bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological and Morphological Changes Induced by Nutrient Limitation of
Pseudomonas fluorescens
378 in Continuous Culture
Authors: Persson Anders , Molin Göran , Weibull Claes
Published: January 2020

Pseudomonas fluorescens
378 was studied in continuous culture at a dilution rate of 0.05 or 0.15 h
−1
and under a limitation of carbon/energy, nitrogen, phosphorus, iron(III), or oxygen. Cultures were examined for nutritional consumption, production of biosurfactant AP-6 and lipase, and electron microscopy morphology. Morphological features were lysis and plasmolysis of the cells, vacuoles in the cells, granules in cell nuclei, and DNA coagulation during transmission electron microscopy preparation. Biosurfactant and lipase production were lost after 8 to 15 retention times, but under iron limitation and at low dilution rate they were maintained for more than 30 retention times. Consumption of nutrients varied between different cultures. Between 2.4 and 6.0 g of succinic acid per g (dry weight) was consumed; the highest value was obtained under phosphorus limitation. The uptake of nitrogen was mostly about 0.16 g/g (dry weight), and that of phosphorus varied between 13 and 58 mg/g (dry weight). Phosphorus-limited cells reduced their phosphorus consumption by at least 50% compared with other limitations. Cell morphology varied among different cultures. Up to 25% cell lysis occurred at the higher dilution rate. The frequencies of plasmolysis varied between 0 and 85%. Granules in nuclei were found in 65 to 100% of the cells. Vacuoles appeared mostly in low numbers, but at the lower dilution rate under phosphorus or iron limitation the frequencies increased to between 25 and 85%. At high dilution rate, the DNA coagulated in 30 to 70% of the cells. Multivariate data analysis demonstrated a general difference between the two tested dilution rates; i.e., both nutritional and morphological features differed more between the two tested dilution rates than between the different limitations. Cultures at the lower dilution rate changed more with time; this was especially pronounced for phosphorus or iron limitation. The data analysis also showed a correlation between plasmolysis or vacuoles in the cells and an increased carbon uptake under phosphorus limitation.
Reduction of Cupric Ions with Elemental Sulfur by
Thiobacillus ferrooxidans
Authors: Sugio Tsuyoshi , Tsujita Yoshihiko , Inagaki Kenji , Tano Tatsuo
Published: January 2020

In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu
2+
) was reduced enzymatically with elemental sulfur (S
0
) by washed intact cells of
Thiobacillus ferrooxidans
AP19-3 to give cuprous ion (Cu
+
). The rate of Cu
2+
reduction was proportional to the concentrations of S
0
and Cu
2+
added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70°C. The activity of Cu
2+
reduction with S
0
by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as α,α′-dipyridyl, 4,5-dihydroxy-
m
-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(
N, N
-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S
0
with Fe
3+
or Mo
6+
as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu
2+
by S
0
, and the Michaelis constant of SFORase for Cu
2+
was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe
3+
and Mo
6+
but also Cu
2+
.
Acetic Acid Bacterial Biota of the Pink Sugar Cane Mealybug,
Saccharococcus sacchari
, and Its Environs
Authors: Ashbolt Nicholas J. , Inkerman Peter A.
Published: January 2020

Saccharococcus sacchari
is the primary colonizer of the developing “sterile” tissue between the leaf sheath and stem of sugar cane. The honeydew secreted by the mealybugs is acidic (about pH 3) and supports an atypical epiphytic microbiota dominated by acetobacter-like bacteria and acidophilic yeast species. However,
Erwinia
and
Leuconostoc
species predominate within the leaf sheath pocket region when the mealybugs die out. The unidentified acetobacters were readily isolated from
S. sacchari
throughout its life cycle and from other genera of mealybugs on sugar cane and various other plants, both above and below ground. No other insect present on sugar cane was a significant vector of acetic acid bacteria. The major factors restricting microbial diversity within the environs of mealybugs were considered to be yeast activity along with bacterial production of acetic acid, ketogluconic acids, and gamma-pyrones, in association with their lowering of pH. The microbial products may aid in suppressing the attack by the parasitic mold
Aspergillus parasiticus
on mealybugs but could act as attractants for the predatory fruit fly
Cacoxenus perspicax.
Biological Control of Damping-Off of Alfalfa Seedlings with
Bacillus cereus
UW85
Authors: Handelsman Jo , Raffel Sandra , Mester Ellen H. , Wunderlich Lynn , Grau Craig R.
Published: January 2020

