- Volume 136, Issue 6, 1990

Glycerol dehydrogenase, NADP+-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an M r of approximately 38000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and d-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6·0, whereas inhibition by NADP+ was less (K i = 45 µm vs 13 µm). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.

Monoclonal antibodies (m Abs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.

Lucifer yellow (LY), an impermeable fluorescent dye used as a marker for fluid phase endocytosis, was internalized by Candida albicans. As observed by fluorescence microscopy, incubation of C. albicans with LY in potassium phosphate buffer (pH 6·0) and glucose (2%, w/v) resulted in localization of the dye inside vacuoles. Sodium azide and carbonyl cyanide m-chlorophenylhydrazone, which are inhibitors of energy metabolism, decreased the uptake of the dye. The optimum temperature for uptake was 30 °C; no internalization was observed at 0 °C. Quantification of cell-associated LY by fluorescence spectrometry showed an uptake linear with time and not saturable over a 400-fold range of concentration. Thus, C. albicans internalized LY into vacuoles by a nonsaturable and time-, temperature- and energy-dependent process consistent with fluid phase endocytosis. Both the yeast and mould phase of this dimorphic fungus endocytosed LY. Growth in complex medium appeared to be required to enable the cells to internalize LY. However, addition of peptone or yeast extract to the phosphate buffer/glucose assay medium interfered with LY uptake by causing an apparent increase of exocytosis. These studies provide the first evidence of fluid phase endocytosis in C. albicans and may explain how some large molecules, such as toxins and cationic proteins, enter C. albicans.

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4·7 kb from C. vinosum and 2·3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.

Hyphal extension in the filamentous actinomycete Streptomyces coelicolor A3(2) was shown to occur by addition of newly synthesized wall material in an apical extension zone. Incubation of mycelia with tritiated N-acetyl-d-glucosamine (GlcNAc), a precursor of peptidoglycan, resulted in localized incorporation of label at the apex, as indicated by light microscopic and electron microscopic autoradiography. Within the hyphal extension zone there was a sharp decrease in incorporation with increasing distance from the apex. Hyphal tip shape, examined by low-temperature scanning electron microscopy, approximated to a semi-ellipsoid of revolution and was not hemispherical. Tip shape could be represented accurately by polynomial equations of degree less than seven. The surface stress theory was successfully applied to hyphal tip growth, with tip shape related qualitatively to the inverse of surface tension within the wall of the extension zone. Surface tension was assumed to be inversely proportional to the rate of incorporation of tritiated GlcNAc. Treatment of surface-grown hyphae with β-lactam antibiotics resulted in localized swelling of hyphal tips. Lysozyme caused swelling of tips and of other regions of hyphae, frequently giving a beaded morphology associated with septa.

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.

An anaerobic enrichment from lake mud yielded a pure culture of a facultatively anaerobic bacterium able to grow on malonate under strictly anaerobic conditions. Strain 16mall was identified as a member of the family Enterobacteriaceae, and assigned to the genus Citrobacter on the basis of morphological, metabolic and biochemical characteristics. Malonate was fermented under strictly anaerobic (sulphide-reduced) conditions to acetate and CO2 concomitant with growth. A maximum growth rate of 1·88 generations h−1 (μ = 1·30 h−1) was measured. The dry weight yield of cells from malonate was estimated at 2·5 g mol−1. Yeast extract was required for growth on malonate: other additives, or a vitamin solution, could not replace this requirement. Other dicarboxylic acids were not degraded in the absence or presence of malonate. Malonate was degraded under anaerobic, but not aerobic conditions. Malonate-decarboxylating activity was inducible by malonate under both anaerobic and aerobic conditions, and was not expressed in glucose- or citrate-grown anaerobic cultures. Monensin had no effect on malonate degradation, while 2,4-dinitrophenol decreased the rate of malonate degradation. This, with the lack of a sodium requirement for anaerobic growth on malonate, suggested that ATP generation may not be mediated by a sodium-pumping mechanism.

Total DNAs of 18 strains of Azospirillum from different sources and geographical areas were compared by restriction endonuclease pattern analysis. Fragments obtained with HindIII or BglII were separated by PAGE and stained with silver nitrate. Each strain possessed a unique and reproducible fingerprint with each enzyme, thereby facilitating strain recognition. UPGMA analysis recovered clusters of band patterns that were compared to the distribution of species within the genus Azospirillum.

