bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Expression of a
Clostridium acetobutylicum
Alcohol Dehydrogenase Gene in
Escherichia coli
Authors: Youngleson Jonathan S. , Santangelo Joseph D. , Jones David T. , Woods David R.
Published: January 2020

An alcohol dehydrogenase (ADH) gene from
Clostridium acetobutylicum
was cloned on a recombinant plasmid, pCADH100.
Escherichia coli
HB101, and an allyl alcohol-resistant mutant, HB101-adh1, containing this plasmid were unable to grow aerobically or anaerobically on agar media containing sublethal concentrations of allyl alcohol.
E. coli
HB101 and HB101-adh1 transformed with the plasmid pCADH100 produced increased levels of ethanol when grown anaerobically under alkaline conditions in the absence of nitrate. Cell extracts from aerobically and anaerobically grown
E. coli
HB101(pCADH100) and HB101-adhl(pCADH100) cells exhibited increased levels of NADP-dependent ADH activity with either ethanol or butanol as the substrate. The inability of
E. coli
HB101(pCADH100) to grow in the presence of allyl alcohol correlated with the appearance of an NADP-dependent ADH activity band on nondenaturing polyacrylamide gel electrophoresis with either ethanol or butanol as the substrate. The position of the cloned NADP-dependent ADH activity bands in
E. coli
HB101(pCADH100) cell extracts with either ethanol or butanol as the substrate coincided with the position of a single NADP-dependent ADH activity band in extracts of
C. acetobutylicum
cells.
E. coli
HB101(pCADH100) cell extracts prepared from both aerobically and anaerobically grown cells exhibited an additional protein band with an apparent
M
r
of approximately 33,000 on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis which was absent in cell extracts of
E. coli
HB101. A protein band with a similar apparent
M
r
was observed in cell extracts of
C. acetobutylicum
, and in vitro transcription and translation experiments with pCADH100 produced a major protein product with a similar apparent
M
r
.
Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus
Authors: Häggblom M M , Apajalahti J H , Salkinoja-Salonen M S
Published: January 2020

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.
Effects of Temperature and Relative Humidity on the Growth of and Enzyme Production by
Actinomucor taiwanensis
during Sufu Pehtze Preparation
Authors: Chou Cheng-Chun , Ho Frank-Ming , Tsai Chin-Shiow
Published: January 2020

The growth of and production of protease, α-amylase, α-galactosidase, and lipase by
Actinomucor taiwanensis
in relation to temperature and relative humidity during the preparation of sufu (Chinese cheese) pehtze were investigated. The incubation temperature, humidity, and cultivation time greatly affected the growth of and enzyme production by
A. taiwanensis
on tofu. It grew best at 97% humidity and 30°C. The highest yields of protease (112 U/g of dry tofu) and lipase (1,448 U/g of dry tofu) were found after 60 h of incubation at 97% humidity and 25°C. On the other hand, the highest yield of α-amylase (1,949 U/g of dry tofu) was observed after 48 h of incubation at 96 to 97% humidity and 30°C, and the highest amount of α-galactosidase (387 U/g of dry tofu) was observed at 35°C and 96% humidity after 60 h of growth. The results suggest that the temperature and humidity should be controlled at 25 to 30°C and around 97%, respectively, during the commercial preparation of sufu pehtze for better growth of and production of enzymes by
A. taiwanensis.
DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community
Authors: Holben William E. , Jansson Janet K. , Chelm Barry K. , Tiedje James M.
Published: January 2020

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded,
32
P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect
Bradyrhizobium japonicum
at densities as low as 4.3 × 10
4
cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-μg DNA sample.
Comparison of virulence factors and R plasmids of Salmonella spp. isolated from healthy and ill swine
Authors: Simmons K W , Wooley R E , Brown J
Published: January 2020