We explored the potential of biological control of alfalfa (
Medicago sativa
L.) seedling damping-off caused by
Phytophthora megasperma
f. sp.
medicaginis
by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with
P. megasperma
f. sp.
medicaginis
zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient
P. megasperma
f. sp.
medicaginis
zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as
Bacillus cereus
and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with
P. megasperma
f. sp.
medicaginis
, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.
Inhibition of the Fermentation of Propionate to Methane by Hydrogen, Acetate, and Propionate
Authors: Fukuzaki Satoshi , Nishio Naomichi , Shobayashi Manabu , Nagai Shiro
Published: January 2020

Inhibition of the fermentation of propionate to methane and carbon dioxide by hydrogen, acetate, and propionate was analyzed with a mesophilic propionate-acclimatized sludge that consisted of numerous flocs (size, 150 to 300 μm). The acclimatized sludge could convert propionate to methane and carbon dioxide stoichiometrically without accumulating hydrogen and acetate in a propionate-minimal medium. Inhibition of propionate utilization by propionate could be analyzed by a second-order substrate inhibition model (shown below) given that the substrate saturation constant,
K

s

, was 15.9 μM; the substrate inhibition constant,
K

i

, was 0.79 mM; and the maximum specific rate of propionate utilization,
q

m

, was 2.15 mmol/g of mixed-liquor volatile suspended solids (MLVSS) per day:
q

s

=
q

m

S
/[
K

s

+
S
+ (
S
2
/
K

i

)], where
q

s

is the specific rate of propionate utilization and
S
is the initial concentration of undissociated propionic acid. For inhibition by hydrogen and acetate to propionate utilization, a noncompetitive product inhibition model was used:
q

s

=
q

m

/[1 + (
P/K

p

)
n
], where
P
is the initial concentration of hydrogen or undissociated acetic acid and
K

p

is the inhibition constant. Kinetic analysis gave, for hydrogen inhibition,
K

p
(H
2
)

= 0.11 atm (= 11.1 kPa, 71.5 μM),
q

m

= 2.40 mmol/g of MLVSS per day, and
n
= 1.51 and, for acetate inhibition,
K

p
(HAc)

= 48.6 μM,
q

m

= 1.85 mmol/g of MLVSS per day, and
n
= 0.96. It could be concluded that the increase in undissociated propionic acid concentration was a key factor in inhibition of propionate utilization and that hydrogen and acetate cooperatively inhibited propionate degradation, suggesting that hydrogenotrophic and acetoclastic methanogens might play an important role in enhancing propionate degradation to methane and carbon dioxide.
Radioimmunoassay of nivalenol in barley
Authors: Teshima R , Hirai K , Sato M , Ikebuchi H , Ichinoe M , Terao T
Published: January 2020

Antibodies against nivalenol (NIV) tetraacetate (Tetra-Ac-NIV) were prepared by immunizing rabbits with a hemisuccinate derivative of 8-hydroxy-3,4,7,15-tetraacetyl-12, 13-epoxytrichothece-9-en conjugated to bovine serum albumin. A radioimmunoassay system with one of these sera was developed to measure NIV contamination in barley. The detection limit for Tetra-Ac-NIV was about 0.5 ng/ml. The relative cross-reactivities of the antiserum with Tetra-Ac-NIV, acetyl T-2 toxin, and scirpenol triacetate, which were determined by the competitive radioimmunoassay, were 1, 0.78, and 0.56, respectively. Other derivatives showed no cross-reactivity. For the determination of NIV in a barley sample, NIV was extracted from the sample with acetonitrile-water (7:3), defatted with hexane, and then acetylated with acetic anhydride to form Tetra-Ac-NIV. The reaction mixture was loaded onto a C18 cartridge to remove excess reagents and impurities. Tetra-Ac-NIV was eluted from the cartridge with 50% methanol in water, and the eluate was subjected to radioimmunoassay. Analysis of six naturally contaminated barley samples for NIV revealed that radioimmunoassay results agreed well with gas chromatographic analyses.
Problems associated with identification of Legionella species from the environment and isolation of six possible new species
Authors: Wilkinson I J , Sangster N , Ratcliff R M , Mugg P A , Davos D E , Lanser J A
Published: January 2020

