Showing papers in "Folia Microbiologica in 1998"

Phytase: Sources, preparation and exploitation

Abstract: This review deals with phytase (myo-inositol hexakisphosphate phosphohydrolase) and covers microbiological sources, phytase occurrence in plants and animals, its purification, physico-chemical and molecular properties. Protein engineering of phytase and potential enzyme applications are discussed.

Effect of the culture filtrates of Streptomyces on growth and productivity of wheat plants

Abstract: Streptomyces olivaceoviridis, S. rimosus andS. rochei proved to possess a high capacity for the production of auxins, gibberellins and cytokinin-like substances, together with substantial levels of α-amylase and proteinase. Grain priming with culture filtrates ofS. olivaceoviridis, S. rimosus orS. rochei appeared to enhance growth vigor and crop yield of wheat plants. In the majority of cases, the culture filtrate ofS. olivaceoviridis appeared to be the most effective in this respect. The present results are discussed in relation to the indirect role played by these bacteria in producing plant growth-regulating substances and their effects on growth and yield of wheat.

Colicins : Exocellular lethal proteins of Escherichia coli

Abstract: Colicins are toxic exoproteins produced by bacteria of colicinogenic strains ofEscherichia coli and some related species ofEnterobacteriaceae, during the growth of their cultures They inhibit sensitive bacteria of the same family About 35%E coli strains appearing in human intestinal tract are colicinogenic Synthesis of colicins is coded by genes located on Col plasmids Until now more than 34 types of colicins have been described, 21 of them in greater detail,viz colicins A, B, D, E1–E9, Ia, Ib, JS, K, M, N, U, 5, 10 In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell The classification of colicins is based on differences in the molecular events of these three steps

Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil

Abstract: Soil samples from an agricultural field contaminated with 10 ppm14C-benz(a)anthracene in glass tubes were brought into contact with cultures of wood-rotting fungi, precultivated on wheat straw substrate. Forty-five strains of white-rot fungi and four brown-rot fungi were tested for their ability to colonize the soil and to mineralize14C-benz(a)anthracene to14CO2 within a 20-week incubation time. Twenty-two white-rot fungi and all brown-rot fungi were unable to colonize the soil. Twenty-three strains of white-rot fungi, all belonging to the genusPleurotus, colonized the soil. During the experiment the noncolonizing fungi and their substrate disintegrated more and more to a nonstructured pulp from which water diffused into the soil. The same phenomenon was observed in the control which contained only straw without fungus and contaminated soil. In samples with colonizing fungi the substrate as well as the mycelia in the soil remained visibly unchanged during the entire experiment. Surprisingly, most samples with fungi not colonizing the soil and the control without fungus liberated between 40 and 58 % of the applied radioactivity as14CO2 whereas the samples with the colonizing fungi respired only 15–25 % as14CO2. This was 3–5 times more14CO2 than that liberated from the control (4.9 %) which contained only contaminated soil without straw and fungus. A similar result was obtained with selected colonizing and noncolonizing fungi and soil contaminated with 10 ppm14C-pyrene. However, in pure culture studies in which14C-pyrene was added to the straw substrate,Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3 % of the added radioactivity to14CO2. The noncolonizing fungiDichomitus squalens andFlammulina velutipes liberated only 17.2 or 1.7 %, respectively, as14CO2. These results lead to the hypothesis that the native soil microflora stimulated by the formed products of straw lysis is responsible for high degradation rates found with noncolonizing fungi.

Gentamicin resistance in clinical strains of Enterobacteriaceae associated with reduced gentamicin uptake

Abstract: Seven strains ofEnterobacteriaceae resistant to gentamicin obtained as representatives of the predominant resistance profiles in the clinical laboratories ofRafeidia and Al-Watani Hospitals in Nablus (Palestine) were included. Five strains showed a broad aminoglycoside resistance profile but contained no evidence of gentamicin acetylation, adenylation, or phosphorylation. Gentamicin uptake in two tested strains was significantly reduced, compared to that of gentamicin-sensitiveE. coli (MIC, 0.5 μg/mL.) These strains are likely resistant due to a relative reduction of the amount of gentamicin and other aminoglycosides entering the bacterial cell. Two strains showed evidence of adenyltransferase ANT (2")-I activity.

