Canadian Journal of Microbiology

Published by NRC Research Press
Online ISSN: 1480-3275
Print ISSN: 0008-4166
Publications
Extremely low frequency magnetic field exposure apparatus. From left to right you can see: magnetic B-field (unit 1), electric E-field (unit 2), no field (unit 4), mutually orthogonal E- and B-fields (unit 3). Inferiorly electric supply chain. Faraday shields exclude electric noise in the units. We used unit 1 and unit 4. 
Effect of static magnetic field (SMF) on the expression of some genes involved in hyphal growth. Analyses were performed by quantitative real-time PCR. Data are expressed as the mean Ϯ standard deviation of 6 independent determinations. A Mann–Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
Effect of extremely low frequency magnetic field (ELF- MF) on the expression of some genes involved in hyphal growth. Analyses were performed by quantitative real-time PCR. Data are expressed as the mean Ϯ standard deviation of 6 independent determinations. Mann–Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
Activity levels of some enzymes of carbohydrate metabolism in Tuber borchii mycelia. Control (white), mycelia exposed to a static magnetic field (SMF) (gray), and mycelia exposed to extremely low frequency magnetic field (ELF-MF) (black). HK, hexokinase; PFK, phosphofructokinase; GPI, glucose phosphate isomerase; PK, pyruvate kinase; G6PD, glucose-6- phosphate dehydrogenase. Data are expressed as the mean Ϯ standard deviation of 11 independent determinations. A Mann– Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.
 
Extremely low frequency magnetic field exposure apparatus. From left to right you can see: magnetic B-field (unit 1), electric E-field (unit 2), no field (unit 4), mutually orthogonal E- and B-fields (unit 3). Inferiorly electric supply chain. Faraday shields exclude electric noise in the units. We used unit 1 and unit 4. 
Primer pairs used in quantitative real-time PCR.
Effect of static magnetic field (SMF) on the expression of some genes involved in hyphal growth. Analyses were performed by quantitative real-time PCR. Data are expressed as the mean Ϯ standard deviation of 6 independent determinations. A Mann–Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
Effect of extremely low frequency magnetic field (ELF- MF) on the expression of some genes involved in hyphal growth. Analyses were performed by quantitative real-time PCR. Data are expressed as the mean Ϯ standard deviation of 6 independent determinations. Mann–Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
Activity levels of some enzymes of carbohydrate metabolism in Tuber borchii mycelia. Control (white), mycelia exposed to a static magnetic field (SMF) (gray), and mycelia exposed to extremely low frequency magnetic field (ELF-MF) (black). HK, hexokinase; PFK, phosphofructokinase; GPI, glucose phosphate isomerase; PK, pyruvate kinase; G6PD, glucose-6- phosphate dehydrogenase. Data are expressed as the mean Ϯ standard deviation of 11 independent determinations. A Mann– Whitney U test was performed to compare exposed samples with control samples. *, P Ͻ 0.05; **, P Ͻ 0.001. 
The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.
 
Maximum concentration of detergent that permits bacterial growth in Luria-Bertani broth. 
In liquid culture, eight typical gram-negative bacteria were ca. 10,000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate. Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01%. Four pseudomonads able to form biofilms were ca. 1000-fold more resistant to CTAB on Luria-Bertani agar plates than they were in liquid culture. A lasI mutant of Pseudomonas aeruginosa was only able to tolerate 0.1% CTAB on Luria-Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants.
 
An endonuclease activity has been purified from human adenovirus type 5 (HAd5) virions and HAd5-infected cell extracts. The endonuclease activity is associated with a monomeric protein of molecular weight approximately 33 000. The endonuclease activity is more active at pH 4.5 than pH 7.2. Incubation of the enzyme at room temperature for periods of longer than 96 h results in a substantial increase in activity. The endonuclease activity is sensitive to EDTA at concentrations 10mM or greater but is insensitive to 500 mM NaCl. Immunological analysis with endonuclease-specific antiserum indicates that the endonuclease may be a host cell derived, viral modified, and incorporated protein.
 
( a ) Recovery of bacteria equivalents of the Lactobacillus paracasei group in ileal samples of 4 subjects before a unique dose of fermented milk ingestion containing Lactobacillus casei DN-114 001 Rif (H0) and during 8 h (H2, H4, H6, H8) after the ingestion. ( b ) Recovery of bacteria equivalents of the L. paracasei group in fecal samples of 7 subjects before (D7), during (D11 and D15), and after (D18 and D22) the end of the step of 8 days of daily ingestion of 300 mL of fermented milk containing L. casei DN-114 001 Rif . The bar indicates mean values obtained at each measurement time for the subjects. 
16S rRNA-targeted oligonucleotide probes used in this study.
Temporal temperature gradient gel electrophoresis analysis of amplicons generated by Lactobacillus group-specific PCR with primers Lac1 and Lac2GC. Ileal samples were collected before a unique dose of fermented milk containing Lactobacillus casei DN-114 001 Rif ingestion (H0) and during the 8 h (H2, H4, H6, H8) after the ingestion at D0. Fecal samples were collected 7 days later at (D7), then at D11, D15, D18,and D22. Day 7 is before a daily ingestion of 300 mL of the test product (a step of 8 days). Days 11 and 15 are during the ingestion, and days 18 and 22 are after the end of the ingestion step. Lane Ac, fermented product (Actimel). Lane M, marker. 
Lactobacillus casei DN-114 001 is a probiotic strain able to interact with the immune system and to interfere with gastrointestinal pathogens. The derived strain DN-114 001Rif was studied during its transit through the upper and distal intestine of human volunteers. Seven volunteers participated in the study, which involved intestinal intubation to sample ileal contents and collection of fecal samples, with a wash-out period of 8 days between the 2 steps. The retrieval of the probiotic was analyzed in the ileum every 2 h for 8 h following the ingestion of one dose of the test product and in the feces prior to, during, and after daily consumption of the test product for 8 days. Persistence of the probiotic amplifiable DNA was assessed using temporal temperature gradient gel electrophoresis and real-time PCR. Fluorescent in situ hybridization allowed analysis of the composition of the dominant digestive microbiota. The ingestion of L. casei DN-114 001Rif led to a significant and transient increase of its amplifiable DNA in ileal and fecal samples. This is related to a high stability in the composition of dominant groups of the gut microbiota. Data from ileal samples are scarce and our study confirms the potentiality for interaction between probiotics and the human immune system.
 