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.
Toxicity of Irradiated Media for
Xenorhabdus
spp
Authors: Xu Jimin , Hurlbert Ronald E.
Published: January 2020

Bacterial isolates of the genus
Xenorhabdus
were shown to be extremely sensitive to photoproducts produced in a number of common media irradiated by fluorescent light. Two forms of toxic oxygen, hydrogen peroxide and superoxide radical, were produced in the media upon exposure to fluorescent light. The addition of pyruvate or catalase to the irradiated media eliminated the toxicity. The poor plating efficiencies previously reported for
Xenorhabdus
spp. are likely due to the uncontrolled exposure of media to ambient lighting.
Use of Robotics To Transfer Cultures on Agar-Containing Media to Fresh Media
Authors: Brewer D. , Schwartzentruber K. , Taylor A.
Published: January 2020

The automation of transfer of microorganisms to fresh growth medium is described. The system was used to transfer our collection of about 1,500 bacteria and fungi without detectable contamination in a total time of 125 h.
A Translation Fusion Product of Two Different Insecticidal Crystal Protein Genes of
Bacillus thuringiensis
Exhibits an Enlarged Insecticidal Spectrum
Authors: Honée Guy , Vriezen Wim , Visser Bert
Published: January 2020

Two truncated
Bacillus thuringiensis
crystal protein genes, belonging to the classes
cryIA(b)
and
cryIC
and both coding for insecticidal N-terminal fragments of the corresponding crystal proteins, were translationally fused. Expression of the gene fusion in
Escherichia coli
showed a biologically active protein with a toxicity spectrum that overlapped those of both contributing crystal proteins.
Microbial degradation of trichloroethylene in the rhizosphere: potential application to biological remediation of waste sites
Authors: Walton B T , Anderson T A
Published: January 2020

The possibility that vegetation may be used to actively promote microbial restoration of chemically contaminated soils was tested by using rhizosphere and nonvegetated soils collected from a trichloroethylene (TCE)-contaminated field site. Biomass determinations, disappearance of TCE from the headspace of spiked soil slurries, and mineralization of [14C]TCE to 14CO2 all showed that microbial activity is greater in rhizosphere soils and that TCE degradation occurs faster in the rhizosphere than in the edaphosphere. Thus, vegetation may be an important variable in the biological restoration of surface and near-surface soils.
Isolation and characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis
Authors: Mueller J G , Chapman P J , Blattmann B O , Pritchard P H
Published: January 2020

A soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth was purified from a seven-member bacterial community previously isolated from a creosote waste site for its ability to degrade polycyclic aromatic hydrocarbons. By standard bacteriological methods, this bacterium was characterized taxonomically as a strain of Pseudomonas paucimobilis and was designated strain EPA505. Utilization of fluoranthene by strain EPA 505 was demonstrated by increase in bacterial biomass, decrease in aqueous fluoranthene concentration, and transient formation of transformation products in liquid cultures where fluoranthene was supplied as the sole carbon source. Resting cells grown in complex medium showed activity toward anthraquinone, benzo[b]fluorene, biphenyl, chrysene, and pyrene as demonstrated by the disappearance of parent compounds or changes in their UV absorption spectra. Fluoranthene-grown resting cells were active against these compound as well as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and phenanthrene. These studies demonstrate that organic compounds not previously reported to serve as growth substrates can be utilized by axenic cultures of microorganisms. Such organisms may possess novel degradative systems that are active toward other compounds whose biological degradation has been limited because of inherent structural considerations or because of low aqueous solubility.
Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments
Authors: Zhang X , Wiegel J
Published: January 2020