Comparison of static and shake culture in the decolorization of textile dyes and dye effluents byPhanerochœte chrysosporium

Abstract: Decolorization of several dyes (Red HE-8B, Malachite Green, Navy Blue HE-2R, Magenta, Crystal Violet) and an industrial effluent with growing cells ofPhanerochœte chrysosporium in shake and static culture was demonstrated. All the dyes and the industrial effluent were decolorized to some extent with varying percentages of decolorization (20–100%). The rate of decolorization was very rapid with Red HE-8B, an industrial dye. Decolorization rates for all the dyes in static condition were found to be less than the shake culture and also dependent on biomass concentration.

The effectiveness of biological treatment of wheat straw by white-rot fungi

Abstract: Out of 13 species ofBasidiomycetes growing on wheat straw, 9 species enhanced thein vitro dry matter digestibility of the substrate. The detergent fiber content (acid and neutral) of the substrate was significantly reduced by most of the fungi tested. Hemicellulose showed the largest proportionate loss, whereas lignin the smallest one.

Biocontrol of fungal root rot diseases of crop plants by the use of rhizobia and bradyrhizobia

Abstract: Twenty-oneRhizobium andBradyrhizobium strains were testedin vitro against the mycelial growth of three pathogenic fungi on solid and liquid media All tested rhizobia and bradyrhizobia significantly suppressed the growth of the three soil-borne root-infecting fungi (Fusarium solani, Macrophominia phasolina andRhizoctonia solani) either in the absence or presence of iron This indicates that the siderophore played a minor role in the biocontrol potential ofRhizobium andBradyrhizobium against pathogenic fungi Pot experiments revealed that the numbers of propagules causing disease after 4 weeks of planting varied with species and host plant The three most activeRhizobium andBradyrhizobium strains (R leguminosarum bvphaseoli TAL 182,B japonicum TAL 377 andBradyrhizobium sp (lupin) WPBS 3211 D) tested under greenhouse conditions for their ability to protect one leguminous (soybean) and two non-leguminous (sunflower and okra) seedlings from root rot caused byFusarium solani, Macrophominia phaseolina andRhizoctonia solani provided significant suppression of disease severity compared with nonbacterized control in both leguminous and non-leguminous seedlingsBradyrhizobium sp (lupin) WPBS 3211 D provided the lowest degree of resistance against all the tested pathogens with all host plants *** DIRECT SUPPORT *** A00EN058 00013

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

Abstract: A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.

Molecular and cellular mechanisms of invasion of the intestinal barrier by enteric pathogens the paradigm of Shigella

Abstract: The pathogenesis of bacillary dysentery can be studied at different levels of integration of the cellular components that constitute the colonic mucosal barrier. We considered the interaction ofShigella flexneri in three experimental systems that provide complementary information and a scheme of events occurring in human colorectal mucosa asShigella invasion proceeds. Interaction ofS. flexneri with individual epithelial cells shows a series of events in which the bacterium, upon contact with the cell surface, releases a set of Ipa proteins (i.e. invasins) through a specialized, activable, type-III secretory apparatus (i.e. Mxi/Spa).Via a complex signaling process, these invasins cause major rearrangements of the subcortical cytoskeletal network which allow bacterial entry by a macropinocytotic event. Then the bacterium lyses its phagocytotic vacuole and initiates intracytoplasmic movement, due to polar assembly of actin filaments caused by a bacterial surface protein, IcsA. This allows very efficient colonization of the host cell cytoplasm and passage to adjacent cellsvia protrusions which are engulfed by a cadherin-dependent process. However, when invasiveShigella are deposited on the apical side of polarized monolayers of human colonic cells, they appear unable to invade, indicating that bacteria need to reach the subepithelial area to invade the epithelium. In this system, it has been shown that transepithelial signaling caused by apical bacteria induces adherence and transmigration of basal polymorphonuclears (PMN), thus disrupting the monolayer permeability and facilitating bacterial invasion. LPS accounts for a large part of this transepithelial signalization to PMN. Such a process could account for invasion in intestinal crypts. Finally, models of infection, such as the rabbit ligated intestinal loop show that initial bacterial entry occurs essentiallyvia M cells of the follicular associated epithelium. It then causes apoptosis of macrophages located in the follicular dome, inducing release of IL-1β which, in turn, initiates inflammation, leading to destabilization of the epithelial structures as modeled above. These data can now be used to understand the mechanisms of mucosal protection against bacillary dysentery.

Biological effects of macrotetrolide antibiotics and nonactic acids.