A commonly occurrring chemoorganotrophic diatom, Nitzschia alba, clone Link 001, stores primarily fatty acids and utilizes the hexose monophosphate shunt as a major pathway in hexose oxidation. The presence and activity of this pathway in exponentially growing N. alba was demonstrated by the growth on potassium gluconate as a sole carbon source, in vitro assay of 6-phosphogluconate dehydrogenase (EC 1.1.1.44), and in vivo radiorespirometric evaluation using [14C]-glucose or -gluconate. Radiorespirometry demonstrated that the hexose monophosphate and Embden--Meyerhoff--Parnas pathways account for 46 and 54% of hexose oxidation, respectively. The predominance of fatty acid storage plus the probable utilization of some C4--C5 hexose monophosphate intermediates in biosynthetic activities support the relatively high hexose monophosphate activity in these exponentially growing cells. Radiorespirometric values are supported by the absence of Enter-Doudoroff activity as determined by the lace of 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.12.14) and 6-phosphogluconate dehydratase (EC 4.2.1.14) activity.
 
Enterohemorrhagic Escherichia coli (EHEC) causes a wide range of systematic diseases in human and animals in 2 main ways: (1) production of Shiga toxin (Stx) and (2) induction of actin polymerization characterized by attaching and effacing (A/E) lesions. Stx is commonly targeted in the development of drugs and vaccines to control EHEC infection for its indispensible contribution to EHEC pathogenesis. In this study, we isolated a Stx-producing EHEC O157:H7 isolate 00B015 and found that its ability to induce actin polymerization was impaired. In addition, it reduces pathogenicity and decreases mortality in mice. Our results report a Stx-producing but virulence-attenuated EHEC isolate 00B015 and suggest that the formation of actin polymerization may help Stx-induced pathogenesis and have a more important contribution in EHEC infections.
 
Toxigenic and nontoxigenic strains of Vibrio cholerae 01 occur in the natural aquatic environment. It is not clear whether V. cholerae 01 lose toxigenicity and become nontoxigenic during survival in the aquatic environment as a result of the effect of various biophysicochemical conditions (e.g., sunlight, pH, temperature, competition with other bacteria for nutrients, etc.). Five toxigenic strains were exposed to artificial aquatic environments in the presence of a filamentous green alga. Rhizoclonium fontanum, and recovered after different time intervals (0 and 0.5 h, 3, 6, 9, and 15 days). This experimental system was exposed to sunlight and the V. cholerae 01 were in competition for nutrients with resident bacterial flora from R. fontanum. The toxigenicity of Vibrio cholerae 01 that were recovered at different time intervals was assessed by tissue culture assay using Vero cells. The toxigenicity of recovered strains was compared with that of the parent strains. The results demonstrated that toxigenic V. cholerae 01 are unlikely to lose their toxigenicity in aquatic environments as a result of the effects of various biophysicochemical conditions. These results are consistent with the hypothesis of environmental reservoirs of V. cholerae.
 
Monoclonal mouse antibodies specific for the 0 antigen of Citrobacter 036, a homopolymer of beta (1----2)-linked 4-deoxy-D-arabinohexose, were generated by the hybridoma technique. Balb/c mice were immunized with killed whole-cell vaccine and initial selection of active clones was based on enzyme-linked immunosorbent assay (ELISA) employing purified lipopolysaccharide (LPS). Concentrated culture supernatants from selected hybrid cultures were used to identify 10 0-antigen specific monoclonal antibodies using the multiple criteria of immunoprecipitation of 0 chains and LPS, inhibition by acid hydrolyzed 0 chains in the screening ELISA, and antibody class analysis. Four monoclonal antibodies were chosen for further study using dose-dependent 0-chain inhibition of ELISA and passive hemagglutination, passive hemolysis, and bacterial agglutination titres. When screened with Citrobacter serotypes known to contain the sugar 4-deoxy-D-arabinose, passive hemagglutination tests showed that the two monoclonal antibodies examined possessed titres which could be correlated with the reported 4-deoxy-D-arabinohexose content of the respective LPS's. This sugar is an antigenically important unit of several Citrobacter serotypes as defined by these well-characterized monoclonal antibodies.
 
7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.159) production by Escherichia coli strain 080 was highest when the organism was grown in brain heart infusion broth at pH 6.5 for 72-96 h with shaking at 37 degrees C. The oxygen consumption rate had a strong effect on the production of this constitutive enzyme. Glucose and lactose at 0.2-0.4%, detergents, and ethylenediaminetetra-acetic acid were found to increase the enzyme production.
 
Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.
 
A nonaxenic sewage culture metabolized 1,1-diphenylethylene by two different pathways involving hydration to form 2,2-diphenylethanol and oxidation and fission of one of the benzene rings to form atropic acid. Five different bacterial strains which grew on 1,1-diphenylethylene were isolated; all were gram-negative rods comprising the genera Pseudomonas, Acetomonas, and Acinetobacter. A fungal isolate, Cladosporium, which used diphenylethylene as a sole carbon source, was also obtained from the same enrichment procedure.
 
