in animal infections, a total of 576 strains of Escherichia coli isolated from cattle, chickens, sheep and pigs were screened by colony hybridization to determine the presence of the aerobactin genetic determinants, and by a bioassay to detect aerobactin secretion in iron-limited conditions. Results obtained by the two complementary methods correlated well. The incidence of the aerobactin system was very high among septicaemia isolates, particularly those from cattle and chickens, an observation that strongly suggests an important role for this mechanism of iron assimilation in pathogenesis. On the other hand, the incidence of the aerobactin system among mastitis strains was not significantly higher than among faecal isolates from healthy animals. No classical enterotoxigenic E. coli strains tested carried the aerobactin genetic determinants. Although most strains that produced aerobactin were also able to make colicin V, the fact that the two characteristics existed separately in a significant minority of isolates suggested that colicin testing alone could not be reliably used to determine the presence of the aerobactin system.

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.

As a model system for analysing interactions between chlamydiae and myeloid cells and their precursors, we have studied binding, ingestion and destruction of Chlamydia trachomatis (L2 serovar) by the human promyelocytic cell line HL-60. HL-60 cells were induced by phorbol myristate acetate (PMA) and dimethyl sulphoxide (DMSO) to differentiate along either the macrophage or the granulocyte pathway, respectively. Using an immunofluorescence assay and electron microscopy, we have shown that induced (differentiated) HL-60 cells, but not uninduced (undifferentiated) HL-60 or other cell lines treated with PMA or DMSO, exhibit increased binding, ingestion and elimination of C. trachomatis; these activities are associated with specific histochemical and antigenic markers of myeloid differentiation. These results suggest that myeloid cells acquire the ability to interact with and kill chlamydiae during cell development.

In situ DNA hybridization using biotin-labelled leptospiral DNA was performed on clinical specimens to investigate its usefulness as a technique for the identification of Leptospira interrogans. The applicability of this test in blood, urine and liver smears was demonstrated. In situ DNA hybridization can be completed in only 4 h and it combines the advantage of visualization of the leptospiral morphology with the specificity of the hybridization reaction. No cross-hybridization was observed with other bacteria. This study shows that hybridization in situ can be simple to perform and may contribute to a rapid diagnosis.

SUMMARY: The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.