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.
Degradation of halogenated aliphatic compounds by the ammonia- oxidizing bacterium Nitrosomonas europaea
Authors: Vannelli T , Logan M , Arciero D M , Hooper A B
Published: January 2020

Suspensions of Nitrosomonas europaea catalyzed the ammonia-stimulated aerobic transformation of the halogenated aliphatic compounds dichloromethane, dibromomethane, trichloromethane (chloroform), bromoethane, 1,2-dibromoethane (ethylene dibromide), 1,1,2-trichloroethane, 1,1,1-trichloroethane, monochloroethylene (vinyl chloride), gem-dichloroethylene, cis- and trans-dichloroethylene, cis-dibromoethylene, trichloroethylene, and 1,2,3-trichloropropane, Tetrachloromethane (carbon tetrachloride), tetrachloroethylene (perchloroethylene), and trans-dibromoethylene were not degraded.
Dechlorination of Chloroform by
Methanosarcina
Strains
Authors: Mikesell Mark D. , Boyd Stephen A.
Published: January 2020

Dehalogenation of carbon tetrachloride, chloroform, and bromoform in pure cultures of
Methanosarcina
sp. strain DCM and
Methanosarcina mazei
S6 was demonstrated. The initial dechlorination product of chloroform was methylene chloride (dichloromethane), which accumulated transiently to about 70% of the added chloroform; trace amounts of chloromethane were also detected. The amount of chloroform dechlorinated per mole of methane produced was approximately 10 times greater than the ratio observed previously for tetrachloroethene dechlorination by these strains. The production of
14
CO
2
from [
14
C]chloroform and the absence of
14
CH
4
imply that processes in addition to reductive dechlorination operate.
Purification and Characterization of Three Chitosanase Activities from
Bacillus megaterium
P1
Authors: Pelletier A. , Sygusch J.
Published: January 2020

Bacillus megaterium
P1, a bacterial strain capable of hydrolyzing chitosan, was isolated from soil samples. Chitosan-degrading activity was induced by chitosan but not by its constituent
d
-glucosamine. Extracellular secretion of chitosanase reached levels corresponding to 1 U/ml under optimal conditions. Three chitosan-degrading proteins (chitosanases A, B, and C) were purified to homogeneity. Chitosanase A (43 kilodaltons) was highly specific for chitosan and represented the major chitosan-hydrolyzing species. Chitosanases B (39.5 kilodaltons) and C (22 kilodaltons) corresponded to minor activities and possessed comparable specific activities toward chitosan, chitin, and cellulose. Chitosanase A was active from pH 4.5 to 6.5 and was stable on the basis of activity up to 45°C. The optimum temperature for enzymatic chitosan hydrolysis was 50°C. Kinetic studies on chitosanase A suggest that the enzyme is substrate inhibited. The apparent
K

m

and
V
max
determined at 22°C and pH 5.6 were 0.8 mg/ml and 280 U/mg, respectively. End products of chitosan hydrolysis by each of the three chitosanases were identified as glucosamine oligomers, similar to those obtained for previously reported chitosanase digestions.
Conversion of Wine Strains of
Saccharomyces cerevisiae
to Heterothallism
Authors: Bakalinsky Alan T. , Snow Richard
Published: January 2020

A general method to convert homothallic strains of the yeast
Saccharomyces cerevisiae
to heterothallism is described which is applicable to genetically well-behaved diploids, as well as to strains that sporulate poorly or produce few viable and mating-competent spores. The heterothallic (
ho
) allele was introduced into three widely used wine strains through spore × cell hybridization. The resultant hybrids were sporulated, and heterothallic segregants were isolated for use in successive backcrosses. Heterothallic progeny of opposite mating type and monosomic for chromosome III produced by sixth-backcross hybrids or their progeny were mated together to reconstruct heterothallic derivatives of the wine strain parents. A helpful prerequisite to the introduction of
ho
was genetic purification of the parental strains based on repeated cycles of sporulation, ascus dissection, and clonal selection. A positive selection to isolate laboratory-wine strain hybrids requiring no prior genetic alteration of the industrial strains, coupled with a partial selection to reduce the number of spore progeny needed to be screened to isolate heterothallic segregants of the proper genotype made the procedure valuable for genetically intractable strains. Trial grape juice fermentations indicated that introduction of
ho
had no deleterious effect on fermentation behavior.
Partial Purification and Characterization of Two Hydrogenases from the Extreme Thermophile
Methanococcus jannaschii
Authors: Shah Nilesh N. , Clark Douglas S.
Published: January 2020