Pseudomonas stutzeri will grow on glucose with either the coordination compound bis(ethylenediamine)-1,10-phenanthrolinecobalt(III) bromide ([Co(en)2(phen)]Br3) or the ligand ethylenediamine as the sole source of nitrogen. The utilization of [Co(en)2(phen)]Br3 by P. stutzeri is presumed to involve initial reduction of the cobalt(III) complex to a labile cobalt(II) species which is in equilibrium with free ethylenediamine. After removal of ethylenediamine by the cells, the cobalt(II) and 1,10-phenanthroline remaining appear to inhibit ethylenediamine oxidation. Resting cells previously grown on ethylenediamine as the sole source of nitrogen are able to catalyze the degradation of ethylenediamine and ethanolamine. Ethanolamine-grown cells degrade ethanolamine but not ethylenediamine.
 
There is now direct and indirect evidence fully documenting the frequent persistence following primary infection of all of the human herpesviruses. Immunosuppression among other factors can bring about reactivation of these viruses in many individuals, and the resulting infections may be severe and occasionally fatal; i.e. with VZV, CMV, and to a lesser extent HSV. The persistence of EBV on the other hand and its reactivation and detection in saliva does not seem to lead to any disease. If we knew more and could monitor the right parameters, we could probably detect and follow the slow stepwise events which lead after long incubation periods to virus-related neoplastic disease in a few individuals. The review of the literature and observations that we have made in AT, BL, and in a family with multiple Burkitt's tumors and NPC suggest that one of the important factors in the development of an EBV-associated tumor could be the target cell itself and its genetics. Studied involving two EBV-transformed lymphoid cell lines of one AT patient have disclosed a chromosome 14 translocation which is a marker found exclusively in lymphomatous cells. The response to HC of the EBV antigen in AT cell lines was similar to that seen in BL cell lines. BL cell lines (6 of 9) and AT cell lines (3 of 3 to date) responded to HC by an increase in EBV-EA synthesis in contrast to 0 of 18 EBV-transformed lymphoid cell lines established from the peripheral blood of normal individuals. Finally, the prevalence of EBV-EA antibodies in normal individuals did not exceed 5 to 10%, whereas in AT and BL it was found to be as high as 50 to 100%. Target cell defectiveness, therefore, as a primary factor, and immunodeficiency, as a secondary factor, may play an important part in the observed increased incidence of tumors, particularly lymphomas in AT, in other congenital or acquired immunodeficiency syndromes and in families with multiple tumors.
 
1,2-Dihydrosantonin is the first stable product in the degradative pathway of alpha-santonin by Pseudomonas cichorii S. Its formation is catalyzed by an oxidoreductase, which is NADH or NADPH dependent and has an apparent Km value of 66.66 microM for santonin and 44.33 microM for NADH. The enzyme activity is stable at pH 6.0, 7.0, and 8.0, and is not affected by EDTA and divalent metal ions. It is postulated that the enzymic reduction of santonin occurs via formation of a transient zwitterionic intermediate, which undergoes nonenzymatic 1,4-sigmatropic rearrangement to yield lumisantonin during the solvent extraction process. Lumisantonin is, thus, not a true metabolic intermediate but an artifact.
 
Microbial degradation of [beta-14C]polystyrene and 1,3-diphenylbutane, a compound structurally representing the smallest repeating unit of styrene (dimer), was investigated in soil and liquid enrichment cultures. Degradation rates in soil, as determined by 14CO2 evolution from applied [14C]polystyrene, varied from 1.5 to 3.0% for a 4-month period. Although relatively low, these percentages were 15 to 30 times greater than values previously reported. Enrichment cultures, containing 1,3-diphenylbutane as the only carbon souce, were used to determine the mechanisms of microbial oxidation of the polymer chain ends. Metabolism of 1,3-diphenylbutane appeared to involve the attack by a monooxygenease to form 2-phenyl-4-hydroxyphenylbutane followed by a further oxidation and subsequent fission of the benzene ring to yield 4-phenylvaleric acid and an unidentified 5-carbon fragment via the classic meta-fission pathway. Phenylacetic acid was probably formed from 4-phenylvaleric acid by subsequent beta-oxidation of the side chain, methyl-oxidation and decarboxylation. An initial examination of the population of microorganisms in the diphenylbutane enrichment cultures indicated that these oxidative reactions are carried out by common soil microorganism of the genera Bacillus, Pseudomonas, Micrococcus, and Nocardia.
 
A study was conducted to investigate the possibility of involvement of chitinase and beta-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani. Fluorescent Pseudomonas isolates GRC(3) and GRC(4) were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC(3). The involvement of chitinases, beta-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and beta-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.
 
Two different strains of Trichoderma pseudokoningii (SE1 A8 and SE1 D81) and Trichoderma viride QM 9123 release into the medium different proportions of the total beta-glucosidase activity produced. This observation correlates with the degree of beta-1,3-glucanase binding to the cell wall found for each strain. DEAE-Sephadex ion-exchange chromatography revealed three peaks of beta-1,3-glucanase activity. These three enzymes (enzyme I, enzyme II, and enzyme III) differ in their extent of binding to the cell walls, their activity on isolated cell walls and Trichoderma beta-glucan, and their affinity for beta-glucan. Of these enzymes, enzyme II shows the largest variation in relative importance among the three strains and is located predominantly within the mural compartment. Enzyme II has the highest activity on and affinity for Trichoderma beta-glucan. Enzyme II is also the most active in releasing beta-glucosidase from cell walls of strain SE1 A8 (the strain excreting a high proportion of its beta-glucosidase into the culture fluid) as well as from strain SE1 D81 (little beta-glucosidase activity in the culture fluid). It is concluded that the action of beta-1,3-glucanase II on cell wall beta-glucan may be responsible for the in vivo release of cell wall bound beta-glucosidase into the culture fluid.
 
