bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel method for monitoring genetically engineered microorganisms in the environment
Authors: Chaudhry G R , Toranzos G A , Bhatti A R
Published: January 2020

A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of DNA) and has an advantage over the plate count technique, which can detect only culturable microorganisms. The method may be useful for monitoring GEMs in complex environments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.
Regulation of Glycolytic Flux and Ethanol Production in
Saccharomyces cerevisiae
: Effects of Intracellular Adenine Nucleotide Concentrations on the In Vitro Activities of Hexokinase, Phosphofructokinase, Phosphoglycerate Kinase, and Pyruvate Kinase
Authors: Alterthum Flavio , Dombek K. M. , Ingram L. O.
Published: January 2020

The progressive decline in the glycolytic activity of
Saccharomyces cerevisiae
during batch fermentation is accompanied by changes in adenine nucleotide pools. The relative activities of four glycolytic enzymes were examined in vitro in the presence of nucleotide concentrations equivalent to intracellular pools. Phosphofructokinase and pyruvate kinase were not inhibited. Phosphoglycerate kinase was inhibited by AMP but was judged unlikely to be of physiological consequence owing to enzyme abundance. Both isoenzymes of hexokinase were strongly inhibited by AMP. The degree of hexokinase inhibition was sufficient to account for the observed decline in glycolytic activity during batch fermentation.
Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis
Authors: Ohashi H , Katsuta Y , Nagashima M , Kamei T , Yano M
Published: January 2020

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.
Effects of cadmium, copper, magnesium, and zinc on the decomposition of citrate by a Klebsiella sp
Authors: Brynhildsen L , Rosswall T
Published: January 2020

The effects of Cd2+, Cu2+, Mg2+, and Zn2+ on the decomposition of citric acid by a Klebsiella sp. were studied by monitoring the degradation of [14C]citrate. The carbon concentration used was 10 micrograms of C liter-1, and the media were designed to provide at least 95% of the citrate complexed to the metal studied. After 72 h of incubation, 80% of the uncomplexed citric acid and 76% of the magnesium citrate had been decomposed. A marked inhibition was observed when Cd2+, Cu2+, or Zn2+ was bound to the organic anion; only 23% of the cadmium citrate, 14% of the zinc citrate, and 5% of the cuprous citrate had been decomposed. The effects were not the result of toxicity, since experiments run with [14C]glucose (nonchelating compound) instead of citrate resulted in similar decomposition rates regardless of the presence of the metal. To examine whether the binding of a metal to citrate enhanced its uptake by the Klebsiella sp., we studied the relative uptake of 65Zn in citrate- and in glucose-containing media. No such effect could be observed, with the uptake of Zn2+ being higher in the glucose-containing media. The study shows that metals may render low-molecular-weight organic acids, such as citric acid, resistant to bacterial degradation. This stresses the importance of metals in influencing microbial decomposition of organic compounds, not only as a result of toxicity.
Seasonal Biotransformation of Naphthalene, Phenanthrene, and Benzo[
a
]pyrene in Surficial Estuarine Sediments
Authors: Shiaris Michael P.
Published: January 2020

Transformation rates of naphthalene, phenanthrene, and benzo[
a
]pyrene in oxidized surficial sediments of a polluted urban estuary, Boston Harbor, Mass., were determined over a period of 15 months. Three sites characterized by muddy sediments were selected to represent a >300-fold range of ambient polycyclic aromatic hydrocarbon (PAH) concentration. Transformation rates were determined by a trace-level radiolabel PAH assay which accounted for PAH mineralization, the formation of polar metabolites, residue, and recovered parental PAHs in sediment slurries. Transformation rates of the model PAHs increased with increasing ambient PAH concentrations. However, turnover times for a given PAH were similar at all sites. The turnover times were as follows: naphthalene, 13.2 to 20.1 days; phenanthrene, 7.9 to 19.8 days, and benzo[
a
]pyrene, 53.7 to 82.3 days. At specific sites, rates were significantly affected by salinity, occasionally affected by temperature, but not affected by pH over the course of the study. Seasonal patterns of mineralization were observed for each of the PAHs at all sites. The timing of seasonal maxima of PAH mineralization varied from site to site. Seasonal potential heterotrophic activities as measured by acetate and glutamate mineralization rates did not always coincide with PAH mineralization maxima and minima, suggesting that the two processes are uncoupled in estuarine sediments.
Volatilization of Selenium by
Alternaria alternata
Authors: Thompson-Eagle E. T. , Frankenberger W. T. , Karlson U.
Published: January 2020