Abstract: Published results as well as patent applications on biological effects of macrotetrolides and nonactic acids are reviewed. Their antimicrobial, antiprotozoan (coccidiostatic), antiparasitic (anthelminthic), insecticidal and acaricidal (miticidal) effects and also newly described immunosuppressive and plant growth stimulating activities are described. Both theoretical papers and practical applications including the effects of macrotetrolides on the environment are included; a particular target organism and precise dosage (e.g. LD50) are reported, in agreement with the original papers. It appears that macrotetrolides and their homologs are very prospective bioactive compounds that find application in agriculture, forestry, human and veterinary medicine while their negative effects on the environment are restricted to a minimum (biological quality of soil and water etc.).

Siderophore production in relation to N2 fixation and iron uptake in pigeon pea-Rhizobium symbiosis

Abstract: Thirty-one bradyrhizobial and rhizobial strains infecting pigeon pea were screened for siderophore production using Chrome Azurol S (CAS) agar plate as well as a CAS assay solution. Of a total of 31 strains only 23 showed siderophore production. Of the 23 siderophore-positive strains, 21 strains showed the production of hydroxamate while 6 strains showed the presence of catechol type of siderophore. A large variation in the quantity of hydroxamate and catechol produced by different rhizobial strains was observed (1.03–3.73 μg hydroxamate N per mg protein; 0.19–3.43 μmol/L of catechol per mg protein). Maximum nodule biomass was produced by strain PP-11 (CC-1020); strain G-14 formed minimum nodule biomass. Nitrogen contents of low, moderate and high siderophore-producing strains were 11.4, 15.4, 20.9 mg per plant, respectively, iron contents were 1445, 1768 and 2003 ppm, respectively. Siderophore production was related to N2-fixing efficiency.

Nutritional and cultural conditions for production of poly-3-hydroxybutyric acid byAzotobacter chroococcum.

Abstract: Free-living nitrogen-fixing bacteria have been identified as a potential source of poly-3-hydroxybutyric acid (PHB). Systematic study of this ability of N2-fixing organisms has lead to the isolation of an efficient strain, identified asAzotobacter chroococcum. Nutritional requirements and cultural conditions for optimal production of PHB by this strain under laboratory conditions were determined. In N-free liquid medium containing 2% glucose, the strain accumulated PHB up to 68% of its cell dry mass. Glucose and mannitol were found to be the best carbon sources, while organic nitrogen compounds were preferred as nitrogen source. Maximum yield (3.3 g/L) was obtained with 0.2% bactopeptone supplementation. Inorganic phosphate at a concentration suboptimal for growth had some growth-promoting effect. Under oxygen limiting conditions, biomass production was enhanced but a different response was obtained for PHB production.

Differences in development of lymphocyte subpopulations from gut-associated lymphatic tissue (GALT) of germfree and conventional rats: Effect of aging

Abstract: The aim of the study was to compare the phenotype of lymphocyte subpopulations of the GALT (gut-associated lymphatic tissue) in germfree (GF) and conventionally (CV) reared rats, i.e. to analyze the effect of microbial colonization on the development of intestinal lymphocyte subsets. Surface marker characteristics were studied in cell suspensions isolated from Peyer's patches, mesenteric lymph nodes, spleen and the intraepithelial lymphocyte compartment of 2- and 12-month old inbred AVN rats. The pattern of T lymphocyte phenotypes in Peyer's patches, mesenteric lymph nodes and spleen determined by FACS analysis did not reveal differences between GF and CV rats. In contrast, a 2-month conventionalization of GF rats led to substantial changes in the composition of intestinal intraepithelial lymphocyte subsets (IELs): increase of CD4+, CD8 alpha+, CD8 beta+, TcR alpha/beta+ bearing lymphocytes was observed after colonization of rats with normal microflora. Surprisingly, the relative numbers of lymphocytes bearing TcR gamma/delta+ did not change during conventionalization. The effect of aging was also studied and differences in IELs composition of aged (GF) and (CV) rats were found to be more pronounced: 6.6% and 30% of lymphocytes bearing TcR alpha/beta were present among IELs in two-month old GF and CV rats, respectively. 30% of IELs in 2-month old GF rats, 80% of IEL from 12-month old CV rats were found to bear TcR alpha/beta. This finding demonstrates that during conventionalization and aging the TcR alpha/beta bearing population of IELs substantially expands. It suggests that mainly this lymphocyte subset responds to microflora stimuli and is probably involved in the protection of the epithelial cell layer of intestinal mucosa.