In the autolytic phase of growth Schizophyllum commune lost 62% of its dry weight in 70 days of incubation. The variations in the activity of some lytic enzymes were studied in the culture fluid and mycelial extracts during growth and autolysis of this fungus. The enzymes 1,3-beta-glucanase (exoglucanase), 1,3(4)-beta-glucanase (endoglucanase), alpha-amylase, and invertase behaved in the same way in culture fluid and mycelial extract, but their activities were much higher in the culture fluid. The enzyme activities increased during autolysis, but then decreased at the end of this period except in the case of alpha-amylase which remained high. It was only possible to detect 1,6-beta-glucanase, cellulase, and polygalacturonase activities at certain times during the autolytic phase of growth. The enzyme chitinase was not detected and 1,3-alpha-glucanase (S-glucanase) occurred in the mycelial extract at a higher concentration than in the culture fluid. A decrease in the activity of this enzyme in the mycelial extract and an increase in the culture fluid occurred during autolysis. The enzyme 1,3-alpha-glucanase exhibited two optima pH, one at 6.0 and the other at 8.0. The Km value for the latter was 0.02 M at pH 5.5 in borate-citrate-phosphate buffer.
 
Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC50 3-45 microM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 degrees C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 microM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 microM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTP gamma S.
 
Immunoblotting analysis of Stachybotrys elegans 1,3-β-glucanases. Samples were separated by SDS-PAGE (12:1%), electrophoretically transferred onto a polyvinylidene difluoride membrane, and immunodetected with purified polyclonal antibodies raised against the 1,3-β-glucanases. Colorimetric detection was performed with alkaline phosphatase conjugated to a goat anti-rabbit antibody. (A) Immunoblot of the 94-kDa 1,3-β-glucanase. Lane 1, 0.5 µg of purified 75-kDa glucanase; lane 2, 0.5 µg of purified 94-kDa glucanase; lane 3, 25 µg of F I. (B) Immunoblot of the 75-kDa 1,3-β-glucanase. Lane 1, 0.25 µg of purified 94-kDa glucanase; lane 2, 0.25 µg of purified 75-kDa glucanase; lane 3, 25 µg of F I. 
Western blot analysis of proteins extracted from six dual culture plates of Stachybotrys elegans and R. solani grown on minimal synthetic medium and immunodetected with the purified 94-kDa 1,3-β-glucanase antibody. Lane 1, 0.2 µg of purified 94-kDa 1,3-β-glucanase; lane 2, 0.2 µg of purified 75-kDa 1,3-β-glucanase; lane 3, 3 µL of protein standard; lanes 4 and 5, total proteins obtained from regions far away from the interaction site; lanes 6-9, total proteins extracted from the interaction site 24 h after contact (lane 6), 48 h after contact (lane 7), 72 h after contact (lane 8), and 120 h after contact (lane 9); 25 µg of protein was loaded in each of lanes 4-9. The numbers on the left are molecular mass markers. 
Morphological changes induced in Rhizoctonia solani hyphae by the 75-kDa 1,3-β-glucanase of Stachybotrys elegans. (A) Complete lysis (big arrow) or swelling (inset, small arrow) of R. solani hyphal tips when grown on water agar and exposed to 0.054 U of 1,3-β-glucanase. (B) Rhizoctonia solani hyphae exposed to boiled 1,3-β-glucanase. Scale bar = 50 µm. 
The mycoparasite Stachybotrys elegans produces, in addition to a previously purified 94-kDa 1,3-beta-glucanase, at least three extracellular 1,3-beta-glucanases (75, 110, and 180 kDa) when grown on purified cell wall of Rhizoctonia solani. We purified to homogeneity an endo-1,3-beta-glucanase of 75 kDa which possesses a low K(m) value of 20 micrograms laminarin.mL-1 and is most active at pH 5.0 and 40 degrees C. Polyclonal antibodies raised against both the 75- and 94-kDa 1,3-beta-glucanases indicate that they are immunologically related but do not cross-react with the 110- and 180-kDa glucanases. Exposure of growing hyphal tips of R. solani to the pure 75-kDa 1,3-beta-glucanase caused them to swell and lyse. A transient increase of the 75-kDa 1,3-beta-glucanase with a concomitant decrease of the 94-kDa 1,3-beta-glucanase and the appearance of a 20-kDa protein were observed at the point of interaction between R. solani and Stachybotrys elegans on plates. Evidence suggesting a precursor-product relationship between the two 1,3-beta-glucanases is provided. Our results indicate that the 75-kDa 1,3-beta-glucanase may be involved in Stachybotrys elegans mycoparasitism.
 
