bluuebunny/crossref_metadata_2025 huggingface dataset:
Three Genes for Metabolism of the Phytoalexin Maackiain in the Plant Pathogen
Nectria haematococca
: Meiotic Instability and Relationship to a New Gene for Pisatin Demethylase
Authors: Miao V. P. W. , Vanetten H. D.
Published: January 2020

Some isolates of the plant-pathogenic fungus
Nectria haematococca
mating population (MP) VI metabolize maackiain and medicarpin, two antimicrobial compounds (phytoalexins) synthesized by chickpea (
Cicer arietinum
L.). The enzymatic modifications by the fungus convert the phytoalexins to less toxic derivatives, and this detoxification has been proposed to be important for pathogenesis on chickpea. In the present study, loci controlling maackiain metabolism (
Mak
genes) were identified by crosses among isolates of
N. haematococca
MP VI that differed in their ability to metabolize the phytoalexin. Strains carrying
Mak1
or
Mak2
converted maackiain to 1a-hydroxymaackiain, while those with
Mak3
converted it to 6a-hydroxymaackiain.
Mak1
and
Mak2
were unusual in that they often failed to be inherited by progeny.
Mak1
was closely linked to
Pda6
, a new member in a family of genes in
N. haematococca
MP VI that encode enzymes for detoxification of pisatin, the phytoalexin synthesized by garden pea. Like
Mak1, Pda6
was also transmitted irregularly to progeny. Although the unusual meiotic behaviors of some
Mak
genes complicate genetic analysis, identification of these genes should afford a more through evaluation of the role of phytoalexin detoxification in the pathogenesis of
N. haematococca
MP VI on chickpea.
Construction of Novel
Bacillus thuringiensis
Strains with Different Insecticidal Activities by Transduction and Transformation
Authors: Lecadet M. M. , Chaufaux J. , Ribier J. , Lereclus D.
Published: January 2020

The shuttle vector pHT3101 and its derivative pHT408, bearing a copy of a
cryIA
(
a
) δ-endotoxin gene, were transferred into several
Bacillus thuringiensis
subspecies through phage CP-54Ber-mediated transduction, with frequencies ranging from 5 × 10
-8
to 2 × 10
-6
transductant per CFU, depending on the strain and on the plasmid. In Cry
-
and Cry
+
native recipients, the introduction of the
cryIA
(
a
) gene resulted in the formation of large bipyramidal crystals that were active against the insect
Plutella xylostella
(order Lepidoptera). In both cases, high levels of gene expression were observed. Transductants displaying a dual specificity were constructed by using as recipients the new isolates LM63 and LM79, which have larvicidal activity against insects of the order Coleoptera. It was not possible, however, to introduce pHT7911 into
B. thuringiensis
subsp.
entomocidus, aizawai
, or
israelensis
by transduction. However, electrotransformation was successful, and transformants expressing the toxin gene
cryIIIA
, carried by pHT7911, were obtained. Again, high levels of expression of the cloned gene were observed. The results indicate that CP-54Ber-mediated transduction is a useful procedure for introducing cloned crystal protein genes into various
B. thuringiensis
recipients and thereby creating strains with new combinations of genes. Finally it was also shown that pHT3101 is a very good expression vector for the cloned δ-endotoxin genes in the different recipients.
Reduction of uranium by Desulfovibrio desulfuricans
Authors: Lovley D R , Phillips E J
Published: January 2020

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.
Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium
Authors: Schmidt J E , Macario A J , Ahring B K , Conway de Macario E
Published: January 2020

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.
Isolation of New
Aureobasidium
Strains That Produce High-Molecular-Weight Pullulan with Reduced Pigmentation
Authors: Pollock Thomas J. , Thorne Linda , Armentrout Richard W.
Published: January 2020

