The Chitinolytic Activity of Bacillus Cohn Bacteria Antagonistic to Phytopathogenic Fungi

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, -1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r _{x , y} = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

Comparative study of some microbial arabinan-degrading enzymes

Summary A variety of thermophilic organisms andBacillus species were screened in shake flask culture for arabanase andp-nitrophenyl--l-arabinosidase activities. Highest arabanase activity was produced by strains ofThielavia terrestris andSporotrichum cellulophilum. Thermoascus aurantiacus and severalBacillus species were most active producers of arabinosidase. Arabinosidases fromBacillus strains had pH optima in the range 5.9–6.7. pH optima of fungal arabinosidases ranged from 2.9 to 6.7.Bacillus arabanases had neutral pH optima, whereas fungal arabanases had pH optima in the range 3.7–5.1. In general, arabinosidases were found to be relatively thermostable, retaining >70% activity for 3 h at 60°C. TheT. aurantiacus enzyme retained 98% activity at 70°C after 3 h.Bacillus arabanases were relatively unstable. All fungal arabanases except theT. aurantiacus enzyme were fully denatured at 70°C after 3 h.     

Chlorophenol and chlorobenzoic acid co-metabolism by different genera of soil bacteria

1. FourPseudomonas strains, anAchromobacter sp., threeNocardia strains,Mycobacterium coeliacum and aBacillus sp., grown on either phenol or benzoic acid as sole carbon source, were tested for co-oxidation of monochlorophenols and monochlorobenzoates respectively.
2. ThePseudomonas spp.,Nocardia spp.,M. coeliacum and theBacillus sp. showed O₂-uptakes in the presence ofo-, m- orp-chlorophenol and oxidised them to either 3- or 4-chlorocatechol.
3. Benzoate-grown cells of the threeNocardia spp. and three of the pseudomonads absorbed O₂ only in the presence ofm-chlorobenzoate, 4-chlorocatechol being identified as the oxidation product in two cases. One pseudomonad gave some O₂-uptake also in presence ofp-chlorobenzoate.
4. Benzoate-grownBacillus cells gave O₂-uptakes with eitherm- orp-chlorobenzoate and the former was oxidised to 5-chloro-2,3-dihydroxybenzoic acid via 5-chlorosalicylic acid, thus representing a novel metabolic pathway.

     

Isolation and Characterization of Thermophilic Bacillus spp. from Geothermal Environments on Deception Island,South Shetland Archipelago

Abstract Ten bacterial strains isolated from water and sediment samples taken from geothermal areas of Deception Island, in the South Shetland archipelago, were found to represent six distinct types of thermophilic, Gram-positive, aerobic, catalase positive, endospore-forming rods, identified as Bacillus sp. Six representative strains were subjected to routine phenotypic characterization, numerical taxonomic, and chemotaxonomic analyses. Two isolates were identified as thermophilic strains of B. licheniformis and B. megaterium, but the four other strains could not be identified as known species of Bacillus and, hence, may represent new ones. Received: 28 March 1996; Accepted: 29 August 1996     

Acidiphilium aminolytica sp. nov.: An acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment

Acidiphilium aminolytica is proposed for a species of the genusAcidiphilium. Acidiphilium aminolytica can be phenotypically differentiated from all other species of the genusAcidiphilium. The seven strains of this species that have been studied are Gram-negative, aerobic, mesophilic, non-sporeforming, motile, and rod-shaped bacteria. They grow between pH 3.0 and 6.0, but not at pH 6.5. They yield positive results in tests for hippuric acid hydrolysis, catalase and urease production. Oxidase, esculin hydrolysis, and -galactosidase tests are negative. They can used-glucose,d-galactose, inositol, sorbitol,l-lysine,l-glutamate,l-arginine, -alanine,dl-4-aminobutyrate,dl-5-aminovalerate, sperimine, or diaminobutane as a sole carbon source, but cannot use elemental sulfur and ferrous iron as an energy source. The DNA base composition is 58.7–59.2 G+C mol%. The major isoprenoid quinone is ubiquinone with ten isoprene unit (Q-10). The major fatty acid is the C_18:1 fatty acid. Two ornithine amide lipids, the C_18:1 fatty acid esters of -N-3-hydroxystearylornithyltaurine and -N-3-hydroxystearylornithine, are detected as the polar aminolipid. DNA relatedness between this species and the other species ofAcidiphilium, the generaAcidomonas, andAcidobacterium was 29 to 2%. These results indicate, that this new species should be placed in the genusAcidiphilium. The type strain (strain 101) ofA. aminolytica is JCM 8796.     