Clonostachys rosea f. catenulata (syn. Gliocladium catenulatum) is an effective fungal biological agent against Fusarium root and stem rot and Pythium damping-off diseases on cucumber plants. Both chitinase and beta-1,3-glucanase enzymes were produced when C. rosea was grown on a synthetic medium containing chitin or laminarin as a sole carbon source, respectively. Chitinase production was also induced by Fusarium cell walls, while beta-1,3-glucanase activity was induced by both Fusarium and Pythium cell walls, as well as by growth on homogenized cucumber roots and on low-carbon media. Mycelial growth of Fusarium and Pythium, when exposed to C. rosea culture filtrates that contain glucanase activity, was significantly reduced compared with the controls, and cell walls of both pathogens were degraded. On excised cucumber roots, hyphae of C. rosea formed appressorium-like structures and coiled around hyphae of Pythium. In culture, C. rosea caused localized degradation of Fusarium hyphae. Cucumber root tissues colonized by C. rosea showed higher levels of beta-1,3-glucanase activity at 7 days post-application compared with untreated controls. To determine if this activity was derived from C. rosea, glucanase isoforms were separated on activity gels. Fungal culture filtrates and root extracts contained the same predominant 20 kDa isoform. Reverse-transcription polymerase chain reaction (RT-PCR) using primers designed to amplify a beta-1,3-glucanase gene in C. rosea confirmed glucanase expression on roots. These results show that C. rosea produces beta-1,3-glucanase in situ, which can degrade hyphae of Fusarium and Pythium and contribute to biological control efficacy.
 
Previously, we isolated a strain of Bacillus that had antifungal activity and produced lytic enzymes with fungicidal potential. In the present study, we identified the bacterium as Paenibacillus ehimensis and further explored its antifungal properties. In liquid co-cultivation assays, P. ehimensis IB-X-b decreased biomass production of several pathogenic fungi by 45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in hyphal morphology. Residual medium from cultures of P. ehimensis IB-X-b inhibited fungal growth, indicating the inhibitors were secreted into the medium. Of the 2 major lytic enzymes, chitinases were only induced by chitin-containing substrates, whereas beta-1,3-glucanase showed steady levels in all carbon sources. Both purified chitinase and beta-1,3-glucanase degraded cell walls of macerated fungal mycelia, whereas only the latter also degraded cell walls of intact mycelia. The results indicate synergism between the antifungal action mechanisms of these enzymes in which beta-1,3-glucanase is the initiator of the cell wall hydrolysis, whereas the degradation process is reinforced by chitinases. Paenibacillus ehimensis IB-X-b has pronounced antifungal activity with a wide range of fungi and has potential as a biological control agent against plant pathogenic fungi.
 
The in situ degradation of the two nitramine explosives, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), was evaluated using a mixture of RDX and HMX, incubated anaerobically at 10 degrees C with marine sediment from a previous military dumping site of unexploded ordnance (UXO) in Halifax Harbor, Nova Scotia, Canada. The RDX concentration (14.7 mg.L-1) in the aqueous phase was reduced by half in 4 days, while reduction of HMX concentration (1.2 mg.L-1) by half required 50 days. Supplementation with the carbon sources glucose, acetate, or citrate did not affect the removal rate of RDX but improved removal of HMX. Optimal mineralization of RDX and HMX was obtained in the presence of glucose. Using universally labeled (UL)-[14C]RDX, we obtained a carbon mass balance distributed as follows: CO2, 48%-58%; water soluble products, 27%-31%; acetonitrile extractable products, 2.0%-3.4%; and products covalently bound to the sediments and biomass, 8.9% (in the presence of glucose). The disappearance of RDX was accompanied by the formation of the mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and formaldehyde (HCHO) that subsequently disappeared. In the case of HMX, mineralization reached only 13%-27% after 115 days of incubation in the presence or absence of the carbon sources. The disappearance of HMX was also accompanied by the formation of the mononitroso derivative. The total population of psychrotrophic anaerobes that grew at 10 degrees C was 2.6 x 10(3) colony-forming units.(g sediment dry mass)-1, and some psychrotrophic sediment isolates were capable of degrading RDX under conditions similar to those used for sediments. Based on the distribution of products, we suggest that the sediment microorganisms degrade RDX and HMX via an initial reduction to the corresponding mononitroso derivative, followed by denitration and ring cleavage.
 
Nitroreductase activity with TNT and RDX as sub­ strates. 
Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.
 
The extent to which lactic acid bacteria, intestinal bacteria, and yeast from the gastrointestinal tract of rats suppress the absorption of 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) was investigated. Trp-P-1 was absorbed from the small intestine very rapidly, but in the stomach it was slowly absorbed, requiring 1 or 2 h after administration. When mixtures of Trp-P-1 and freeze-dried microorganisms were administered to rats for 1 h, the amounts of Trp-P-1 absorbed from the small intestine were significantly reduced, and the levels of Trp-P-1 in blood decreased by 40.4-64.7% compared with a control in which only Trp-P-1 was administered. There were no significant differences between the organisms used. In vitro, freeze-dried cells of the strains tested bound 51-97% of Trp-P-1. The Trp-P-1 bound to cells was effectively extracted by aqueous methanol, ethanol, ammonia (50 g/L), and solutions of MgCl2 and CaCl2 (100 mM/mL), but little was extracted by water and solutions of KCl, NaCl, and buffers at various pH values.
 
Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.
 
Polyporic acid, atromentin, bovinone, and oosporein are common metabolic products of a number of species of fungi. The related compound cochliodinol and its congeners are produced by several Chaetomium spp. These quinonoid metabolites have been shown to inhibit the growth and metabolism of a range of bacterial genera. The antibiotic activity of the quinones depends on the substituents at the 3 and 6 positions of the 2,5-dihydroxy-1,4-benzoquinone ring; in aerobic systems the activity appears to be inversely proportional to the polarity of the metabolite. It has been shown that reduction of the quinone to the hydroquinone changes the antibiotic activity of these metabolites but does not abolish it. Contrary to previous reports, the activity of these hydroquinones is not reversed by cysteine.
 
The endo-beta-1,4-glucanase (carboxymethylcellulase) activity in cell extracts prepared from Bacteroides succinogenes S85 was almost unaffected by prolonged incubation at 39 degrees C in the presence of merthiolate, a sulfhydryl inhibitor. The beta-1,4-glucosidase (cellobiase) activity, however, was rapidly inactivated by the same treatment. The cellobiase was also inactivated by exposure to air, but was stabilized by dithiothreitol in a nitrogen atmosphere. These results suggest that the cellobiase required reduced sulfhydryl groups for activity.
 