New isolates of
Aureobasidium pullulans
were obtained from plant leaf surfaces gathered in San Diego County. The new fungal isolates were identified as
A. pullulans
on the basis of the appearance of polymorphic colonies formed on agar plates, the electrophoretic profiles of repeated genomic DNA sequences, and the production of pullulan in shake flask cultures. The isolates showed different degrees of pigmentation. One of the natural isolates was nonpigmented under mock production conditions in liquid culture, but was still able to synthesize a reduced amount of pigment on agar plates at late times. A mutagenic treatment with ethidium bromide produced derivatives of normally pigmented natural isolates that exhibited an increased tendency toward yeastlike growth and reduced pigmentation. Additionally, some of the new isolates and mutant derivatives accumulated pullulan of relatively high molecular weight in the culture broths.
Characterization of an 8.7-kilobase thiostrepton resistance-encoding plasmid (pGIF3) of Streptomyces incarnatus
Authors: Malina H , Robert-Gero M
Published: January 2020

The low-copy-number 8.7-kb plasmid pGIF3 of Streptomyces incarnatus was studied after cloning in the Escherichia coli vector pBR322; a restriction map was constructed. Southern blot analysis showed that pGIF3 in S. incarnatus occurs predominantly as integrated in a larger replicon. The plasmid carries a gene for thiostrepton resistance having no homology with the known thiostrepton resistance gene from Streptomyces azureus.
Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae
Authors: Thanabalu T , Hindley J , Brenner S , Oei C , Berry C
Published: January 2020

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.
Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1
Authors: Omori T , Monna L , Saiki Y , Kodama T
Published: January 2020

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.
Kinetics of Escherichia coli destruction by microwave irradiation
Authors: Fujikawa H , Ushioda H , Kudo Y
Published: January 2020

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.
Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers
Authors: Berman M H , Frazer A C
Published: January 2020

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.
Induction of ploidy level increments in an asporogenous industrial strain of the yeast Saccharomyces cerevisiae by UV irradiation
Authors: Sasaki T
Published: January 2020

Cells of an asporogenous industrial strain of the yeast Saccharomyces cerevisiae were irradiated with UV light by using a method that was developed previously (T. Sasaki and Y. Ohshima, Appl. Environ. Microbiol. 53:1504-1511, 1987). This treatment gave rise to large-cell clones among the surviving cells, from which colonies consisting of cells with a normal morphology and a prototrophic property were obtained. The large-cell trait of these was stably inheritable, with the cell volumes being about twice that of the parent for 7 years on a slant agar medium at 4 degrees C with repeated transfers. The cellular DNA content of these clones, in comparison to those of two authentic haploid strains, was determined by chemical analysis. The ratio of the DNA contents showed that the parent and its large-cell derivatives were a diploid and tetraploids, respectively. No abnormality was found in the chromosomal DNA patterns of the large-cell clones, at least as determined by agarose gel electrophoresis with a CHEF-DR II pulsed-field electrophoresis system. These findings led to the conclusion that our UV light method is applicable for inducing ploidy level increments in the widely used yeast species S. cerevisiae.
Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan
Authors: Croux C , Canard B , Goma G , Soucaille P
Published: January 2020

An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.
Extracellular Polysaccharide Is Not Responsible for Aluminum Tolerance of
Rhizobium leguminosarum
bv. Phaseoli CIAT899
Authors: Kingsley Mark T. , Bohlool B. Ben
Published: January 2020

Strain UHM-5, a pSym
-
Exo
-
derivative of the aluminum-tolerant
Rhizobium leguminosarum
bv. phaseoli strain CIAT899, was equally tolerant of aluminum (Al) as the parental culture. Dialyzed culture supernatants of the wild-type cells grown in YEM broth (10
9
cells ml
-1
) contained 185 μg of glucose equivalents ml
-1
whereas UHM-5 culture supernatants yielded 2 μg of glucose ml
-1
. The Exo
-
derivative and the parental strain gave essentially similar growth in medium containing from 0 to 300 μM Al, indicating that the pSym of CIAT899, and extracellular polysaccharide, were not involved in the aluminum tolerance of this strain. However, increasing the level of Al from 80 to 150 μM increased the lag phase, induced a slight killing of the inoculum, and depressed the final populations by about fivefold. Doubling the aluminum concentration from 150 to 300 μM presented a severe aluminum stress to CIAT899 and UHM-5: the inoculum level dropped 10-fold, indicating killing of the inoculum, and remained depressed for ca. 4 days before continuing to grow slowly; the final population was decreased 15-fold relative to that of cultures grown in medium containing 80 μM Al. The production by CIAT899 of other extracellular or intracellular aluminum tolerance factors was investigated in culture by using aluminum-sensitive rhizobia as stress indicators. These experiments, conducted at 80 μM Al, demonstrated that CIAT899 produced neither extracellular nor intracellular products that could alleviate toxicity for the Al-sensitive indicator rhizobia.
Differential bioavailability of soil-sorbed naphthalene to two bacterial species
Authors: Guerin W F , Boyd S A
Published: January 2020