Degradation of polychlorinated biphenyl mixtures by the lignin-degrading fungusPhanerochœte chrysosporium

Abstract: Degradation of lower-chlorinated and higher-chlorinated PCB congeners (Delor 103 and Delor 105 as equivalents of Aroclor 1242 and Aroclor 1254, respectively) by the white-rot fungusPhanerochœte chrysosporium was investigated in N-limited and non-limited media. No degradation of either Delor 103 or Delor 105 was found in a N-limited medium 9 d after their addition whereas in the non-limited medium during the same period their levels dropped by 55 and 58%, respectively. The degradation was non-specific and no significant differences in the degradation of di-, tri-, tetra-, penta-, hexa-, and hepta-congeners were found. No activity of Mn-dependent peroxidase (MnP) or lignin peroxidase (LiP) was detectable in the non-limited medium. We assume that the degradation of PCBs byP. chrysosporium is relatively non-specific, takes place under non-limited conditions and is independent of the activities of MnP or LiP.

Lipase production by Aspergillus and Penicillium species.

Abstract: Forty each of aspergilli and penicillia were screened for extracellular lipase production on agar plates and in liquid medium containing olive oil as substrate. Twenty-nine aspergilli and twenty-six penicillia produced lipase. Out of these, 19 aspergilli and 22 penicillia showed activity both on Nile blue sulfate and glycerol tributyrate agar plates while only 10 aspergilli and 4 penicillia showed a positive response to glycerol tributyrate agar alone. The screening revealed 11 Aspergillus spp. and 15 Penicillium spp. as new lipase producers. Pig fat as an economic substrate for lipase production was also investigated.

γ-ray induced mutagenesis ofCellulomonas biazotea for improved production of cellulases

Abstract: A rifampin-resistant mutant ofCellulomonas biazotea secreted elevated levels of cellulasesin vivo. The cellulase production in the mutant was not inhibited in the presence of 5% glucose, cellobiose or glycerol in the solid medium. The mutant exhibited approximately two- to three-fold enhanced product yields and productivity of cellular β-glucosidase over the wild parent in shake-flask culture studies when grown on either cellulosic or lignocellulosic substrates. Extracellular production of filter paper cellulase (FPase) and endo-glucanase (CMCase) were also significantly (p≤0.05) altered. During growth of the mutant on α-cellulose, the maximum volumetric productivities for CMCase, FPase and β-glucosidase were 52, 23.3, and 15.2 IUL−1 h−1,i.e 118, 121, and 229% their respective values for the parental strain. Some enzyme properties of the mutant cellulases were altered. Mutant-derived cellulases produced higher yields of glucose arising by degradation of bagasse, wheat straw, and α-cellulose (1.53-, 1.57-, and 1.75-fold, respectively).

Sequence of archaealMethanococcus jannaschii α-amylase contains features of families 13 and 57 of glycosyl hydrolases: A trace of their common ancestor?

Abstract: Two sequentially different, seemingly unrelated α-amylase families exist, known as family-13 and family-57 glycosyl hydrolases Despite the common enzyme activity, it has as yet been impossible to detect any sequence similarity between the two families The detailed analysis of the recently determined sequence of the α-amylase from methanogenic archaeonMethanococcus jannaschii using the sensitiveHydrophobic Cluster Analysis method revealed that this α-amylase contains features of both families of α-amylases Thus theM jannaschii α-amylase is similar to thePyrococcus furiosus α-amylase from family 57 while it obviously contains most of the sequence fingerprints characteristic for α-amylase family 13 Importantly, a glutamic acid residue equivalent with the family-13 catalytic glutamate positioned in the β5-strand segment was identified in members of family 57 The results presented in this report indicate that the two families, 13 and 57, are either the products of a very distant common ancestor or have evolved from each other, although at present they can represent two different α-amylase families with evolved different catalytic mechanisms, catalytic machinery and folds

Use of propolis and ultragriseofulvin to inhibit aflatoxigenic fungi

Abstract: Propolis ethanolic extract (PEE) at 3 and 4 g/L and ultragriseofulvin (UG) at 0.75 and 1 g/L reduced the percentage of conidia germination in twoAspergillus flavus isolates. PEE at 1–4 g/L decreased the mycelial dry mass ofA. flavus isolates by 11–80%, and aflatoxin B1 production by 34–100%. UG concentrations of 0.25–1 g/L reduced the growth and aflatoxin B1 production of the isolates by 16–88 and 48–98%, respectively. Any increase in PEE and UG concentration was accompanied by a clear decrease in the per cent conidia germination, growth and aflatoxin B1 production. At equal concentration, UG was about 4-times more effective than PEE.