Seleniferous water continues to be a serious problem to wildlife in the central valley of California. Water samples collected from Kesterson Reservoir, Peck Ranch, and Lost Hills evaporation pond facilities contained between 0.005 and 5 mg of Se per liter. The objective of this study was to isolate Se-methylating organisms in evaporation pond water and to assess, through enrichment and manipulation of their optimal growth parameters, the environmental factors which govern microbial Se methylation.
Alternaria alternata
was isolated as an active Se-methylating organism. The volatile product was identified as dimethylselenide. The effects of pH, temperature, Se substrates, and methyl donors on the ability of
A. alternata
to methylate Se were investigated in liquid medium containing 100 mg of Se per liter. The optimum pH and temperature for methylation were 6.5 and 30°C, respectively. Selenate and selenite were methylated more rapidly than selenium sulfide and various organic Se compounds (6-selenoguanosine, 6-selenoinosine, seleno-
dl
-methionine, and 6-selenopurine).
l
-Methionine and methyl cobalamine (0.1 μM) stimulated dimethylselenide production. This study demonstrates that Se-methylating organisms are present in evaporation pond water and are capable of liberating substantial quantities of Se in the volatile dimethylselenide form. By determining the optimum environmental conditions which stimulate volatilization, it may be possible to design a way to remove Se from seleniferous water in situ.
Isolation and Ultrastructure of the Flagella of
Methanococcus thermolithotrophicus
and
Methanospirillum hungatei
Authors: Cruden Diana , Sparling Richard , Markovetz A. J.
Published: January 2020

The flagella of the archaebacteria
Methanococcus thermolithotrophicus
and
Methanospirillum hungatei
enter the cells in regions with ultrastructure resembling that of the polar organelles found in a variety of eubacteria. Flagella of both organisms consist of a filament, a hook, and a basal body with two rings similar to those of gram-positive eubacteria. The integrity of the flagella of
M. thermolithotrophicus
is lost in the absence of high salt concentrations, and those of both organisms are unstable at high pH. The flagellar filaments of
M. hungatei
are composed of two flagellins of 24 and 26 kilodaltons.
Bacterial Colonization of Cod (
Gadus morhua
L.) and Halibut (
Hippoglossus hippoglossus
) Eggs in Marine Aquaculture
Authors: Hansen Geir Høvik , Olafsen Jan A.
Published: January 2020

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (
Gadus morhua
L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (
G. morhua
L.) and halibut (
Hippoglossus hippoglossus
) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera
Pseudomonas, Alteromonas, Aeromonas
, and
Flavobacterium
were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium
Leucothrix mucor
was found on eggs from both species. While growth of
L. mucor
on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed.
Vibrio fischeri
could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera
Moraxella
and
Alcaligenes
were found only on halibut eggs.
Caulobacter
and
Seliberia
spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.
Evaluation of the 7-h membrane filter test for quantitation of fecal coliforms in water
Authors: Barnes R , Curry J I , Elliott L M , Peter C R , Tamplin B R , Wilcke B W
Published: January 2020

The 7-h fecal coliform (FC) test for detection of FC organisms in water was evaluated to establish its validity and usefulness for emergency and disaster situations. The waters tested consisted of routine samples collected for public health surveillance and enforcement purposes. A total of 984 water samples from throughout California were assayed. These included samples from coastal salt waters, rivers, canals, and reservoirs, in addition to potable and miscellaneous freshwater sources. A portion of each sample was tested concurrently by both the 7-h FC test and the most-probable-number FC five-tube test. The 7-h FC test samples were incubated for 7 to 7.25 h at 41.5 degrees C. Overall, greater than 90% agreement was obtained between the methods in determining whether the water quality was acceptable or unacceptable. Statistical analysis of the 984 samples confirmed that the 7-h FC method was a suitable alternative to the most-probable-number FC method for evaluation of freshwater samples. During emergencies or disasters, the 7-h FC test could provide a means for detection of fecal contamination of water with results available in less than 1 day.
Evaluation of different approaches for identification of xenobiotic- degrading pseudomonads
Authors: Busse H , El-Banna T , Auling G
Published: January 2020