Prediction of the fate of hydrophobic organic contaminants in soils is complicated by the competing processes of sorption and biodegradation. To test the hypothesis that sorbed naphthalene is unavailable to degradative microorganisms, we developed a simple kinetic method to examine the rates and extents of naphthalene degradation in soil-free and soil-containing systems in a comparison of two bacterial species. The method is predicated on the first-order dependence of the initial mineralization rate on the naphthalene concentration when the latter is below the Michaelis-Menten half-saturation constant (Km) for naphthalene for the organism under study. Rates and extents of mineralization were estimated by nonlinear regression analysis of data by using both a simple first-order model and a three-parameter, coupled degradation-desorption model described for the first time here. Bioavailability assays with two bacterial species (Pseudomonas putida ATCC 17484 and a gram-negative soil isolate, designated NP-Alk) gave dramatically different results. For NP-Alk, sorption limited both the rate and extent of naphthalene mineralization, in accordance with values predicted on the basis of the equilibrium aqueous-phase naphthalene concentrations. For strain 17484, both the rates and extents of naphthalene mineralization exceeded the predicted values and resulted in enhanced rates of naphthalene desorption from the soils. We conclude that there are important organism-specific properties which make generalizations regarding the bioavailability of sorbed substrates inappropriate.
Microsensor Measurements of Sulfate Reduction and Sulfide Oxidation in Compact Microbial Communities of Aerobic Biofilms
Authors: Kühl Michael , Jørgensen Bo Barker
Published: January 2020

The microzonation of O
2
respiration, H
2
S oxidation, and SO
4
2-
reduction in aerobic trickling-filter biofilms was studied by measuring concentration profiles at high spatial resolution (25 to 100 μm) with microsensors for O
2
, S
2-
, and pH. Specific reaction rates were calculated from measured concentration profiles by using a simple one-dimensional diffusion reaction model. The importance of electron acceptor and electron donor availability for the microzonation of respiratory processes and their reaction rates was investigated. Oxygen respiration was found in the upper 0.2 to 0.4 mm of the biofilm, whereas sulfate reduction occurred in deeper, anoxic parts of the biofilm. Sulfate reduction accounted for up to 50% of the total mineralization of organic carbon in the biofilms. All H
2
S produced from sulfate reduction was reoxidized by O
2
in a narrow reaction zone, and no H
2
S escaped to the overlying water. Turnover times of H
2
S and O
2
in the reaction zone were only a few seconds owing to rapid bacterial H
2
S oxidation. Anaerobic H
2
S oxidation with NO
3
-
could be induced by addition of nitrate to the medium. Total sulfate reduction rates increased when the availability of SO
4
2-
or organic substrate increased as a result of deepening of the sulfate reduction zone or an increase in the sulfate reduction intensity, respectively.
Corrosion and Electrochemical Oxidation of a Pyrite by
Thiobacillus ferrooxidans
Authors: Mustin C. , Berthelin J. , Marion P. , de Donato P.
Published: January 2020

The oxidation of a pure pyrite by
Thiobacillus ferrooxidans
is not really a constant phenomenon; it must be considered to be more like a succession of different steps which need characterization. Electrochemical studies using a combination of a platinum electrode and a specific pyrite electrode (packed-ground-pyrite electrode) revealed four steps in the bioleaching process. Each step can be identified by the electrochemical behavior (redox potentials) of pyrite, which in turn can be related to chemical (leachate content), bacterial (growth), and physical (corrosion patterns) parameters of the leaching process. A comparison of the oxidation rates of iron and sulfur indicated the nonstoichiometric bacterial oxidation of a pure pyrite in which superficial phenomena, aqueous oxidation, and deep crystal dissolution are successively involved.
Localization of Glucose Oxidase and Catalase Activities in
Aspergillus niger
Authors: Witteveen Cor F. B. , Veenhuis Marten , Visser Jaap
Published: January 2020