It has been shown that 2,5-dihydroxy-1,4-benzoquinones decrease vegetative growth and inhibit spore germination of 12 species of fungi belonging to six diverse genera. The nature of are substituents at the 3 and 6 positions of the quinone ring also affected their growth-inhibitory properties; generally those substituents of lower polarity inhibited growth at lower concentrations. As in the case of cochiliodinol, chemical modification of the quinone group, or the hydroxyl groups of the quinone ring, in compounds of the polyporic acid series, also led to loss of biological activity.
 
The antimicrobial effect of 5 naphthoquinones was tested against the phytopathogenic bacteria Erwinia carotovora. Disk diffusion tests and determination of minimal inhibitory concentrations (MIC) indicate that the compound naphthazarin (NTZ) has the best antibacterial activity among the naphthoquinones tested. Studies on the mode of action indicate the effect of NTZ was bactericidal at 10 microg/mL. When cultivation was done in the presence of sodium ascorbate, the restoration of E. carotovora growth was observed with 3 microg/mL NTZ, but not when a 10 microg/mL dose was used. The incubation of NTZ with bacterial suspension of E. carotovora resulted in important changes in the absorption spectra of this naphthoquinone, indicating that a redox reaction takes place. These results may suggest that NTZ induces an increase of reactive oxygen species that are toxic to the cell. The compound NTZ was also effective in preventing E. carotovora growth on potato tubers, inhibiting the soft rot development at a concentration of 2 mg/mL.
 
Three mycobacterial strains isolated from fish were tested for their ability to metabolize various amines, supplied individually as sources of carbon. A series of six aliphatic and two cyclic amines was used. Growth occurred only with putrescine. The ability to oxidize putrescine was enhanced by growing the cells in the presence of the compound. Putrescine did not appear to influence the rate of endogenous respiration and therefore O2 uptake values were corrected for endogenous O2 uptake. During the oxidation of putrescine by washed cells, O2 uptake curves "broke" at a point corresponding to 41–44% of the maximum level of oxidation. Thereafter, very small increases in O2 uptake occurred so that on the completion of experiments, the level of oxidation corresponded to 45–47%. At this point, 69–76% of the amino nitrogen of putrescine was released as ammonia, the rest being assimilated. Using putrescine-1,4-14C under CO2-free conditions, approximately 75% of the terminal carbons were evolved as 14CO2. Since the data suggested that carbons 2 and 3 were assimilated, CO2 production accounted for a loss of 37.5% of putrescine carbon. When washed cells oxidized putrescine-1,4-14C, cell fractions containing lipid, nucleic acid, and protein were rapidly labelled.
 
A genomic library of Ruminococcus albus 8 DNA was constructed in Escherichia coli using bacteriophage lambda ZapII. This library was screened for cellulase components and several Ostazin brilliant red/carboxymethyl cellulose positive clones were isolated. All of these clones contained a common 3.4-kb insert, which was recovered as a plasmid by helper phage excision. The carboxymethyl cellulase coding region was localized to a 1.4-kb region of DNA by nested deletions, and a clone containing the entire celA gene was sequenced. Analysis of the sequence revealed a 1231-bp open reading frame, coding for a protein of 411 amino acids with a predicted molecular weight of 45 747. This protein, designated CelA, showed extensive homology with family 5 endoglucanases by both primary amino acid sequence alignment and hydrophobic cluster analysis. Cell-free extracts of E. coli containing the celA clone demonstrated activity against carboxymethyl cellulose and acid swollen cellulose but not against any of the p-nitrophenol glycosides tested, indicating an endo-beta-1,4-glucanase type of activity. In vitro transcription-translation experiments showed that three proteins of 48,000, 44,000, and 23,000 molecular weight were produced by clones containing the celA gene. Northern analysis of RNA extracted from R. albus 8 grown on cellulose indicated a celA transcript of approximately 2700 bases, whereas when R. albus 8 was grown on cellobiose, celA transcripts of approximately 3000 and 600 bases were detected. Primer extension analysis of these RNAs revealed different transcription initiation sites for the celA gene when cells were grown with cellulose or cellobiose as the carbon source. These two sites differed by 370 bases in distance. A model, based on transcription and sequence data, is proposed for celA regulation.
 
Growth-inhibitory activities of some or all of 98 1,4-naphthoquinones and 16 related compounds on Escherichia coli and two strains of Staphylococcus aureus were determined alone or in combination. These values, when plotted against their polarographic half-wave potentials and those of their C2-n-butylthio analogs support the hypothesis that these compounds, or the products resulting from their reaction with a protein nucleophile, function by short-circuiting one or other of the quinones present in the electron-transport chain.
 