Different approaches were evaluated to identify aerobic, gram-negative, biodegradative isolates assumed to be pseudomonads. Quinone and polyamine analysis allowed rapid identification to the genus level, i.e., allocation of those isolates belonging to the Pseudomonas fluorescens complex which represents the phylogenetically defined core of the heterogeneous genus Pseudomonas. Subsequent studies concentrated only on these true pseudomonads. The multiple-test system API 20NE, determination of the moles percent G+C content, and polyacrylamide gel electrophoresis of soluble proteins aided in identification on the species level. Polyacrylamide gel electrophoresis of both soluble proteins and whole-cell lipopolysaccharides allowed recognition of identical strains and double isolates, which were confirmed by DNA-DNA hybridization.
Survival and Activity of a 3-Chlorobenzoate-Catabolic Genotype in a Natural System
Authors: Fulthorpe Roberta R. , Wyndham R. Campbell
Published: January 2020

A chlorobenzoate-degrading
Alcaligenes
strain, BR60, was introduced to flowthrough lake microcosms and exposed to 3-chlorobenzoate (3Cba) concentrations from 0 to 25 μM. A DNA probe specific for BR60 chlorobenzoate catabolic genes was used with the most probable number (MPN) technique to enumerate bacteria harboring this genetic information. This MPN-DNA hybridization method combined with [U-
14
C]3Cba uptake rate measurements allowed the correlation of the size and activity of a specific catabolic population in a natural mixed community for the first time. An experiment involving the release of a streptomycin-resistant strain of BR60 indicated that estimates of bacteria carrying the introduced catabolic genotype often outnumbered plate count estimates of viable BR60 by as much as 3 orders of magnitude, particularly when 3Cba inputs were high. The MPN-DNA hybridization method provided catabolic population estimates highly correlated to 3Cba exposure levels and the [U-
14
C]3Cba uptake rates in the microcosms. Plate counts of BR60 were poorly correlated with both 3Cba exposure levels and uptake rates. In the absence of chlorobenzoate selection, the catabolic genotype declined to very low levels by the MPN-DNA hybridization technique after 8 weeks in the microcosms.
Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4
Authors: Shields Malcolm S. , Montgomery Stacy O. , Chapman Peter J. , Cuskey Stephen M. , Pritchard P. H.
Published: January 2020

o
-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of
18
O- and
16
O-labeled 3-methylcatechol from toluene in an atmosphere of
18
O
2
and
16
O
2
established that G4 catabolizes toluene by successive monooxygenations at the
ortho
and
meta
positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the
ortho
position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.
Rapid detection and identification of Legionella pneumophila by a membrane immunoassay
Authors: Bérubé A , Trudel M , Payment P
Published: January 2020

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.
Isolation and Characterization of an
Enterobacter cloacae
Strain That Reduces Hexavalent Chromium under Anaerobic Conditions
Authors: Wang Pi-Chao , Mori Tsukasa , Komori Kohya , Sasatsu Masanori , Toda Kiyoshi , Ohtake Hisao
Published: January 2020

An
Enterobacter cloacae
strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the
E. cloacae
isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed.
E. cloacae
is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of
E. cloacae
HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in
E. cloacae
HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).
Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri
Authors: Körner H , Zumft W G
Published: January 2020