The subcellular localization of glucose oxidase (EC 1.1.3.4) in
Aspergillus niger
N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were demonstrated by nondenaturing polyacrylamide gel electrophoresis of crude extracts of induced and noninduced cells. Comparison of both protoplast and mycelial extracts indicated that, of two constitutive catalases, one is located outside the cell membrane whereas the other is intracellular. Parallel with the induction of glucose oxidase, two other catalases are also induced, one located intracellularly and one located extracellularly. Furthermore, lactonase (EC 3.1.1.17) activity, catalyzing the hydrolysis of glucono-δ-lactone to gluconic acid, was found to be exclusively located outside the cell membrane, indicating that gluconate formation in
A. niger
occurs extracellularly.
Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914
Authors: Macfarlane G T , Macfarlane S
Published: January 2020

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.
Glyphosate degradation by immobilized bacteria: field studies with industrial wastewater effluent
Authors: Hallas L E , Adams W J , Heitkamp M A
Published: January 2020

Immobilized bacteria have been shown in the laboratory to effectively remove glyphosate from wastewater effluent discharged from an activated sludge treatment system. Bacterial consortia in lab columns maintained a 99% glyphosate-degrading activity (GDA) at a hydraulic residence time of less than 20 min. In this study, a pilot plant (capacity, 45 liters/min) was used for a field demonstration. Initially, activated sludge was enriched for microbes with GDA during a 3-week biocarrier activation period. Wastewater effluent was then spiked with glyphosate and NH4Cl and recycled through the pilot plant column during start-up. Microbes with GDA were enhanced by maintaining the pH at less than 8 and adding yeast extract (less than 10 mg/liter). Once the consortia were stabilized, the column capacity for glyphosate removal was determined in a 60-day continuous-flow study. Waste containing 50 mg of glyphosate per liter was pumped at increasing flow rates until a steady state was reached. A microbial GDA of greater than 90% was achieved at a 10-min hydraulic residence time (144 hydraulic turnovers per day). Additional studies showed that microbes with GDA were recoverable within (i) 5 days of an acid shock and (ii) 3 days after a 21-day dormancy (low-flow, low-maintenance) mode. These results suggest that full-scale use of immobilized bacteria can be a cost-effective and dependable technique for the biotreatment of industrial wastewater.
Self-similar colony morphogenesis by gram-negative rods as the experimental model of fractal growth by a cell population
Authors: Matsuyama T , Matsushita M
Published: January 2020

The ability to form a fractal colony was shown to be common among several species of the family Enterobacteriaceae. Bacterial spreading growth in a two-dimensional field of nutrient concentration was indicated to be important for this experimental self-similar morphogenesis. As a basic analogy, the diffusion-limited aggregation model was suggested. Fractal dimensions of colonies were mostly in the range of values from 1.7 to 1.8, similar to those of the two-dimensional diffusion-limited aggregation model. Bacterial characteristics and culture conditions inducing changes in fractal patterns and growth rates were identified. The contribution of the bacterial multicellular nature to fractal morphogenesis is discussed.
Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms
Authors: Pipke R , Wagner-Döbler I , Timmis K N , Dwyer D F
Published: January 2020

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.
Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols
Authors: Kiyohara H , Hatta T , Ogawa Y , Kakuda T , Yokoyama H , Takizawa N
Published: January 2020

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.
Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10
Authors: Hill K E , Weightman A J , Fry J C
Published: January 2020

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.
Physicochemical and structural investigation of the surfaces of some anaerobic subgingival bacteria
Authors: Cowan M M , van der Mei H C , Rouxhet P G , Busscher H J
Published: January 2020