The Streptomyces aureofaciens homologue of the whiB gene is essential for sporulation; its expression correlates with the developmental stage.

Abstract: In previous experiments, aStreptomyces aureofaciens gene highly similar to the sporulation-specificwhiB gene ofStreptomyces cœlicolor was identified. By intergrative transformationvia double cross-over, a stable null mutant of thewhiB-homologous gene ofS. aureofaciens was obtained. The disruption blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores, producing white aerial hyphae without septation. Expression of thewhiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared fromS. aureofaciens in various developmental stages. Two putative promoters were identified upstream of thewhiB coding region. The stronger promoter,whiB-P2, was induced at the beginning of aerial mycelium formation, and the weaker promoter,whiB-P1, was expressed fairly constantly during differentiation. No differences in the expression of thewhiB promoters were detected in anrpoZ-disruptedS. aureofaciens strain. The promoter bearing DNA fragment was inserted into the promoter-probe vector pARC1 to produce an expression pattern consistent with the results of direct RNA analysis.

Stimulation of Conidiation by Derivatives of cAMP in Trichoderma viride

Abstract: Derivatives of cyclic-3′,5′-adenosine monophosphate, N6,2′-O-dibutyryl-cAMP, 8-(chlorophenylthio)-cAMP and 8-bromoadenosine-cAMP added at 0.1–10 μmol/L concentrations into the growth medium have markedly stimulated conidiation inTrichoderma viride. Their stimulatory effect on conidiation was best observed in colonies that were constantly kept in the dark but was also marked in colonies illuminated by sub-saturating doses of light. The relative stimulation of conidiation depended not only on the concentration of exogenous cyclic nucleotides but also on the concentration of glucose in the medium. It was more pronounced in glucose-rich media than in media where the concentration of the sugar was low. Concentrations of cAMP analogues equal to 50 μmol/L and higher inhibited the conidiation.

The Yersinia Yop virulon, a bacterial system to subvert cells of the primary host defense

Abstract: The Yop virulon enables yersinias (Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It is an integrated system allowing extracellular bacteria to communicate with the host cell's cytosol by injection of effector proteins. It is composed of four elements: (1) a contact or type III secretion system called Ysc, devoted to the secretion of Yop proteins. This secretion apparatus, made of some 22 proteins, recognizes the Yops by a short N-terminal signal that is not cleaved off during secretion; (2) a system designed to deliver bacterial proteins into eukaryotic target cells. This system is made of YopB, YopD and LcrV; (3) a control element (YopN) and (4) a set of effector Yop proteins designed to disarm these cells or disrupt their communications (YopE, YopH, YopM, YpkA/YopO, YopP). The whole virulon is encoded by a 70-kb plasmid called pYV. Transcription of the genes is controlled both by temperature and by contact with a eukaryotic cell.

Studies on toxigenic fungi in roasted foodstuff (salted seed) and halotolerant activity of emodin-producing Aspergillus wentii

Abstract: Commercial roasted salted peanuts (3% NaCl), popcorn (1% NaCl), summer-squash (9% NaCl), sunflower (3% NaCl) and wild-melon (3% NaCl) seeds are polluted with fungi, mostlyAspergillus flavus, A. niger, Penicillium chrysogenum, P. corylophilum andRhizopus stolonifer. Contamination of popcorn with the fungi is about 10 times higher than in the other foods. These fungi, common also on unsalted seeds, are significantly inhibited in seeds (30% moisture content) treated with 9–21% NaCl. The halotolerantA. wentii represents the main fungus recovered from seeds treated by 15–21% NaCl. 9% NaCl stimulated emodin production byA. wentii on peanut and citrinin production byP. chrysogenum on popcorn and sunflower. Aflatoxin, citrinin and emodin production on popcorn persisted up to 15% NaCl. Popcorn is thus strongly susceptible to fungal invasion and toxin pollution. The halotolerance ofA. wentii was confirmed by its strong permanent growth in liquid medium at up to 15% NaCl. At 3% NaCl the mycelial growth and nitrogen content increased while the level of emodin and lipid production decreased. CO2 evolution strongly increased at 9–15% NaCl as a characteristic ofA. wentii salt tolerance. Emodin inhibited seed viability and the inhibition dose for 50% reduction (LD50) was 65 mg/L for popcorn and 45 mg/L for sunflower.