F
420
-nonreactive and F
420
-reactive hydrogenases have been partially purified from
Methanococcus jannaschii
, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F
420
-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F
420
-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F
420
-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F
420
-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F
420
-nonreactive hydrogenase was maximal at 80°C but was still detectable at 103°C. The maximum activities of F
420
-reactive hydrogenase for F
420
and methyl viologen were measured at 80 and 90°C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103°C. Moreover, the half-life of the F
420
-nonreactive hydrogenase at 70°C was over 9 h, and that of the F
420
-reactive enzyme was over 3 h.
Characterization of an
endo
-Acting Amylopullulanase from
Thermoanaerobacter
Strain B6A
Authors: Saha Badal C. , Lamed Raphael , Lee Chan-Yong , Mathupala Saroj P. , Zeikus J. Gregory
Published: January 2020

A thermoanaerobe (
Thermoanaerobacter
sp.) grown in TYE-starch (0.5%) medium at 60°C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75°C and pH 5.0, respectively. Both the enzyme activities were stable up to 70°C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70°C and 45 min at 75°C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by β- and γ-cyclodextrins but not by α-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in α1,6 linkages and produced multiple saccharides from cleavage of α-1,4 linkages in starch. The
K

m

s for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.
Molecular characterization of promoters of the Lactococcus lactis subsp. cremoris temperate bacteriophage BK5-T and identification of a phage gene implicated in the regulation of promoter activity
Authors: Lakshmidevi G , Davidson B E , Hillier A J
Published: January 2020

DNA fragments from the temperate lactococcal bacteriophage BK5-T were cloned into the promoter-detecting plasmid pMU1328. Five DNA fragments conferring promoter activity were selected by transformation of Streptococcus sanguis and were functional in Escherichia coli, S. sanguis, and Lactococcus lactis subspp. lactis and cremoris. The nucleotide sequences of these fragments were determined, and primer extension analysis was used to locate the site of initiation of transcription from each promoter in both E. coli and S. sanguis. Transcription was initiated from the same nucleotide in these two organisms, and the promoters contained -10 and -35 regions similar to the consensus sequence for E. coli promoters. The activities of three of the five promoters were decreased two- to threefold when a compatible plasmid containing a 3.8-kilobase-pair EcoRI fragment (EcoRI-f) of BK5-T was coresident with the promoter-containing plasmid in either L. lactis subsp. cremoris or E. coli. Data from Tn5 mutagenesis, subcloning experiments, and DNA sequence analysis indicate that this decrease in promoter activity requires a region of EcoRI-f that contains a 621-base-pair open reading frame. This region has been designated bpi (for BK5-T promoter inhibitor).
Identification of Francisella species and discrimination of type A and type B strains of F. tularensis by 16S rRNA analysis
Authors: Forsman M , Sandström G , Jaurin B
Published: January 2020