Characterization of thermophilic Campylobacter. II. Enzymatic profiles

The enzymatic profiles of 234 wild strains of thermophilic Campylobacters, seven type strains ofCampylobacter species, and 18 reference strains ofCampylobacter species and Campylobacter-like organisms were studied by use of API strips. These strips allow the detection of 56 arylamidases, one transpeptidase, and ten esterases.Forty enzymes were present at least once. The mean number of enzymes per strain was 13. The enzymatic activity was usually weak. Three enzymes were present in all the strains: esterases of butyric and valeric acids, andl-phenylalanine-l-proline arylamidase. A combination of three enzymes provided a good predictive value for the species differentiation ofC. jejuni andC. coli. There were no differences in relation to the geographical origin of the strain nor to the animal species from which it was isolated. The -glutamyl transpeptidase could be used for the biotyping of the strains.A portion of this work was presented at the Second Workshop of Campylobacter Infections, held in Brussels, Belgium, in September 1983.     

On the morphology of trichophyton quinckeanum

Summary The cultivation of four old non-sporulating strains ofTrichophyton quinckeanum on solid nutrient medium with 15% CO₂ (Sabouraud dextrose agar) gave an increased production of macroconidia. The macroconidia are multilocular with smooth walls. It is believed that the polymorphism of the macroconidia — some with pointed tip and other with round terminal cell — is characteristic of the species and has a taxonomic significance. Macroconidia with a pointed end cell are not of theMicrosporon type, since they have smooth walls and are not pointed on both ends Filiform appendages from the terminal cell occur regularly. Small and large macroconidia were seen. The microconidia are pyriform or elongated. No rounded microconidia withBotrytis type of arrangement were observed. The so-calledT. niveum is probably identical withT. quinckeannum.Together with the characteristics of the species as a parasite it is to be concluded thatT. quinckeanum is a peculiar dermatophyte species, corresponding toQuincke's -Pilz, but cannot be assimilated by the group ofT. mentagrophytes (Ctenomyces s. microides).     

Klebsiella ornithinolytica sp. nov., formerly known as ornithine-positiveKlebsiella oxytoca

The nameKlebsiella ornithinolytica sp. nov. is proposed for a group ofKlebsiella strains referred to previously as NIH Group 12 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, encapsulated, nonmotile rods with the general characteristics of the familyEnterobacteriaceae and of the genusKlebsiella. They give positive results in tests for indole production, Voges-Proskauer, citrate utilization, lysine and ornithine decarboxylases, urease, -galactosidase, malonate utilization, growth in KCN, and esculin hydrolysis, and they produce acid and gas fromd-glucose, and acid froml-arabinose, cellobiose, lactose, melibiose, raffinose, rhamnose, sucrose, trehalose,d-xylose, adonitol,d-arabitol, myo-inositol, sorbitol, arbutin, salicin, -methyl-d-glucoside, and mucate. They give negative drolysis, DNase, pectinase, and acid production fromd-arabinose, melezitose, and dulcitol. They can grow at 4°C and 42°C, and do not produce any pigment. DNA relatedness of eight strains ofKlebsiella ornithinolytica to three strains including the type strain of this species averaged 88% in reaction at 75°C. DNA relatedness to the already recognizedKlebsiella species inEnterobacteriaceae was 1 to 20%. Phenotypic and DNA relatedness data also indicated that a group of organisms referred to as Enteric Group 47 orKlebsiella Group 47 at the Centers for Disease Control (Atlanta, Georgia) was identical withK. ornithinolytica. The type strain ofK. ornithinolytica is NIH 90-72 (JCM 6096).     