The lacZ gene of Escherichia coli was fused to several positions downstream from the 5′ end of the Bacillus subtilis sacB gene, which encodes levansucrase (LS), a sucrose-inducible extracellular enzyme. Effects of hybrid protein expression in B. subtilis were studied. Several fusions were tested, and two significantly interfered with growth of cells and with LS secretion when induced with sucrose. Chromosomal amplification of the fusions, leading to strong expression of the hybrid proteins, completely blocked LS secretion and was lethal for B. subtilis when expression was induced.

A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. Using this ‘surface slide system’(SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined. In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (= colony density) and comparable to those obtained in liquid culture. For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer was similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats. In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density. In contrast, liquid matings were virtually unaffected by initial cell density. The transfer advantage of the permanently derepressed over the repressed plasmid was much less apparent for lower colony densities. I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.

The ability of Saccharomyces boulardii to protect mice against intestinal pathology caused by toxinogenic Clostridium difficile was studied. Different regions of the intestine of experimental mice were prepared for observation by scanning electron microscopy or homogenized for C. difficile enumeration and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The test group was treated for 6 d with an S. boulardii suspension in drinking water and challenged with C. difficile on day 4. The three control groups were: axenic mice, mice treated with only S. boulardii and mice only challenged with C. difficile. The results showed that: (i) 70% of the mice infected by C. difficile survived when treated with S. boulardii; (ii) the C. difficile-induced lesions on the small and large intestinal mucosa were absent or markedly less severe in S. boulardii-treated mice; and (iii) there was no decrease in the number of C. difficile but rather a reduction in the amount of toxins A and B in S. boulardii-treated mice.

Cytoskeleton inhibitors were used to study morphogenesis in the pathogenic and dimorphic fungus Candida albicans Nocodazole is a specific microtubule inhibitor and chloropropham (CIPC), at high concentrations, is an inhibitor of microtubules and microfilaments. Distribution of microtubules and microfilaments was studied by immunofluorescence techniques using anti-tubulin antibody with FITC-conjugated secondary antibody, and by staining with Rh-phalloidin. Nocodazole did not arrest apical cell elongation at a concentration (20 µg ml−1) that inhibited nuclear division and migration. Cytoplasmic and nuclear microtubules disappeared within 30 min in filamentous cells under these conditions. However, the Rh-phalloidin-stained actin granules which were localized in the tips of filamentous cells, and the microfilaments, were arranged normally at this concentration of nocodazole. Growth, and normal distribution of microtubules and microfilaments, were inhibited by a high concentration (200 µg ml−1) of CIPC. At a concentration (100 µg ml−1) of CIPC that permitted nuclear division, apical cell elongation was arrested, and filamentous growth was converted into yeast growth. At this concentration of CIPC, microtubules were distributed normally in filamentous cells. Long microfilaments were not observed, and actin granules did not localize in the tips of filamentous cells, but were distributed throughout the cytoplasmic cortex. Our results show that cytoplasmic microtubules are not essential for the elongation of filamentous cell tips but that microfilaments are apparently essential for this process.

We demonstrate that cathepsin G, derived from human neutrophils, exhibits potent in vitro antimicrobial activity against Listeria monocytogenes. Cathepsin G listericidal activity was by a non-enzymic mechanism and was dependent on the cationic nature of the molecule. The listericidal activity of cathepsin G occurred in a manner that was both time-dependent and concentration-dependent.

Lipids constituted around 5% of the dry weight in Candida albicans 3153, while sterols and phospholipids accounted for 1·2% and 1·1% respectively. Phospholipids were mainly localized in the microsomal fraction; phosphatidylserine (PS), phosphatidylcholine (PC), phospharidylethanolamine (PE) and phosphatidylinositol (PI) were the major phospholipids. Incorporation studies with [14C]acetate and [32P]orthophosphoric acid demonstrated that PS was synthesized at the highest rate followed by PC, PE and PI. There was little difference in either the content or the rate of biosynthesis of PC, PE and PI. Incorporation of labelled serine, ethanolamine and choline revealed serine to be a precursor for PC, PE and PS, ethanolamine for PC and PE, and choline for PC biosynthesis only.