The antibiotic resistance and virulence profiles of Salmonella spp. isolated from healthy (group 1) and ill (group 2) swine were compared. Parameters studied included colicin and siderophore production; mannose-sensitive hemagglutination of erythrocytes; resistance to the lethal effect of serum complement; resistance to antibiotics; and the transmissibility of these characteristics to recipient organisms. Group 1 (19 isolates) had 14 serotypes, and group 2 (20 isolates) had 2 serotypes. Isolates from group 2 were resistant to more antibiotics and had a greater ability to hemagglutinate erythrocytes and transfer R plasmids to recipient organisms, but a lesser ability to produce siderophore than group 1. All 39 isolates resisted the lethal effects of serum complement. Colicin was produced by 1 of 19 from group 1 and 0 of 20 from group 2. A donor Escherichia coli isolated from a pig with enteritis transferred R plasmids to 62% of group 1 and 0% of group 2 Salmonella spp. when they were used as recipient organisms. A transconjugant from the mating of donor E. coli to a group 1 Salmonella spp. was further able to pass an R plasmid to recipient E. coli and salmonellae. Plasmid isolation from group 1 yielded 1 of 19 strains with a 56-megadalton plasmid, while 20 of 20 strains from group 2 contained three to five plasmids from 2.4 to 60 megadaltons in size.
Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake
Authors: Hoppe Hans-Georg , Kim Sang-Jin , Gocke Klaus
Published: January 2020

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [
3
H]leucine as a marker for the leucine pool and
l
-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (
T

r

, percent per hour) and hydrolysis rates of Leu-MCA (
H

r

, percent per hour), as well as the quotient
T

r

/
H

r

, reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient
T

r

/
H

r

is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.
Isolation and Characterization of Marine Caulobacters and Assessment of Their Potential for Genetic Experimentation
Authors: Anast Nick , Smit John
Published: January 2020

A total of 25 marine caulobacters were isolated from littoral marine sources. Several aspects of their physiology and morphology were examined, as well as their suitability for genetic manipulation in laboratory cultivation. Caulobacters were readily isolated from all sources, including samples from areas containing pollution-related organic compounds. All isolates grew best in media containing seawater, but eight strains grew if sea salts were replaced with NaCl alone, three strains grew at 1/10 the normal sea salt concentration, and one isolate grew, albeit poorly, in freshwater medium. Of the marine isolates, 12 strains grew under anaerobic conditions, indicating that some caulobacters are not obligately aerobic bacteria, as they are currently categorized. Although some freshwater caulobacters are able to oxidize manganese, this capability was not found in these marine caulobacters. Of the marine isolates, 10 strains were resistant to mercury chloride concentrations 10- to 20-fold greater than that tolerated by sensitive bacteria. However, a mercury reductase gene comparable with that found in R100-type plasmids was not detected by gene hybridization. With respect to the potential for genetic experimentation, most strains grew rapidly (3- to 4-h generation time at 30°C), producing colonies on solid media in 2 to 3 days. The isolates were sensitive to antibiotics commonly used in recombinant DNA experiments, and spontaneous drug-resistant mutants were selectable. Conjugal transfer of plasmids from
Escherichia coli
to several marine caulobacters was demonstrated for four broad-host-range plasmid incompatibility groups, by using both self-transmissible plasmids and cloning-oriented plasmids that require a helper plasmid. Conjugal transfer of broad-host-range plasmids between freshwater and marine caulobacters was also demonstrated in both directions. Native plasmids of approximately 100- to 150-kilobase sizes were found in 2 of the 25 marine
Caulobacter
strains. The native plasmids were present in relatively high copy number and appeared stable in laboratory culture. In short, the marine caulobacters appeared appropriate as candidates for genetic manipulation and the expression of selected genes in the marine environment.
Fermentation of methanol in the sheep rumen
Authors: Pol A , Demeyer D I
Published: January 2020

Sheep fed a hay-concentrate diet were adapted to pectin administration and ruminal infusion of methanol. Both treatments resulted in a strong increase in the rate of methanogenesis from methanol. Quantitative data show that methanol was exclusively converted into methane. Treatments did not influence ruminal volatile fatty acid percentages.
Isolation and Characterization of Methanogenic Bacteria from Landfills
Authors: Fielding Elizabeth R. , Archer David B. , de Macario Everly Conway , Macario Alberto J. L.
Published: January 2020

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of
Methanobacterium formicicum, Methanosarcina barkeri
, several different immunotypes of
Methanobacterium bryantii
, and a coccoid methanogen distinct from the reference immunotypes were identified.
Aflatoxin contamination of some common drug plants
Authors: Roy A K , Sinha K K , Chourasia H K
Published: January 2020