The onset and cessation of the synthesis of denitrification enzymes of Pseudomonas stutzeri were investigated by using continuous culture and defined dissolved oxygen levels covering the full range of transition from air saturation to complete anaerobiosis. Expression of nitrate reductase, nitrite reductase (cytochrome cd1), and N2O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration. N2O reductase was synthesized constitutively at a low level; for enhanced expression, oxygen concentrations were required to decrease below 5 mg of O2 per liter. The threshold values for synthesis of nitrate reductase and cytochrome cd1 in the presence of nitrate were ca. 5 and ca. 2.5 mg of O2 per liter, respectively. With nitrous oxide as the respiratory substrate, nitrite reductase was again the most sensitive to oxygen concentration; however, thresholds for all denitrification enzymes shifted to lower oxygen levels. Whereas the presence of nitrate resulted in maximum expression and nearly uniform induction of all reductases, nitrite and nitrous oxide stimulated preferably the respective enzyme catalyzing reduction. In the absence of a nitrogenous oxide, anaerobiosis did not induce enzyme synthesis to any significant degree. The accumulation of nitrite seen during both the aerobic-anaerobic and anaerobic-aerobic transition phases was caused by the differences in onset or cessation of synthesis of nitrate and nitrite reductases and an inhibitory effect of nitrate on nitrite reduction.
Isolation and Characterization of Aminopeptidase-Deficient
Lactobacillus bulgaricus
Mutants
Authors: Atlan Danièle , Laloi Patrick , Portalier Raymond
Published: January 2020

Lactobacillus bulgaricus
CNRZ 397 is able to hydrolyze many amino-acyl- and dipeptidyl-β-naphthylamides. Analysis of heat inactivation kinetics, protease inhibitor effects, and the subcellular location of aminopeptidase (AP) activities from the parental strain and mutant derivatives dificient in alanyl- or leucyl-β-naphthylamide hydrolysis pointed out the existence of four APs. All mutants isolated were totally deficient in AP II, a cell wall metallo-enzyme with a broad substrate specificity but that is specifically responsible for lysyl-AP activity and is characterized by a molecular mass of 95,000 daltons. AP I and AP III are cytoplasmic enzymes that exhibit arginyl-AP activity; both enzymes are inducible during growth in rich peptide MRS medium (Difco Laboratories, Detroit, Mich.). The existence of a fourth AP (AP IV) that is involved in leucyl-AP activity was suggested. Moreover, we showed that X-prolyl-dipeptidyl-AP activity, which was not catalyzed by an AP, involved an enzyme(s) that is controlled by a regulatory mechanism that is common to that of AP II.
Effect of Na
+
Concentration and Nutritional Factors on the Lag Phase and Exponential Growth Rates of the Marine Bacterium
Deleya aesta
and of Other Marine Species
Authors: Berthelet Marc , MacLeod Robert A.
Published: January 2020

Growth of the marine bacterium
Deleya aesta
in a succinate minimal medium showed increasingly long lag phases as Na
+
was decreased below the optimum (200 to 500 mM). The minimum Na
+
concentration permitting growth consistently was 15 mM. Supplementation of the medium with KHCO
3
(as a source of CO
2
) or yeast extract, especially in combination, reduced the lag phase, increased the rate of exponential growth, and allowed growth at 8 mM Na
+
. KHCO
3
did not reduce the lag period but did increase the rate of exponential growth of
Deleya venusta, Deleya pacifica
, and
Alteromonas haloplanktis
214. Yeast extract was active for all three. The effect of yeast extract on
D. aesta
could be reproduced by a mixture of amino acids approximating its amino acid composition.
l
-Alanine,
l
-aspartate, and
l
-methionine, in combination, were the most effective in reducing the lag phase, although not as effective as the complete mixture. Succinate,
l
-aspartate, and
l
-alanine were transported into the cells by largely independent pathways and oxidized at rates which were much lower at 10 than at 200 mM Na
+
.
l
-Methionine was transported at a low rate in the absence of Na
+
and at a higher rate at 10 mM but was not oxidized. Above 25 mM Na
+
, the rate of transport of the carbon source was not the rate-limiting step for growth. It is concluded that a combination of transportable carbon sources reduced the lag period and increased the rate of exponential growth because they can be taken up independently and at low Na
+
utilized simultaneously.
Effects of Fixation on Cell Volume of Marine Planktonic Protozoa
Authors: Choi Joon W. , Stoecker Diane K.
Published: January 2020