The surfaces of nine clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Peptostreptococcus micros and that of laboratory strain P. gingivalis W83 were studied by using contact angle measurements, X-ray photoelectron spectroscopy, infrared spectroscopy, microelectrophoresis of whole cells, and transmission electron microscopy of whole and sectioned cells. P. intermedia strains were hydrophilic, as judged from their small water contact angles, and had highly negative zeta potentials, consistent with the presence of a prominent ruthenium red (RR)-staining layer and fibrillar appendages which are probably partly carbohydrate. The two clinical isolates of P. gingivalis were also hydrophilic and highly negatively charged despite the presence of prominent fibrils, which usually yield less negative zeta potentials. This finding suggests that the RR-staining layer dominates the suspension characteristics of P. gingivalis and P. intermedia strains. P. gingivalis W83 had no demonstrable fibrils and a morphologically distinct RR-staining layer, and it was more hydrophobic than the two clinical isolates of P. gingivalis. P. micros isolates were hydrophobic and much less negatively charged than the other species. The A. actinomycetemcomitans strains displayed long, prominent fibrils and a very thin RR-staining layer, which resulted in high hydrophobicity but distinctly different zeta potentials for the two. Physicochemical data on microbial cell surfaces usually have clear and predictable relationships with each other. For the strains in this study that did not follow these relationships, their aberrant behavior could be explained as due to a masking effect caused by specific surface architecture. We conclude that this combined analysis provides a detailed image of subgingival bacterial surface architecture.
Influence of pH, Oxygen, and Humic Substances on Ability of Sunlight To Damage Fecal Coliforms in Waste Stabilization Pond Water
Authors: Curtis Thomas P. , Mara D. Duncan , Silva Salomao A.
Published: January 2020

Simple beaker experiments established that light damages fecal coliforms in waste stabilization ponds by an oxygen-mediated exogenous photosensitization. Wavelengths of up to 700 nm were able to damage bacteria. The ability of wavelengths of >425 nm to damage fecal coliforms was dependent on the presence of dissolved sensitizers. The sensitizers were ubiquitous in raw sewage, unaffected by sewage treatment, not derivatives of bacteriochlorophyll or chlorophyll, absorbed well in UV light, and had a slight yellowish color; they are therefore believed to be humic substances. The ability of light to damage fecal coliforms was highly sensitive to, and completely dependent on, oxygen. Scavengers of H
2
O
2
and singlet oxygen could protect the bacteria from the effects of sunlight, but scavengers of hydroxyl radicals and superoxides could not. Light-mediated damage of fecal coliforms was highly sensitive to elevated pH values, which also enabled light with wavelengths of >425 nm (in the presence of the sensitizer) to damage the bacteria. We conclude that humic substances, pH, and dissolved oxygen are important variables in the process by which light damages microorganisms in this and other environments and that these variables should be considered in future research on, and models of, the effects of light.
Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli
Authors: Barbosa M F , Beck M J , Fein J E , Potts D , Ingram L O
Published: January 2020

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.
Cloning, expression, and nucleotide sequence of a lipase gene from Pseudomonas fluorescens B52
Authors: Tan Y , Miller K J
Published: January 2020

In this study, we report the cloning and expression of lipase gene from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. Sequence analysis revealed one major open reading frame of 1,428 nucleotides that was predicted to encode a protein with a molecular weight of 50,241. The predicted enzyme was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date.
Lactococcus lactis release from calcium alginate beads
Authors: Champagne C P , Gaudy C , Poncelet D , Neufeld R J
Published: January 2020

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.
Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus
Authors: Tajana E , Fiechter A , Zimmermann W
Published: January 2020

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.
Rapid detection of members of the family Enterobacteriaceae by a monoclonal antibody
Authors: Levasseur S , Husson M O , Leitz R , Merlin F , Laurent F , Peladan F , Drocourt J L , Leclerc H , Van Hoegaerden M
Published: January 2020

Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria.
Comparison of an in vitro method and an in vivo method of Giardia excystation
Authors: Isaac-Renton J L , Shahriari H , Bowie W R
Published: January 2020