This paper deals with the natural occurrence of aflatoxins in some common drug plants collected from storehouses in Bihar, India. Of 15 samples analyzed, 14 were aflatoxin positive. The highest level of aflatoxin contamination was detected in the seeds of Piper nigrum (1.20 micrograms/g), followed by the level detected in the seeds of Mucuna prurita (1.16 micrograms/g), and the lowest level was detected in the bark of Acacia catechu (0.09 micrograms/g). Of 158 isolates of Aspergillus flavus obtained from as many samples of drug plants, 49 were found to be toxigenic. The amount of aflatoxin B1 elaborated by the toxigenic isolates was in the range of 0.86 to 5.24 micrograms/ml of culture filtrate.
Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in
Rhodococcus rhodochrous
J1
Authors: Mathew Caluwadewa Deepal , Nagasawa Toru , Kobayashi Michihiko , Yamada Hideaki
Published: January 2020

The nitrilase which occurs abundantly in cells of
Rhodococcus rhodochrous
J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.
Colony Dimorphism in
Bradyrhizobium
Strains
Authors: Sylvester-Bradley Rosemary , Thornton Philip , Jones Peter
Published: January 2020

Ten isolates of
Bradyrhizobium
spp. which form two colony types were studied; the isolates originated from a range of legume species. The two colony types differed in the amount of gum formed or size or both, depending on the strain. Whole 7-day-old colonies of each type were subcultured to determine the proportion of cells which had changed to the other type. An iterative computerized procedure was used to determine the rate of switching per generation between the two types and to predict proportions reached at equilibrium for each strain. The predicted proportions of the wetter (more gummy) or larger colony type at equilibrium differed significantly between strains, ranging from 0.9999 (strain CIAT 2383) to 0.0216 (strain CIAT 2469), because some strains switched faster from dry to wet (or small to large) and others switched faster from wet to dry (or large to small). Predicted equilibrium was reached after about 140 generations in strain USDA 76. In all but one strain (CIAT 3030) the growth rate of the wetter colony type was greater than or similar to that of the drier type. The mean difference in generation time between the two colony types was 0.37 h. Doubling times calculated for either colony type after 7 days of growth on the agar surface ranged from 6.0 to 7.3 h. The formation of two persistent colony types by one strain (clonal or colony dimorphism) may be a common phenomenon among
Bradyrhizobium
strains.
New enrichment method for isolation of pathogenic Yersinia enterocolitica serogroup O:3 from pork
Authors: Wauters G , Goossens V , Janssens M , Vandepitte J
Published: January 2020

A new enrichment medium for the recovery of pathogenic Yersinia enterocolitica serogroup O:3 from naturally infected meat products based on three selective agents, Irgasan, ticarcillin, and potassium chlorate (ITC), was compared with several other one- or two-step enrichments. Y. enterocolitica serogroup O:3 was recovered from 96.5% of 29 pork tongues, 24% of 50 ground pork samples, 16% of 25 masseter muscle samples, and 61% of tonsils. ITC was by far the most sensitive method for the recovery of Y. enterocolitica O:3, especially from ground meat and masseter muscles, while cold and two-step enrichments yielded better results for nonpathogenic strains. Plating of ITC enrichments onto SS-deoxycholate-calcium agar gave overall better results than plating onto cefsulodin-Irgasan-novobiocin agar for serogroup O:3.
Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens
Authors: Stack R J
Published: January 2020

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.
Ethanol-Induced Leakage in
Saccharomyces cerevisiae
: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity
Authors: Salgueiro Sancha P. , Sá-Correia Isabel , Novais Júlio M.
Published: January 2020

Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of
Saccharomyces cerevisiae
. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration:
k

e

X

=
k

e

Xm

e

E (X-X

m

)

, where
k

e

X

and
k

e

Xm

are the efflux rate constants, respectively, in the presence of a concentration
X
of ethanol or the minimal concentration of ethanol,
X

m

, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol.
E
is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (
X

m

) was lower. The exponential constants for the enhancement of the rate of leakage (
E
) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts,
Kluyveromyces marxianus, Saccharomyces cerevisiae
, and
Saccharomyces bayanus
. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by
S. cerevisiae
and
S. bayanus
. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.
Mineralization of phenanthrene by a Mycobacterium sp
Authors: Guerin W F , Jones G E
Published: January 2020