The effect of proteolytic enzymes upon endoglucanase activity in a mixed membrane fraction from Achlya ambisexualis. All enzymes were used at a strength of 4 U/mg membrane protein . By class, the proteases are organized as follows: (i) cysteine proteases: papain (Pap), bromelain (Brom), and ficin (Fic); (ii) metalloproteases: collagenase type I from Clostridium histolyticum (Coll), and carboxypeptidase A from bovine pancreas (C-A); (iii) serine proteases: α chymotrypsin from bovine pancreas (Chym), trypsin type IX from porcine pancreas (Tryp), and carboxypeptidase Y from baker's yeast (C-Y); and (iv) aspartyl protease: pepsin from porcine stomach mucosa (Pep). Buffers (20 mM) were Tris-HCl (pH 7.5), used with C-A, C-Y, Chym, and Tryp; Tris-HCl (pH 7.5) with 2 mM CaCl 2 , used with Coll; Na–MES (morpholineethanesulfonic acid) (pH 6.3) with 1 mM DTT (dithiothreitol), used with Fic and Pap; Tris-glycine (pH 3.0) used with Pep; and Na-acetate (pH 5.0) with 1mM DTT, used with Brom. Reactions were carried out as described in the text and terminated by the addition of 10 mM EPNP (1,2-epoxy-3-[p-nitrophenoxy]propane) for aspartyl proteases, 2 mM PMSF (phenylmethanesulfonyl fluoride ) for serine proteases, 10 mM EDTA (ethylene diamine tetraacetic acid) for metalloproteases, or 10 mM iodoacetamide for cysteine proteases. Error bars are ± one standard deviation.  
The distribution of endoglucanase activity in the soluble () and insoluble () phases as a function of papain digestion of a mixed membrane fraction from Achlya ambisexualis. The " membrane-bound " activity in this figure is latent activity that has been activated by Triton X-100 (see text). Error bars are ± one standard deviation.  
The effect of protease inhibitors on endogenous activation of endoglucanase activity of a mixed membrane fraction from Achlya ambisexualis. Reactions were carried out as described in the text, in the presence of either 2 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM iodoacetamide (IODO), 10 mM 1,2- epoxy-3-[p-nitrophenoxy]propane (EPNP), or 10 mM ethylene diamine tetraacetic acid (EDTA). Error bars are ± one standard deviation.  
Electrophoretic activity staining of endoglucanase activities from Achlya ambisexualis. Lane 1, mixed membrane fraction (MMF) proteins solubilized by lithium dodecyl sulfate; lane 2, MMF proteins solubilized by endogenous activation; lane 3, MMF proteins solubilized by treatment with papain (4 U/mg membrane protein) for 30 min; lane 4, MMF proteins solubilized by treatment with papain (8 U/mg membrane protein) for 30 min; lane 5, secreted proteins from culture medium. Numbers on the right indicate the calculated molecular masses (kDa) of the major activity bands in culture medium. Numbers on the left indicate the calculated molecular masses of activity bands released by the listed treatments and that have no near equivalent in culture medium.  
Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-beta-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.
 
Albino mice were infected intravenously with Mycobacterium tuberculosis var. bovis (Ravenel strain). The animals treated with the given antihistaminic substance died significantly sooner than the non-treated control animals. In a similar experiment, the deteriorating effect of antihistamine drug on experimental tuberculosis in guinea pigs was demonstrated. On the basis of previous experiments, it is supposed that the physiological stimulation of the defense mechanism by histamine has been hampered in its function. The administration of 1,4-dimethyl-7-isopropylazulene, which is believed to be a non-toxic agent simulating histamine production, prolonged the life of infected animals. When antihistamine was given in addition to 1,4-dimethyl-7-isopropylazulene, the deteriorating effect of the antihistamine was inhibited. Results are discussed in terms of whether the host–parasite relationship can be favorably influenced by means of a stimulant of the reticulo-endothelial system.
 
Three mycobacterial strains isolated from fish degraded putrescine by a pathway in which γ-aminobutyraldehyde (Δ′-pyrroline), γ-aminobutyric acid, succinic semialdehyde, and succinic acid were intermediates. These results agree substantially with those of other workers using different microorganisms. Intact cells utilized γ-aminobutyric acid in a transaminase reaction with endogenously supplied α-ketoglutarate to produce succinic semialdehyde and glutamate. Studies with arsenite-poisoned cells showed that a significant proportion of putrescine was metabolized via pyruvate and alanine. When putrescine-1,4-14C was substrate, HCl extracts of cells contained radioactive aspartate and glutamate in addition to alanine. The further metabolism of succinate therefore proceeded in two directions: one yielding oxalacetate and α-ketoglutarate by way of the tricarboxylic acid cycle, and the other branching off the cycle to yield pyruvate. Studies with cell-free extracts suggested that putrescine nitrogen was assimilated via glutamate, which served as the amino-group donor to yield alanine and aspartate.
 
This study was designed to assay changes in glycogen synthesis which may occur as a result of cortisol treatment of chlamydial-infected cells. Monolayers of baby hamster kidney (BHK) cells, unlabeled and prelabeled with [6-14C]glucose, were treated with various concentrations of cortisol before (pretreated) or during (post-treated) infection with Chlamydia trachomatis. At designated times after absorption, the cells were harvested and assayed for total glycogen and 14C accumulation in glycogen. The total amount of glycogen accumulated in cells during the period of greatest chlamydial glycogen synthesis (36 h) was not affected by cortisol treatment. Cortisol treatment appeared to have retarded the accumulation of glycogen in treated infected cells until 30 h after infection. Treated infected cells prelabeled with [6-14C]glucose accumulated a greater amount of 14C in glycogen than untreated infected cells. All cortisol-treated, infected cells exhibited elevated levels of fructose-1,6-bisphosphatase activity, whereas untreated infected cells did not. The hypothesis that cortisol affects chlamydia multiplication by altering the intracellular environment of the host cell is compatible with the results obtained.
 
A new Salmonella serotype classified in the Kauffman sub-genus I (Kauffman 1963) has been isolated in Canada from a stock of cacao beans from Nigeria. Salmonella sherbrooke shares the antigenic structure 16:d:1,6.
 