The effects of fixation on the cell volume of marine heterotrophic nanoflagellates and planktonic ciliates were investigated. Decreases in cell volume depended on the combination of the protozoan taxa and the particular fixative. For a particular fixative and protozoan species, degree of shrinkage was independent of physiological state. The volume of fixed cells was found to be approximately 20 to 55% lower than the cell volume of live organisms. For the heterotrophic microflagellates, the fixatives ranked, in order of decreasing effect on cell volume, as glutaraldehyde, formaldehyde, acid Lugol's solution, and modified van der Veer solution. With oligotrichous ciliates and a tintinnid ciliate, formaldehyde caused less shrinkage than glutaraldehyde or acid Lugol's solution. With the aldehyde fixatives, the microflagellates were found to shrink more than the ciliates. Differential effects of fixation on cell volumes may result in an underestimation of the biomass of certain protozoan taxa in natural samples.
Protozoan Grazing and Bacterial Production in Stratified Lake Vechten Estimated with Fluorescently Labeled Bacteria and by Thymidine Incorporation
Authors: Bloem Jaap , Ellenbroek Frank M. , Bär-Gilissen Marie-José B. , Cappenberg Thomas E.
Published: January 2020

In stratified Lake Vechten, The Netherlands, protozoan grazing was estimated on the basis of uptake of fluorescently labeled bacteria and compared with bacterial production estimated on the basis of thymidine incorporation. By using a grazer-free mixed bacterial population from the lake in continuous culture, an empirical relationship between cell production and thymidine incorporation was established. Thymidine incorporation into total cold-trichloroacetic-acid-insoluble macromolecules yielded a relatively constant empirical conversion factor of ca. 10
18
(range, 0.38 × 10
18
to 1.42 × 10
18
) bacteria mol of thymidine
−1
at specific growth rates (μ) ranging from 0.007 to 0.116 h
−1
. Although thymidine incorporation has been assumed to measure DNA synthesis thymidine incorporation appeared to underestimate the independently measured bacterial DNA synthesis by at least 1.5- to 13-fold, even if all incorporated label was assumed to be in DNA. However, incorporation into DNA was found to be insignificant as measured by conventional acid-base hydrolysis. Methodological problems of the thymidine technique are discussed. Like the cultures, Lake Vechten bacteria showed considerable thymidine incorporation into total macromolecules, but no significant incorporation into DNA was found by acid-base hydrolysis. This applied not only to the low-oxygen hypo- and metalimnion but also to the aerobic epilimnion. Thus, the established empirical conversion factor for thymidine incorporation into total macromolecules was used to estimate bacterial production. Maximum production rates (141 × 10
6
bacteria liter
−1
h
−1
; μ, 0.012 h
−1
) were found in the metalimnion and were 1 order of magnitude higher than in the epi- and hypolimnion. In all three strata, the estimated bacterial production was roughly balanced by the estimated protozoan grazing. Heterotrophic nanoflagellates were the major consumers of the bacterial production and showed maximum numbers (up to 40 × 10
6
heterotrophic nanoflagellates liter
−1
) in the microaerobic metalimnion.
Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species
Authors: Lemke P A , Davis N D , Creech G W
Published: January 2020

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.
Turnover of Extracellular DNA in Eutrophic and Oligotrophic Freshwater Environments of Southwest Florida
Authors: Paul John H. , Jeffrey Wade H. , David Andrew W. , DeFlaun Mary F. , Cazares Lisa H.
Published: January 2020

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 × 10
9
cells per liter and 28.6 μg of chlorophyll
a
per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 ± 3.6 h in the Crystal River and 10.5 ± 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.
Production and Utilization of Ethanol by the Homoacetogen
Acetobacterium woodii
Authors: Buschhorn Heike , Dürre Peter , Gottschalk Gerhard
Published: January 2020