A Mycobacterium sp., designated strain BG1, able to utilize the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon and energy source was isolated from estuarine sediment following enrichment with the hydrocarbon. Unlike other phenanthrene degraders, this bacterium degraded phenanthrene via 1-hydroxy-2-naphthoic acid without accumulating this or other aromatic intermediates, as shown by high-performance liquid chromatography. Degradation proceeded via meta cleavage of protocatechuic acid. Different nonionic surfactants (Tween compounds) solubilized the phenanthrene to different degrees and enhanced phenanthrene utilization. The order of enhancement, however, did not correlate perfectly with increased solubility, suggesting physiological as well as physicochemical effects of the surfactants. Plasmids of approximately 21, 58, and 77 megadaltons were detected in cells grown with phenanthrene but not in those which, after growth on nutrient media, lost the phenanthrene-degrading phenotype. Given that plasmid-mediated degradations of aromatic hydrocarbons generally occur via meta cleavages, it is of interest that the addition of pyruvate, a product of meta cleavage, supported rapid mineralization of phenanthrene in broth culture; succinate, a product of ortho cleavage, supported growth but completely repressed the utilization of phenanthrene. The involvement of plasmids may have given rise to the unusual degradation pattern that was observed.
Carbon metabolism of the cryptoendolithic microbiota from the Antarctic desert
Authors: Vestal J R
Published: January 2020

The carbon metabolism of the cryptoendolithic microbiota of sandstones from the Ross Desert of Antarctica was studied in situ and in vitro. Organic and inorganic carbon compounds were metabolized by the microbiota, with bicarbonate incorporation into community lipids occurring primarily in the light. Light intensity affected the photometabolism of carbon with a photosynthesis-intensity response optimum at about 200 to 300 micromoles of photons per m2 per s. Photosynthesis was also affected by temperature, with a minimum activity at -5 degrees C, an optimum activity at 15 degrees C, and complete inhibition at 35 degrees C, indicating that the cryptoendolithic community was psychrophilic. The primary source of CO2 for photosynthesis in situ was the atmosphere. CO2 may also be photometabolized by using the carbon produced from respiration within the endolithic community. Photosynthesis occurred maximally when the microbiota was wet with liquid water and to a lesser extent in a humid atmosphere. This simple microbial community, therefore, exists under extremes of water, light, and temperature stress which affect and control its metabolism.
Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology
Authors: Stahl D A , Flesher B , Mansfield H R , Montgomery L
Published: January 2020

To address the long-standing need for more precise descriptions of natural microbial ecosystems, 16S rRNAs were used to track certain species and phylogenetically coherent groups of microorganisms in their natural setting without culturing. Species- and group-specific 16S rRNA-targeted oligonucleotide hybridization probes were developed to enumerate various strains of Bacteroides succinogenes and Lachnospira multiparus-like organisms in the bovine rumen before, during, and after perturbation of that ecosystem by the addition of the ionophore antibiotic monensin. Based on probe hybridization, relative numbers of L. multiparus-like organisms were depressed about 2-fold during monensin addition and demonstrated a transient 5- to 10-fold increase immediately after removal of the antibiotic from the diet. The most pronounced population changes were observed among different strains of B. succinogenes, as evaluated by three hybridization probes. One probe hybridized to all strains, whereas the other two identified genetically distinct groups represented by strains isolated from the rumen and from the ceca of nonruminants. The rumen-type strains predominated on most days (ca. 0.2 to 0.8% of total ribosome numbers). Their proportion transiently increased about fivefold immediately after the addition of monensin to the feed and then transiently fell below the average premonensin level. During this time (ca. 2 weeks after monensin addition) the cecal type predominated (ca. 0.1 to 0.2%). Cultural enumeration of B. succinogenes on nonselective agar and by observing clearings in cellulose agar media were largely unsuccessful due to the low number of organisms present and the predominance of other cellulolytic species.(ABSTRACT TRUNCATED AT 250 WORDS)
Ferulic Acid Esterase Activity from
Schizophyllum commune
Authors: MacKenzie C. Roger , Bilous Doris
Published: January 2020

Schizophyllum commune
produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase,
p
-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.
Microbial degradation of acenaphthene and naphthalene under denitrification conditions in soil-water systems
Authors: Mihelcic J R , Luthy R G
Published: January 2020