Comparison of ethanol tolerance of free and immobilized Saccharomyces uvarum yeasts

Abstract: O2 consumption and CO2 production of free and immobilizedSaccharomyces uvarum in the presence of ethanol were compared. The protective effect of immobilization on the yeast ethanol tolerance at 5–20% of ethanol was more evident in CO2 production than in O2 consumption. CO2 production by the yeast immobilized in calcium alginate and calcium pectate gel beads was approximately 2.5-times higher than by the free yeast at 5 and 10% of ethanol. 4-Fold increase of CO2 production was observed at 15% ethanol. Immobilization in calcium-containing carriers (alginate, pectate) resulted in enhanced activities of yeasts compared to the κ-carrageenan carrier.

Antibacterial Effects of Some 1-Substituted 1,2,4-Triazoles

Abstract: Seventeen synthetic 1-substituted 1,2,4-triazoles exerted a significant effect on the bacteriaB. subtilis, S. aureus, E. coli andP. aeruginosa. The least sensitive to the effects of the triazoles wasS. aureus. With all triazole derivatives and their combinations,B. subtilis andP. aeruginosa exhibited IC50 and MIC values several times higher than with ampicillin. The most effective triazoles have a N-phenyl ring or benzimidinoyl ring substituted with one or several chlorine atoms. The highest tested concentration of the three most effective triazoles influenced the specific growth rate.

Antitumor action of bovine seminal ribonuclease

Abstract: Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 μg per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally—i.p., subcutaneously—s.c., and intratumorally—i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

Abstract: A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Interactions of the bacterial pathogen Listeria monocytogenes with mammalian cells : Bacterial factors, cellular ligands, and signaling

Abstract: Listeria monocytogenes is a food borne pathogen which has the very unique property of crossing three barriers during infection eliciting meningitis, meningo-encephalitis and abortions with a mortality rate of about 30%. Indeed, after crossing the intestinal barrier,Listeria disseminatesvia the lymph and the blood, to the brain and/or the placenta after crossing the brain-blood barrier and/or the placental barrier. During disease, this organism infects a variety of tissues and cell types in which it is mostly intracellular due to its capacity to induce its own phagocytosis into cells which are normally nonphagocytic. The strategies used byListeria to enter cells are different from those used by other well known invasive pathogens.Listeria thus appears as a fine model to study the molecular and cellular basis of bacterial invasion. In addition, not only during entry into cells but also during intra-and intercellular movement,Listeria exploits mammalian cell functions and is thus a novel tool for elucidating some unsolved fundamental aspects of cell biology, such as ligand receptor signaling and actin cytoskeleton rearrangements. In this review, the molecular and cellular basis of entry ofListeria into cells and of its intracellular motility will be discussed.

Cloning and expression of β-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES 2.0

Abstract: Genes for β-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium,Cellulomonas biazotea, were cloned in pUC18 in itsSacI cloning site and transformed toE. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representativeE. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of β-glucosidase onE. coli. Expression of thebgl genes resulted in overproduction of β-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carryingbgl genes from the representative recombinantE. coli were isolated withSacI ligated in the shuttle vector pYES2.0 in itsSacI site and transformed toE. coli andS. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of β-glucosidase onS. cerevisiae and enabled it to grow on cellobiose and salicin. Thegall promoter of shuttle vector pYES2.0 enabled the organisms to produce twice more β-glucosidase than that supported by thelacZ-promoter of pUC18 plasmid inE. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains ofS. cerevisiae The enzyme produced bybgl + yeast andE. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the β-glucosidases fromS. cerevisiae were substantially the same as those fromC. biazotea.

Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeast Hansenula polymorpha: isolation and study of regulatory mutants.

Abstract: Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeast Hansenula polymorpha. Maltase was shown to be responsible for the growth of H. polymorpha not only on maltose, but also on sucrose. The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (PNPG) < sucrose < maltose. Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained from H. polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-D-glucose. One of the isolated mutants, L63, was studied in more detail. Mutant L63 was recessive and monogenic and it was not deficient in hexokinase. Its analysis revealed that H. polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.