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H₂S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Transepithelial transport in cell culture: Stoichiometry of Na/phlorizin binding and Na/d-glucose cotransport. A two-step,two-sodium model of binding and translocation

Summary The renal cell line LLC-PK₁ cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I _sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm^–2hr^–1, andI _sc, 0.50±0.50(8) eq cm^–2hr^–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI _sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI _sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation.     

Effective production of N-acetyl-β-D-glucosamine bySerratia marcescens using chitinaceous waste

The strain ofSerratia marcescens QM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-β-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of chitin and pretreatment of chitin source were investigated. Also, the effect of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and, ball-milled with a nominal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30°C.     

Ecological study of soil fungi of an agricultural field in Allahabad

Summary The soil fungi from an agricultural field in Allahabad where sugarcane is being grown for many years, have been isolated from various depths during different seasons and were identified. The inter-relations of chemical composition of soil and distribution of fungi is also being shown here.The techniques for the isolation and the study of the fungus flora was that ofGoddard modified bySaksena &Mehrotra. Soil samples were examined from 1–6 depths in three seasons of the year and were mechanically and chemically analysed.For the isolation, soil dilution plate method, a modification ofMenzies' method, direct method ofWaksman, Rossi Cholodny Burried slide technique, and screened immersion plate method, were followed Fifty five different species of microfungi belonging to Phycomycetes, Ascomycetes and Fungi — Imperfecti were isolated and identified. The moisture contents, hydrogen-ion concentrations, carbon, nitrogen, phosphorus, calcium, magnesium and iron, etc., of the soil samples from 1–6 inches depths, were also studied.Out of these 12 species covering 9 genera belonged to the Phycomycetes, nine genera of Ascomycetes and nine genera of Fungi Imperfecti were recorded.P. multicolor Grigorieva-Monoilova &Poradielova, andP. roqueforti Thom.,Gliocladium vermoesoni (Biourge)Thom. andMasoniella grisea (Smith)Smith were recorded for the first time from Indian Soil. A new varietyChaetominum nigricolor Ames var.simplex was also isolated.     

d-Pentose metabolism byBacteroides vulgatus strain 8482

Bacteroides vulgatus strain 8482 metabolizedd-arabinose by a mechanism involving a 32 (top to bottom) cleavage of the arabinose carbon skeleton. During growth in the presence of 1-¹⁴C-d-arabinose, acetate, propionate, and succinate were labeled, but during growth in the presence of 5-labeledd-arabinose, only labeled acetate and succinate were formed. The metabolism ofd-ribose by strain 8482 differed from that ford-arabinose. Strain 8482 converted glycolic acid and glycine to acetate and succinate, but not propionate, by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons and equilibration of carbons 1 and 2 of glycolic acid and glycine with nonequivalent metabolic pools. The metabolism ofd-arabinose,d-ribose,d-glycine, andd-glycolic acid by strain 8482 was similar, in some respects, to that ofBacteroides fragilis strain 2044, but differed substantially from the metabolism of the same substances byBacteroides ruminicola strain B₁4.     

The combined effect of high hydrostatic pressure,heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice

The objective of this study was to combine pressure (345 MPa) with heat (50 C), and bacteriocins (5000 AU/ml sample) for a short time (5 min) for the inactivation of relatively pressure-resistant strains of four foodborne pathogens: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella in pasteurized milk and orange juice. Without bacteriocin addition, 5.5 log-cycle reduction was obtained for S. aureus 485 in milk whereas more than 8 log-cycle reduction was achieved for all the other strains studied. After storage of samples for 24 h at 4 C, S. aureus 765 also gave positive results on selective media, where no growth was observed for all the other micro-organisms assayed. Incubation of the same pressurized samples at 37 C for 48 h showed growth of L. monocytogenes strains in addition to S. aureus strains, where still no growth was observed for E. coli O157:H7 and Salmonella strains in their respective selective media. For orange juice samples, more than 8 log-cycle reduction was achieved for all the bacterial species studied. No growth was seen for these species on their respective selective media agar plates after storage at 4 C for 24 h and at 37 C for 48 h. When a bacteriocin-based biopreservative (BP₁) was combined with pressurization, more than 8 log-cycle reduction in cell population of the resistant strains of S. aureus and L. monocytogenes were achieved in milk after pressurization. Milk samples were stored at 25 C up to 30 days to test the effect of treatment and samples showed no growth whereas all the controls were positive.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