This study examined the microbial degradation of acenaphthene and naphthalene under denitrification conditions at soil-to-water ratios of 1:25 and 1:50 with soil containing approximately 10(5) denitrifying organisms per g of soil. Under nitrate-excess conditions, both acenaphthene and naphthalene were degraded from initial aqueous-phase concentrations of about 1 and several mg/liter respectively, to nondetectable levels (less than 0.01 mg/liter) in less than 9 weeks. Acclimation periods of 12 to 36 days were observed prior to the onset of microbial degradation in tests with soil not previously exposed to polycyclic aromatic hydrocarbon (PAH) compounds, whereas acclimation periods were absent in tests with soil reserved from prior PAH degradation tests. It was judged that the apparent acclimation period resulted from the time required for a small population of organisms capable of PAH degradation to attain sufficient densities to exhibit detectable PAH reduction, rather than being a result of enzyme induction, mutation, or use of preferential substrate. About 0.9% of the naturally occurring soil organic carbon could be mineralized under denitrification conditions, and this accounted for the greater proportion of the nitrate depletion. Mineralization of the labile fraction of the soil organic carbon via microbial denitrification occurred without an observed acclimation period and was rapid compared with PAH degradation. Under nitrate-limiting conditions the PAH compounds were stable owing to the depletion of nitrate via the more rapid process of soil organic carbon mineralization. Soil sorption tests showed at the initiation of a test that the total mass of PAH compound was divided in comparable proportions between solute in the aqueous phase and solute sorbed on the solid phase. The microbial degradation of the PAH compound depends on the interrelationships between (i) the desorption kinetics and the reversibility of desorption of sorbed compound from the soil, (ii) the concentration of PAH-degrading microorganisms, and (iii) the competing reaction for nitrate utilization via mineralization of the labile fraction of naturally occurring soil organic carbon.
High Pressure Enhances the Growth Rate of the Thermophilic Archaebacterium
Methanococcus thermolithotrophicus
without Extending Its Temperature Range
Authors: Bernhardt G. , Jaenicke R. , Lüdemann H.-D. , König H. , Stetter K. O.
Published: January 2020

Temperature and hydrostatic pressure are essential in determining the assemblage of species in their specific biotopes. To evaluate the effect of high pressure on the range of viability of thermophiles, the pressure and temperature dependence of the growth of the methanogenic archaebacterium
Methanococcus thermolithotrophicus
was investigated. High pressure up to 50 MPa enhanced the growth rate without extending the temperature range of viability. The optimum temperature remained unaltered (65°C). Beyond 50 MPa, cell lysis predominated over cell proliferation. Destabilization was also observed at temperatures below and above the optimum growth temperature (<60°C, ≥70°C) and at low substrate concentrations.
Isolation and purification of a hemorrhagic factor (wortmannin) from Fusarium oxysporum (N17B)
Authors: Abbas H K , Mirocha C J
Published: January 2020

An isolate of Fusarium oxysporum Schlecht, emend. Synd. et Hans. N17B isolated from a grassy area in Lakselv, Norway (Arctic region) produced a toxin in culture when grown on rice in the laboratory. This new toxin, which was given the trivial name of H-1 (indicating hemorrhagic factor), caused toxic effects in rats, including food refusal, weight loss, hemorrhage in the stomach, intestines, heart, and thymus, and finally death. The UV spectrum of H-1 showed 210, 254, and 292 nm as absorption maxima. The infrared spectrum showed carbonyl groups at 1,675 and 1,750 cm-1 and an ether group at 1,215 cm-1. H-1 does not fluoresce under short- or long-wavelength UV light and exists as fluffy, white crystals that turn yellow when subjected to basic reagents such as ammonium hydroxide or tetraethylenepentamine. Elemental and accurate mass determinations in both electron impact and positive chemical ionization indicate an empirical formula of C23H24O8. Its mass spectra (electron impact, chemical ionization, and fast atom bombardment [FAB]) show a molecular ion of 428 and major fragments at m/z+ 386, 368, 355, and 295. H-1 was found to be identical to the antibiotic called wortmannin which is produced by Penicillium wortmannii and Myrothecium roridum. This is the first report of the synthesis of wortmannin by species of the genus Fusarium.
Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp
Authors: Nagayama S , Kawamura O , Ohtani K , Ryu J C , Latus D , Sudheim L , Ueno Y
Published: January 2020

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.
Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance
Authors: Casiano-Colón A , Marquis R E
Published: January 2020