Cloning and transcriptional regulation of the KlFBA1 gene that codes for the class II fructose-1,6-bisphosphate aldolase of the yeast Kluyveromyces lactis are described. KlFBA1 mRNA diminishes transiently during the shift from hypoxic to fully aerobic conditions and increases in the reversal shift. This regulation is mediated by heme since expression was higher in a mutant defective in heme biosynthesis. KlFBA1 transcription is not induced by calcium-shortage, low temperature, or at stationary phase. These data suggest that KlFBA1 plays a role in the balance between oxidative and fermentative metabolism and that this gene is differentially regulated in K. lactis and Saccharomyces cerevisiae, i.e., a respiratory vs. fermentative yeast.
 
Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed malanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenlalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but no catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae.
 
Viable counts of L. monocytogenes ATCC 7644 grown in meat broth incubated at high temperature — 45 °C (A) or to which lactic acid — pH 5.2 (B) or NaCl — 10 g/ 100 ml (C) was added following overnight exposure at 35 °C to sublethal concentrations of carvacrol. (■) Control, non-adapted cells; (+): cells pre-adapted at 1/2 MIC — 0.3 μL/mL; (Δ): cells pre-adapted at 1/4 MIC — 0.15 μL/mL.  
Listeria monocytogenes has the capability of adapting to 1 or more antimicrobial compounds or procedures applied by the food industry to control the growth and survival of microorganisms in foods. In this study, the effects of Rosmarinus officinalis essential oil (EO) and the related compound 1,8-cineole on the inhibition of the growth and survival of L. monocytogenes ATCC 7644 were determined. The ability of the R. officinalis EO and 1,8-cineole to induce direct and cross-protection of bacteria against various stresses (lactic acid, pH 5.2; NaCl, 3 g/100 mL; high temperature, 45 °C) was also determined. At all concentrations tested (minimum inhibitory concentration (MIC), ½ MIC, and ¼ MIC), both compounds inhibited the cell viability of L. monocytogenes over 120 min of exposure. Overnight exposure of L. monocytogenes to sublethal amounts of either the R. officinalis EO or 1,8-cineole in meat broth revealed no induction of direct or cross-protection against lactic acid, NaCl, or high temperature. Similarly, cells subjected to 24 h cycles of adaptation with increasing amounts (½ MIC to 2× MIC) of the EO and 1,8-cineole showed no increase in direct tolerance, as they were able to survive in growth medium containing up to ½ MIC of either substance. These results show the antimicrobial efficacy of R. officinalis EO and 1,8-cineole for use in systems, particularly as anti-L. monocytogenes compounds.
 
Biosynthesis of fungal melanin from 1,3,6,8-tetrahydroxynaphthalene (abbreviations used are 1,3,6,8-tetrahydroxynaphthalene, 1,3,6,8-THN; 1,3,8-trihydroxynaphthalene, 1,3,8-THN; and 1,8-dihydroxynaphthalene, 1,8-DHN).  
Isolate SS7 of Sclerotinia sclerotiorum was previously shown to produce and excrete into agar medium copious amounts of the melanin precursor 1,8-dihydroxynaphthalene. Much reduced quantities of this product were produced in the presence of tricyclazole, an inhibitor of pentaketide melanin biosynthesis. In this study, we demonstrate that young cultures of isolate SS7 produce 1,8-dihydroxynaphthalene monoglucoside, a new natural product not previously reported from fungi. When cultured in the presence of tricyclazole, such young cultures also accumulated two new monoglucosides of 1,3,8-trihydroxynaphthalene, which, as well as 1,8-dihydroxynaphthalene monoglucoside, were also obtained from cultures of two other isolates of S. sclerotiorum. It is proposed that rapid glucosylation of 1,3,8-trihydroxynaphthalene in young tricyclazole-inhibited S. sclerotiorum cultures accounts for the failure to observe 2-hydroxyjuglone or other metabolites usually associated with blockage of the pentaketide pathway to melanin in fungi.
 
The efficiency of the adsorption-elution technique using fiber glass filters to concentrate viruses from water was evaluated to detect poliovirus type 1 in drinking, river, and sewage water. At pH 3.5 and with 5 X 10(-4) M aluminium chloride more than 99% were adsorbed at a 0.25-micron filter. Beef extract (3%), pH 9, eluted 85-95% of the adsorbed viruses and organic flocculation at pH 3.5 permitted to reconcentrate the viruses in 1/20 of the elution volume with a 50-72% efficiency. The overall efficiency of the technique for 100 ml to 1000 l of the different types of water using 10(2) to 10(6) PFU was 38 to 58%.
 
L-Cysteine (1.21 mg/ml) inhibited the linear growth of Helminthosporium carbonum 22% at pH 5.5 and 60% at pH 4. The increased toxicity with increasing acidity was attributed, in part, to the increase in the concentration of the cysteine cation [HS—CH2—CH(NH3+)—COOH]. In the presence of chlorogenic acid (1.77 mg/ml), L-cysteine (1.21 mg/ml) was more toxic than when tested alone. This increased toxicity was due to the acidity of chlorogenic acid, which had little or no fungitoxicity when tested alone. Chlorogenic acid did not increase the toxicity of L-cysteine when the increased acidity was neutralized with base. L-Cysteine at 0.6 mg/ml and 0.3 mg/ml inhibited the growth of H. carbonum, respectively, 32% and 10% at pH 4.5, and 5% and 1% at pH 5.5. Since L-cysteine is toxic only at relatively high concentrations, it appears doubtful whether this amino acid contributes significantly to the fungitoxicity of potato peel extracts.
 
Top-cited authors
Joseph Kloepper
  • Auburn University
Bernard R. Glick
  • University of Waterloo
Albrecht von Quadt
Johannes Hallmann
  • Julius Kühn-Institut
Walter F Mahaffee
  • USDA - Agricultural Research Service Corvallis, OR