Tularemia is a zoonotic disease, occurring throughout the Northern Hemisphere. The causative agent, the bacterium Francisella tularensis, is represented by two main types. Type A is found in North America, whereas type B is mainly found in Asia and Europe and to a minor extent in North America. No routine technique for rapid diagnosis of tularemia has been generally applied. We have partially sequenced 16S rRNAs of two F. tularensis strains, as well as the closely related Francisella novicida. Of 550 nucleotides analyzed, only one difference in 16S rRNA primary sequence was found. This 16S rRNA analysis enabled the construction of oligonucleotides to be used as genus- and type-specific probes. Such probes were utilized for the establishment of a method for rapid and selective detection of the organism. This method allowed identification of Francisella spp. at the level of genus and also discrimination of type A and type B strains of F. tularensis. The analysis also permitted the detection of F. tularensis in spleen tissue from mice infected with the bacterium. The results presented will enable studies on the epizootiology and epidemiology of Francisella spp.
L-lysine production at 50 degrees C by mutants of a newly isolated and characterized methylotrophic Bacillus sp
Authors: Schendel F J , Bremmon C E , Flickinger M C , Guettler M , Hanson R S
Published: January 2020

The amino acid L-lysine was produced from homoserine auxotrophic and S-(2-aminoethyl)-L-cysteine-resistant mutants of a newly isolated gram-positive methylotrophic bacterium, capable of growth on methanol at 60 degrees C. The temperature optimum for growth was between 50 and 53 degrees C. These aerobic, gram-positive, endospore-forming, rod-shaped bacteria required biotin and vitamin B12 for growth. Extracts of the bacteria grown on methanol lacked hydroxypyruvate reductase and contained hexulose 6-phosphate synthase activity. Therefore, these bacteria were considered to be type I methylotrophic bacteria of the genus Bacillus. Fed-batch fermentations resulted in cell densities of 50 g of cell dry weight per liter. Biomass yields on carbon, nitrogen, phosphate, and sulfate were determined. Generation of homoserine auxotrophic and amino acid analog-resistant mutants resulted in L-lysine concentrations of nearly 20 g/liter in fed-batch fermentations.
Microbiological aspects of aerobic thermophilic treatment of swine waste
Authors: Beaudet R , Gagnon C , Bisaillon J G , Ishaque M
Published: January 2020

A thermophilic strain (D2) identified as a Bacillus sp. was isolated from an aerobic digestor of swine waste after several months of operation at 55 degrees C. Aerobic thermophilic batch treatment of swine waste inoculated with strain D2 was studied in a 4-liter fixed-bed reactor. Stabilization of the waste was achieved in less than 30 h when the original chemical oxygen demand (COD) was between 15 and 20 g/liter or in less than 48 h when the COD was around 35 g/liter. When the COD was higher than 30 g/liter, the pH of the waste reached 9.2 to 9.5 during the treatment, and periodic adjustment of the pH to 8.5 was necessary to maintain the activity of the biofilm. In this reactor, ammoniacal nitrogen was completely eliminated by desorption in less than 72 h of incubation. The different packing materials used resulted in similar rates of degradation of organic matter. The thermophilic treatment was also efficient in the 75-liter digestor, and stabilization was achieved in approximately 50 h. A bank of 22 thermophilic bacterial strains originating from different environments and adapted to the thermophilic treatment of swine waste was established. This thermophilic treatment allows, in one step, rapid stabilization of the waste, elimination of the bad smell, and complete elimination of ammonia nitrogen by stripping.
Isolation of behavioral mutants of Azospirillum brasilense by using Tn5 lacZ
Authors: van Rhijn P , Vanstockem M , Vanderleyden J , De Mot R
Published: January 2020

Tn5 lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100 or pCIB110. Kanamycin-resistant Azospirillum colonies were obtained with a maximum frequency of 10(-6) per recipient cell. The potential of Tn5 lacZ for random transposon mutagenesis coupled to transcription analysis in A. brasilense 7030 was demonstrated. Sixty percent of all Kmr A. brasilense 7030 mutants expressed beta-galactosidase activity. Mutants affected in motility (Fla-) and general chemotaxis (Che-) were identified. Chromosomal insertions of Tn5 lacZ are involved, except for two Che- mutants. The latter che loci reside on a 90-megadalton plasmid. Expression of an acidic protein (Mr, 110,000) was abolished in these mutants.
Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity
Authors: Fujii T , Kondo K , Shimizu F , Sone H , Tanaka J , Inoue T
Published: January 2020

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.
Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein
Authors: Brittain Andrew M. , Karl David M.
Published: January 2020