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.
Evaluation of the Role of Syringomycin in Plant Pathogenesis by Using Tn
5
Mutants of
Pseudomonas syringae
pv.
syringae
Defective in Syringomycin Production
Authors: Xu Guo-Wei , Gross Dennis C.
Published: January 2020

Syringomycin is a necrosis-inducing phytotoxin produced by
Pseudomonas syringae
pv.
syringae.
To determine whether syringomycin production is a determinant in virulence or pathogenicity, we isolated nontoxigenic (Tox
−
) Tn
5
-containing mutants and then quantitatively evaluated them for the ability to multiply and cause disease in immature sweet-cherry fruits. Transposon Tn
5
was delivered to Tox
+
strain B301D-R by using the suicide vector, pGS9, and the resultant kanamycin-resistant (Km
r
) colonies were screened for changes in syringomycin production by testing for antibiosis against
Geotrichum candidum.
Southern blot analysis of
Kpn
I-and
Eco
RI-digested DNA showed that 15 (0.3%) Tox
−
mutants were isolated which had Tn
5
inserted into 1 of 14 distinct loci. Phenotypic characterization of the Tox
−
mutants identified three major groups, which were differentiated by pathogenicity and ability to cause a tobacco hypersensitive reaction (HR). The eight strains in group A were pathogenic (Path
+
) in cherry fruit assays, but the disease index was 17 to 66% lower (significant at
P
= 0.01) than for the parental Tox
+
strain, B301D-R. The population dynamics of group A strains W4S770 and W4S116 in cherry fruits were, however, indistinguishable from that of strain B301D-R. The remaining seven Tox
−
strains were nonpathogenic; group B strain W4S2545 (Path
−
HR
+
) and group C strain W4S468 (Path
−
HR
−
) developed significantly lower populations (10
5
to 10
7
CFU per cherry fruit) 3 days after inoculation than strain B301D-R did (nearly 10
9
CFU per fruit). The data indicate that syringomycin is not essential for pathogenicity, but contributes significantly to virulence.
Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria
Authors: Dwyer Daryl F. , Weeg-Aerssens Els , Shelton Daniel R. , Tiedje James M.
Published: January 2020

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium
Methanospirillum hungatei
PM-1 or
Desulfovibrio
sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H
2
uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (Δ
G
′) and the butyrate degradation rate (
v
) were greater for NSF-2-
Desulfovibrio
sp. strain PS-1 (Δ
G
′ = −17.0 kJ/mol;
v
= 0.20 mM/h) than for NSF-2-
M. hungatei
PM-1 (Δ
G
′ = −3.8 kJ/mol,
v
= 0.12 mM/h). The Δ
G
′ value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable Δ
G
′ resulted from the close coupling of the rates of butyrate and H
2
oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when
Desulfovibrio
sp. strain PS-1 was added as a new H
2
sink. When the specific activity of H
2
consumption was increased by adding higher densities of the
Desulfovibrio
sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H
2
pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H
2
consumption, not the type of H
2
consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical
V
max
.
Plugging of a Model Rock System by Using Starved Bacteria
Authors: MacLeod F. A. , Lappin-Scott H. M. , Costerton J. W.
Published: January 2020

The effects of starvation on bacterial penetration through artificial rock cores were examined.
Klebsiella pneumoniae
was starved in a simple salts solution for a duration of up to 4 weeks. These cell suspensions were injected into sintered glass bead cores, and the resulting reductions in core permeabilities were recorded. Vegetative cell cultures of
K. pneumoniae
grown in a sodium citrate medium were injected into other, similar cores, and the reductions in core permeabilities were recorded. The starved cell suspensions did not completely block the core pores, whereas the vegetative cultures reduced core permeability to less than 1%. Scanning electron microscopy of core sections infiltrated with either vegetative or starved cells showed that the former produced shallow “skin” plugs and copious amounts of glycocalyx at the inlet face, whereas the latter produced very little glycocalyx and the cells were distributed evenly throughout the length of the core. The use of a DNA assay to produce a cell distribution profile showed that, compared with the vegetative cells, starved bacteria were able to penetrate deeper into the cores. This was due to the smaller size of the cells and the reduction in biofilm production. This ability of starved bacteria to penetrate further into cores than the normal-size vegetative cells can be usefully applied to selective plugging for enhanced oil recovery. To further test the suitability of starved cells for use in selective plugging, the activities of starved cells present within cores were monitored before and after nutrient stimulation. Our data indicate that with nutrient stimulation, the starved cells lose their metabolic dormancy and produce reductions in core permeability due to cell growth and polymer production.
Nutrient Resuscitation and Growth of Starved Cells in Sandstone Cores: a Novel Approach to Enhanced Oil Recovery
Authors: Lappin-Scott H. M. , Cusack F. , Costerton J. W.
Published: January 2020