Acetobacterium woodii
formed ethanol as a fermentation product in addition to acetate when the phosphate concentration of the medium was between 0.2 and 8.4 mM. Considerable amounts of alanine were also found (2 to 11 mM). Supplementation with phosphate caused a shift to acetate as the only end product. Ethanol could also serve as a substrate for
A. woodii.
The fermentation yielded predominantly acetate and was strictly dependent on high bicarbonate concentrations. 1-Propanol, 1-butanol, and 1-pentanol were converted to the corresponding fatty acids but allowed only marginal growth.
A. wieringae
and
A. carbinolicum
grown under identical conditions were also able to form ethanol, and
A. wieringae
could use ethanol as a substrate, too. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were determined in
A. woodii.
Activity stains of polyacrylamide gels with crude extracts allowed the detection of acetaldehyde dehydrogenase but not of alcohol dehydrogenase. Trace amounts of methane were detected during growth of
A. woodii
on glucose and ethanol.
Exopolysaccharide-Deficient Mutants of
Rhizobium fredii
HH303 Which Are Symbiotically Effective
Authors: Kim Choong-Hyun , Tully Raymond E. , Keister Donald L.
Published: January 2020

Nineteen Tn
5
-induced mutants of
Rhizobium fredii
HH303 defective in acidic exopolysaccharide synthesis were isolated by screening for lack of Calcofluor fluorescence. They were grouped by complementation analysis by using
Rhizobium meliloti
cosmids carrying
exo
genes. All of the 19 mutants were symbiotically effective or partially effective, indicating that the major bacterial acidic exopolysaccharide of this strain of
R. fredii
may not be required for symbiotic development in the soybean.
Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil
Authors: Casida L. E.
Published: January 2020

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either
Cupriavidus necator
, a potent bacterial predator, or one of its prey species,
Micrococcus luteus. C. necator
also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of
Arthrobacter globiformis
or
Bacillus thuringiensis
. Therefore, the autochthonous state of
Arthrobacter
species in soil and the survival of
B. thuringiensis
were possibly enhanced by the resistance of these species to protozoa. The addition of
Bacillus mycoides
and
Escherichia coli
cells caused specific responses by soil protozoa. The protozoa that responded to
E. coli
did not respond to
B. mycoides
or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as
E. coli
, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.
Maintenance of Intracellular pH and Acid Tolerance in
Rhizobium meliloti
Authors: O'Hara Graham W. , Goss Thomas J. , Dilworth Michael J. , Glenn Andrew R.
Published: January 2020

The development and function of the
Rhizobium meliloti-Medicago
sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable. The intracellular pH of
R. meliloti
was measured using either radioactively labeled weak acids (5,5-dimethyloxazolidine-2,4-dione and butyric acid) or pH-sensitive fluorescent compounds; both methods gave similar values. Six acid-tolerant strains (WSM419, WSM533, WSM539, WSM540, WSM852, and WSM870) maintained an alkaline intracellular pH when the external pH was between 5.6 and 7.2. In contrast, two Australian commercial inoculant strains (CC169 and U45) and four acid-sensitive strains from alkaline soils in Iraq (WSM244, WSM301, WSM365, and WSM367) maintained an alkaline intracellular pH when the external pH was ≥6.5, but had intracellular pH values of ≤6.8 when the external pH was ≤6.0. Four transposon Tn
5
-induced mutants of acid-tolerant strain WSM419, impaired in their ability to grow at pH 5.6, showed limited control over the intracellular pH. The ability to generate a large pH gradient under acid conditions may be a better indicator of acid tolerance in
R. meliloti
under field conditions than is growth on acidic agar plates.
Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium
Authors: Pace J , Chai T J
Published: January 2020

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.
Nodulating Competitiveness of a Nonmotile Tn
7
Mutant of
Bradyrhizobium japonicum
in Nonsterile Soil
Authors: Liu Ruilong , Tran Van Mai , Schmidt E. L.
Published: January 2020