An in vitro method and an in vivo method of excystation were compared to determine the most useful method for the retrieval of Giardia duodenalis isolates. Cysts from 11 Giardia strains were used. In vitro excystation produced motile trophozoites in 16 sets, while in vivo excystation produced trophozoites in all of the 21 comparative sets of excystations. Few cultures were lost because of contamination by either method (17% of in vitro-derived trophozoites versus 23% of in vivo-derived trophozoites; P greater than 0.05). Both methods demonstrated comparable isolate retrieval rates (15% of in vitro-derived trophozoites adapting to culture compared with 29% of in vivo-derived trophozoites; P greater than 0.05), although analysis of the strains retrieved showed that two isolates were retrieved from in vitro excystation alone, compared with four from in vivo excystation. Analysis that included results of extra in vivo cultures showed that a total of nine isolates were retrieved by using this type of excystation. Despite the disadvantages of cost and labor, in vivo excystation appears to be more useful than in vitro excystation for isolate retrieval at the present time.
Use of polymerase chain reaction for detection of Listeria monocytogenes in food
Authors: Niederhauser C , Candrian U , Höfelein C , Jermini M , Bühler H P , Lüthy J
Published: January 2020

A previously described polymerase chain reaction (PCR) assay (B. Furrer, U. Candrian, C. Höfelein, and J. Lüthy, J. Appl. Bacteriol. 70:372-379, 1991) was used to analyze food for the presence of Listeria monocytogenes. Food samples were artificially contaminated to develop two procedures to detect the organism following enrichment steps. Procedure A was based on dilution of the enrichment broth followed by lysis of the bacteria and direct analysis of the lysate with PCR. With procedure A and artificially contaminated food samples, it was possible to detect fewer than 10 bacteria per 10 g of food. In procedure B, centrifugation was used to concentrate bacteria before lysis and PCR. With procedure A, 330 naturally contaminated food samples of several types were analyzed. Twenty samples were found to be positive for L. monocytogenes, which was in agreement with the classical culture technique. By using procedure B on a subset of 100 food samples, 14 were found to be positive by PCR whereas the classical culture method detected only 13. Analysis times, including enrichment steps, were 56 and 32 h with procedures A and B, respectively.
VIRTUS, a model of virus transport in unsaturated soils
Authors: Yates M V , Ouyang Y
Published: January 2020

As a result of the recently proposed mandatory groundwater disinfection requirements to inactivate viruses in potable water supplies, there has been increasing interest in virus fate and transport in the subsurface. Several models have been developed to predict the fate of viruses in groundwater, but few include transport in the unsaturated zone and all require a constant virus inactivation rate. These are serious limitations in the models, as it has been well documented that considerable virus removal occurs in the unsaturated zone and that the inactivation rate of viruses is dependent on environmental conditions. The purpose of this research was to develop a predictive model of virus fate and transport in unsaturated soils that allows the virus inactivation rate to vary on the basis of changes in soil temperature. The model was developed on the basis of the law of mass conservation of a contaminant in porous media and couples the flows of water, viruses, and heat through the soil. Model predictions were compared with measured data of virus transport in laboratory column studies and, with the exception of one point, were within the 95% confidence limits of the measured concentrations. The model should be a useful tool for anyone wishing to estimate the number of viruses entering groundwater after traveling through the soil from a contamination source. In addition, model simulations were performed to identify parameters that have a large effect on the results. This information can be used to help design experiments so that important variables are measured accurately.
Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity
Authors: Murphy R C , Stevens S E
Published: January 2020

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.
Bioremediation of soils contaminated with the herbicide 2-sec-butyl-4,6-dinitrophenol (dinoseb)
Authors: Kaake R H , Roberts D J , Stevens T O , Crawford R L , Crawford D L
Published: January 2020

A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) from contaminated soils was investigated. One soil contained dinoseb as the major contaminant, although several other hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinoseb was not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by the addition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated the consumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobic conditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbial consortium that degraded dinoseb completely, without the formation of the polymerization products seen under aerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronically contaminated soil, the natural microflora was capable of establishing anaerobic conditions and degrading dinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degrading microorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinoseb degradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates over those of uninoculated controls.