Identification and partial characterization of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin

Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B, showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed chitinase A as the major species. Chitinase B was a minor component of the chitinolytic complex. Whereas some properties were common to the three forms, analysis of other parameters revealed significant catalytic site-related differences. Accordingly, the three chitinases hydrolyzed the fluorogenic substrate 4-methylumbelliferyl chitotriose with typical Michaelian kinetics and K_m values of 4.5, 11.8 and 3.8 M for A, B and B, respectively. Allosamidin strongly inhibited the three enzyme forms with different kinetics. Dixon plots revealed competitive-type inhibition and K_i values of 10.0, 2.3 and 10.8 nM for A, B and B, respectively. K_m/K₁ ratios indicated 450-, 350- and 5130-fold higher affinity for the inhibitor over the substrate for the A, B and B forms, respectively. Results are discussed in terms of the possibility that the three chitinase species correspond to different enzyme proteins.     

Characterization of thermophilicCampylobacter: I. Carbon-substrate utilization tests

The carbon-substrate utlization profile of 234 wild strains of thermophilic campylobacters originating from different animal sources and different part of the world was studied using a microgallery as well as the profile of 25 type strains ofCampylobacter species and reference strains ofCampylobacter-like organisms. Among the 98 substrates tested, succinate, fumarate,d-l-lactate,l-malate, pyruvate,l-glutamate,l-aspartate, andl-serine (with one exception for the last two) were always utilized by the wild strains, and acetate, propionate,d-malate, 2-cetoglutarate, itaconate, citrate, andl-proline by some of the strains. A strong association was found between assimilation ofd-malate and a positive hippurate test.     

Diazotrophic bacilli isolated from the sunflower rhizosphere and the potential of Bacillus mycoides B38V as biofertiliser

The nitrogen fixation by strains belonging to the Bacillus genus remains poorly explored. In this work, the diversity of endospore‐forming bacilli isolated from the rhizosphere of sunflower was evaluated. A total of 101 strains were identified based on the V1‐V2 variable regions of the 16S rRNA gene. Strains belonging to the genera Bacillus and Paenibacillus represented 41.6% and 58.4%, respectively, of total isolates. The production of indolic compounds was a common trait among the isolates, and approximately 75% of them exhibited positive nitrogenase activity; but only 9.2% displayed activities higher than 1 nmol C₂H₄ mg protein h^?1. Within the genus Bacillus, the isolates related to the B. cereus group displayed the highest nitrogenase activity and were the second most frequent group of Bacillus sp. isolated. Plants inoculated with the isolate B38V showed the highest N content, and their shoot dry weights were significantly increased compared with positive control. Our results indicated that B38V, which belongs to the B. mycoides species, has the potential to promote sunflower growth. The data obtained in this study provide additional information concerning the diversity of Gram‐positive diazotrophic within the genera Bacillus and Paenibacillus and their potential for the biofertilisation of sunflower crops.     

l-[3H]lysine binding to rat retinal membrane: II. effect of kainic acid,d,l-α-aminoadipic acid,iodoacetic acid,and modification by dark-exposure

The rat retina and the different brain regions contain membranes sites that bindl-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: I) intraocular injection of kainic acid induces a reduction of the density ofl-lysine binding sites, II)d,l--aminoadipic acid injected into the eye enhances both kinetic parameters (B _max andK _d) ofl-[³H]lysine binding sites, III) the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and IV) the exposure to darkness of the rats reducesl-[³H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites forl-lysine after the exposure to darkness. The results show thatl-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.