A nonmotile mutant of
Bradyrhizobium japonicum
serogroup 127 was generated by Tn
7
mutagenesis and matched with the wild type against a common competitor in studies of soybean nodulation in nonsterile soil. The Tn
7
mutant was very similar to the wild type in growth rate in culture, soybean lectin-binding ability, flagellar morphology, and nodulating capability, but it had a longer lag phase. Competing strains were distributed uniformly in soil in various ratios and at different population densities prior to planting. Mutant and wild type were equally prevalent in the seedling rhizosphere at about the time of nodule initiation, suggesting that motility conferred no advantage in rhizosphere colonization. Nodulation success of the Tn
7
mutant was lower than that of the wild type under all test conditions. Differences were greatest at low soil populations of competitors and much less pronounced at initial populations of 10
7
g
−1
. The longer lag phase of the Tn
7
mutant may have contributed to its decreased competitiveness, especially at the higher inoculation levels. The antibiotic and motility markers were stable, and the rifampin resistance derived from the parent did not affect adversely the competitiveness of the Tn
7
mutant. We found motility to be of limited importance to the competitiveness of a strain in normal nonsterile soil, where the significance, if any, of this ability may be in migration at the immediate root surface in soils sparsely populated with rhizobial symbionts.
In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice
Authors: Barc M C , Tekaia F , Bourlioux P
Published: January 2020

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.
Biosynthesis of zearalenone: a simple and efficient method to incorporate [13C]acetate label by using solid cultures
Authors: Pathre S V , Khadikar P V , Mirocha C J
Published: January 2020

A suitable method was developed to efficiently incorporate 13C-labeled acetate into zearalenone by using solid cultures. Periodic feeding of the label during the zearalenone production phase significantly increased the label incorporation for the singly labeled acetate.
Chloromethane, a Novel Methyl Donor for Biosynthesis of Esters and Anisoles in
Phellinus pomaceus
Authors: Harper David B. , Hamilton John T. G. , Kennedy James T. , McNally Kieran J.
Published: January 2020

Chloromethane (CH
3
Cl), a gaseous natural product released as a secondary metabolite by many woodrotting fungi of the family
Hymenochaetaceae
, has been shown to act as a methyl donor for biosynthesis of methyl esters of benzoic and furoic acid in the primary metabolism of
Phellinus pomaceus.
The broad-specificity methylating system could esterify a wide range of aromatic and aliphatic acids. In addition to CH
3
Cl, both bromo- and iodomethanes acted as methyl donors. Methylation did not appear to proceed via methanol or a coenzyme A intermediate. The initial growth-related accumulation of methyl benzoate during culture of
P. pomaceus
was paralleled by an increase in activity of the methylating system in the mycelium. Changes in percent incorporation of C
2
H
3
from exogenous C
2
H
3
Cl during growth indicated that although utilization of CH
3
Cl was initially closely coupled to biosynthesis of the compound, the system became less tightly channeled later in growth. This phase coincided with release of gaseous CH
3
Cl by the fungus. A biochemically distinct CH
3
Cl-utilizing system capable of methylating phenols and thiophenol was also identified in the fungus, but in contrast with the carboxylic acid-methylating system, it attained maximum activity in the idiophase. Preliminary investigations of a non-CH
3
Cl-releasing fungus,
Fomitopsis pinicola
, have shown the presence of a CH
3
Cl-utilizing system capable of methylating benzoic acid, suggesting that CH
3
Cl biosynthesis may occur in non-hymenochaetaceous fungi. Halogenated compounds hitherto found in nature are mainly stable end products of metabolism. The participation of CH
3
Cl in primary fungal metabolism demonstrates that some halometabolites may have a previously unrecognized role as intermediates in the biosynthesis of nonhalogenated natural products.
Bacterial Community Structure and Function along a Heavy Metal Gradient
Authors: Dean-Ross Deborah , Mills Aaron L.
Published: January 2020