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7·0–7·5, and the temperature optimum 37 °C. The apparent K m for ATP was 60 µm. Phosphoamino acid analysis revealed that the protein kinase transferred the γ-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5–5′-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5–5′-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.

Several linear megaplasmids were detected in the facultatively lithoautotrophic Gram-positive bacterium Nocardia opaca. The wild-type strain MR11 contains, in addition to the cccDNA plasmids pHG31-a and pHG31-b, the linear plasmids pHG201 (270 kb), pHG202 (400 kb) and pHG203 (420 kb). The wild-type strain MR22 contains, in addition to the cccDNA plasmid pHG33, the linear plasmids pHG204 (180 kb), pHG205 (280 kb) and pHG206 (510 kb). After preparation of DNA from cells embedded in agarose, the linear plasmids were demonstrated by pulsed-field electrophoresis. By means of DNA probes for genes of soluble hydrogenase and ribulose-bisphosphate carboxylase, the conjugative plasmids pHG201 and pHG205 were shown to be the carriers of the genetic information for these enzymes. A restriction map of pHG201 for the enzymes AsnI, SpeI, XbaI is presented.

A gene which is unique to the QpRS plasmid from chronic isolates of Coxiella burnetii was cloned, sequenced, and expressed in Escherichia coli. This gene, termed cbbE′ codes for a putative surface protein of approximately 55 kDa, termed the E′ protein. The cbbE′ gene is 1485 bp in length, and is preceded by predicted promoter regulatory sequences of TTTAAT (−35), TATAAT (−10), and a Shine-Dalgarno sequence of GGAGAGA, all of which closely resemble those of E. coli and other rickettsiae. The open reading frame (ORF) of cbbE′ ends with a UAA codon followed by a second in-frame UAG stop codon and a region of dyad symmetry which may act as a rhofactor-independent terminator. The ORF of cbbE′ is capable of coding for a polypeptide of 495 amino acids with a predicted molecular mass of 55893 Da. The E′ protein has a predicted pI of ~8·7, and contains a distinct hydrophobic region of 12 amino acid residues. In vitro transcription/translation and E. coli expression of recombinant plasmids containing cbbE′ produce a protein of approximately 55 kDa. The in vivo expression of cbbE′ yields a novel protein that can be detected on immunoblots developed with rabbit antiserum generated against purified outer membrane from C. burnetii. DNA hybridization analysis shows that cbbE′ is unique to the QpRS plasmid found in chronic isolates of C. burnetii, and is absent in chromosomal DNA and plasmids (QpH1, QpDG) from other isolates of C. burnetii. A search of various DNA and amino acid sequence data bases revealed no homologies to cbbE′.

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5–7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in ~6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.

Desulfovibrio desulfuricans survived exposure to air on the surface of agar plates for at least 48 h. Under continuous culture conditions two effects of low oxygen partial pressures (10–15 mmHg) were noted: (1) a relief of inhibition by H2S; (2) specific inductive responses to oxygen including an increase in cell respiration (two- to threefold) and in levels of cytochrome c (about 20%), and a rise in the activities of NADH oxidase (threefold) and superoxide dismutase (tenfold). At oxygen partial pressures greater than about 15 mmHg, these effects were reversed, growth became progressively inhibited and the cells began to wash out of the system.

As observed for other cyanobacteria, net H2 formation or consumption in photoheterotrophic, N2-fixing cultures of Nostoc sp. strain Cc, a cyanobacterium isolated from Cycas circinalis, was markedly dependent on the nickel ion content of the growth medium. Mean hydrogen consumption in the light of nickel-supplemented cultures, containing DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and glucose (photoheterotrophic conditions), was about 10 times higher than in control cultures supplemented with DCMU, but lacking glucose. Uptake of exogenous H2 did not influence growth or glucose consumption, but significantly increased acetylene reduction and the total N-content of the biomass, as well as ammonia excretion. It is postulated that the H2 uptake capability of the cyanobiont, and hence the nickel ion availability in the plant roots, may be important for the efficiency of N2 assimilation in plant-cyanobacteria symbioses.