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-
3
H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [
3
H]thymidine experiments and compared the degree of nonspecific labeling by [
3
H]adenine with that derived from [
3
H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.
Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates
Authors: Folsom B R , Chapman P J , Pritchard P H
Published: January 2020

Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.
Effects of betaine on enumeration of airborne bacteria
Authors: Marthi B , Lighthart B
Published: January 2020

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.
Carbon- and Nitrogen-to-Volume Ratios of Bacterioplankton Grown under Different Nutritional Conditions
Authors: Nagata Toshi , Watanabe Yasunori
Published: January 2020

Carbon- and nitrogen-to-volume (C/V and N/V) ratios were determined for freshwater bacterial assemblages grown in lake water filtrate or in water enriched with nutrients (aqueous extract of lake seston, glucose, arginine, phosphate, or ammonium). Biovolume was measured by epifluorescence microphotography, and carbon and nitrogen biomasses were measured with a CHN analyzer. Despite large variations of nutritional conditions (i.e., the composition and concentration of the dissolved organic carbon) and different mean cell sizes of the bacterial assemblage (0.17 to 1.8 μm
3
per cell), the C/V, N/V, and carbon-to-nitrogen weight ratios varied little (C/V ratio, 0.14 pg of C per μm
3
[standard deviation, 0.057;
n
= 15]; N/V ratio, 0.027 pg of N per μm
3
[standard deviation; 0.011,
n
= 15]; carbon-to-nitrogen weight ratio, 5.6 [standard deviation, 2.2,
n
= 15]). An average C/V ratio of 0.12 pg of C per μm
3
that was derived from natural and cultured bacterial assemblages is proposed as an appropriate conversion factor for estimation of the biomass of freshwater bacteria.
Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling
Authors: Tannock G W , Fuller R , Pedersen K
Published: January 2020

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.
Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase
Authors: Hartmans S , van der Werf M J , de Bont J A
Published: January 2020

By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.
Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures
Authors: Haugland R A , Schlemm D J , Lyons R P , Sferra P R , Chakrabarty A M
Published: January 2020

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.
Inactivation of human and simian rotaviruses by chlorine dioxide
Authors: Chen Y S , Vaughn J M
Published: January 2020

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.
Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins
Authors: Van Rie J , Jansens S , Höfte H , Degheele D , Van Mellaert H
Published: January 2020

To investigate the biochemical basis of the differences in the insecticidal spectrum of Bacillus thuringiensis insecticidal crystal proteins (ICPs), we performed membrane binding and toxicity assays with three different ICPs and three lepidopteran species. The three ICPs have different toxicity patterns in the three selected target species. Binding studies with these 125I-labeled ICPs revealed high-affinity saturable binding to brush border membrane vesicles of the sensitive species. ICPs with no toxicity against a given species did not bind saturably to vesicles of that species. Together with previous data that showed a correlation between toxicity and ICP binding, our data support the statement that differences in midgut ICP receptors are a major determinant of differences in the insecticidal spectrum of the entire lepidopteran-specific ICP family. Receptor site heterogeneity in the insect midgut occurs frequently and results in sensitivity to more than one type of ICP.
Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts
Authors: Hino A , Mihara K , Nakashima K , Takano H
Published: January 2020

Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast. Kluyveromyces thermotolerans FRI 501 cells showed high survival ability after freezing when their resting cells were fermented for 0 to 180 min in modified liquid medium, and they grew to log and stationary phases. Among the freeze-tolerant strains of Saccharomyces cerevisiae, FRI 413 and FRI 869 showed higher surviving and trehalose-accumulating abilities than other S. cerevisiae strains, but were affected by a prolonged prefermentation period and by growth phases. The freeze tolerance of the yeasts was, to some extent, associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period. In the freeze-sensitive yeasts, the degree of hydrolysis of trehalose may thus be affected by the kind of saccharide, unlike in freeze-tolerant yeasts.
Inoculum size as a factor limiting success of inoculation for biodegradation
Authors: Ramadan M A , el-Tayeb O M , Alexander M
Published: January 2020

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.