Klebsiella pneumoniae
, which was reduced in size (0.25 by 0.5 μm) by carbon deprivation, was injected into a series of sandstone cores and subjected to separate treatments. Scanning electron microscopy of 400-mD cores showed these small starved cells in nearly every core section. The cells were a mixture of small rods and cocci with little or no biofilm production. Continuous or dose stimulation with sodium citrate allowed the cells to grow throughout the sandstone and completely plug the length of the core. The resuscitated cells were larger than the starved cells (up to 1.7 μm) and were encased in glycocalyx. Scanning electron microscopic results of resuscitation in situ with half-strength brain heart infusion broth showed that a shallow “skin” plug of cells formed at the core inlet and that fewer cells were located in the lower sections. Starved cells also penetrated 200-mD cores and were successfully resuscitated in situ with sodium citrate, so that the entire core was plugged. Nutrient resuscitation of injected starved cells to produce full-size cells which grow and block the rock pores may be successfully applied to selective plugging and may effectively increase oil recovery.
Extraction from Natural Planktonic Microorganisms of DNA Suitable for Molecular Biological Studies
Authors: Fuhrman Jed A. , Comeau Dorothy E. , Hagström Åke , Chan Amy M.
Published: January 2020

We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-μm-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100°C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease
Sau
3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.
Sources of Variability in the Measurement of Fungal Spore Yields
Authors: Smith C. S. , Slade S. J. , Nordheim E. V. , Cascino J. J. , Harris R. F. , Andrews J. H.
Published: January 2020

Variability in the production of fungal spores and in the measurement of spore yields was investigated in four species of fungi:
Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides
, and
Acremonium strictum
. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean ± 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.
A method to assess the bacterial content of refrigerated meat
Authors: Perez de Castro B , Asensio M A , Sanz B , Ordoñez J A
Published: January 2020

A new method has been developed to estimate the levels of gram-negative bacteria on refrigerated meat. The method is based on the aminopeptidase activity of these bacteria, which cleaves L-alanine-p-nitroanilide to yield p-nitroaniline, which is easily determined spectrophotometrically. This method allows the determination of levels around 10(6) to 10(7) CFU cm-2 in about 3 h. Because of the yellow color of p-nitroaniline, bacterial loads around 10(7) CFU cm-2 develop a color intense enough to be detected with the naked eye.
Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity
Authors: Nielsen P V , Beuchat L R , Frisvad J C
Published: January 2020

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.
An Acidophilic and a Neutrophilic
Nitrobacter
Strain Isolated from the Numerically Predominant Nitrite-Oxidizing Population of an Acid Forest Soil
Authors: Hankinson T. R. , Schmidt E. L.
Published: January 2020

Two physiologically and serologically distinct strains of chemoautotrophic nitrite-oxidizing bacteria were isolated as numerically predominant members of the nitrite-oxidizer population of an undisturbed forest soil with a pH range of 4.3 to 5.2. One isolate responded as a neutrophile, characteristic of the family
Nitrobacteraceae,
and cross-reacted strongly with fluorescent antibody to
Nitrobacter
strain Engel. The second isolate responded as an acidophile in pure culture, demonstrated maximal nitrite oxidation activity at pH 5.5, and had a pH tolerance range of pH 4.1 to 7.2. Nitrite oxidase in whole cells of the acidophile sustained activity to at least pH 3.5. Cell morphology of both strains typified the genus
Nitrobacter
in all respects when cultured at pH 7. However, under more acidic conditions the acidophile tended to elongate and at times appeared to branch. These data provide the first evidence for the existence of an acidophilic chemoautotrophic nitrifying bacterium. Isolation of the neutrophilic
Nitrobacter
strain reported here complements the earlier isolation of a neutrophilic
Nitrosospira
strain to provide further evidence of a prominent acid-intolerant population of chemoautotrophic nitrifiers in this acid forest soil.