The response of the planktonic, sediment, and epilithic bacterial communities to increasing concentrations of heavy metals was determined in a polluted river. None of the communities demonstrated a pollution-related effect on bacterial numbers (viable and total), heterotrophic activity, resistance to Pb or Cu, or species diversity as determined by either the Shannon-Wiener diversity index or rarefaction. The lack of correlation between concentrations of heavy metals and resistance in the sediment bacterial community was investigated and found to be due at least in part to the high pH of the river water and the resultant reduction in heavy metal toxicity. The three different communities demonstrated characteristic profiles based on the relative abundances of bacterial strains grouped according to functional similarities.
Evaluation of Malolactic Bacteria Isolated from Oregon Wines
Authors: Henick-Kling T. , Sandine W. E. , Heatherbell D. A.
Published: January 2020

Oregon is a cool wine-producing region where grapes characteristically contain high concentrations of organic acids. To reduce the natural acidity and increase the microbiological stability and flavor complexity of the wine, malolactic fermentation is encouraged. In this study, strains of
Leuconostoc oenos
indigenous to Oregon wines were evaluated for their suitability to conduct malolactic fermentation in Oregon wines. Tests determined the malolactic activity of the Oregon isolates in comparison with commercial strains ML-34, PSU-1, MLT-kli, and ens 44-40 under various temperature and pH conditions. Sensitivities to sulfur dioxide, ethanol, and fumaric acid also were determined. Two Oregon strains, Er-1a and Ey-2d, were selected for commercial winemaking tests because they had greater malolactic activity under conditions of low pH (3.0) and low temperature (15 and 8°C), respectively.
Bacteriophage Transport in Sandy Soil and Fractured Tuff
Authors: Bales Roger C. , Gerba Charles P. , Grondin Gerald H. , Jensen Stephen L.
Published: January 2020

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 40% of the void volume but did not adsorb. Dispersion in the field was similiar to that observed in the laboratory. The phage f2 was largely excluded from the porous matrix of the two fractured-rock cores studied, coming through 1.2 and 2.0 times later than predicted on the basis of fracture flow alone. Because of matrix diffusion, nonsorbing solutes were retarded by over a factor of three relative to fracture flow. The time for a solute tracer to equilibrate with the porous matrix of 6.5-cm-diameter by 25-cm-long cores was measured in days. Results of both granular-medium and fractured-rock experiments illustrate the inability of a solute tracer to provide estimates for dispersion and effective porosity that are applicable to a colloid. Bacteriophage can be used to better estimate the maximum subsurface transport rate of colloidal contaminants through a porous formation.
Electrode system for determination of microbial cell populations in polluted water
Authors: Maoyu Y , Zhang Y
Published: January 2020

Microbial cell populations in polluted water were determined by using a fuel cell-type electrode. The electrode was composed of a Pt anode, a Pt-K3Fe(CN)6-K4Fe(CN)6 cathode, and a cation-exchange membrane for separating two electrode compartments. The principle of microbial cell number determination is based on sensing a redox dye reduced by microorganisms with the electrode. Sample solutions containing microorganisms, a redox dye (thionine), and peptone were purged with oxygen-free nitrogen during the determination. A linear relationship was obtained between the increasing rate of current and the number of microbial cells measured by the colony count method above 10(4) cells per ml. The determination time varied with the number of microbial cells determined from 20 to 60 min for 3.6 x 10(6) and 3.6 x 10(4) cells per ml, respectively.
Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media
Authors: Angle J. S. , Chaney R. L.
Published: January 2020

Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [
N
-2-hydroxyethylpiperazine-
N
′ -2-ethanesulfonic acid−2-(
N
-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for
Rhizobium fredii
USDA 201. Arabinose-HEPES-MES was modified by addition of the chelator nitrilotriacetate to buffer the supply of free Cd
2+
ion to maintain a constant Cd activity and by the use of only MES to buffer pH (buffered arabinose-MES medium [BAM]). Ca and Mg were supplied at the normal levels for soil solutions, and other trace elements were supplied at the levels required for normal growth of plants. The concentration of free Cd
2+
ion was calculated by using the computer program GEOCHEM-PC with a corrected data base. The Cd MIC in BAM was 14.0 μM, while that in a tryptone-yeast extract medium was 107 μM. The results indicate that substantial free Cd
2+
is removed from solution in most standard media, resulting in falsely high MICs. The new BAM medium allows for the precise determination of MICs, thus avoiding the uncertainties associated with other media.