bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance
Authors: Verreth C , Cammue B , Swinnen P , Crombez D , Michielsen A , Michiels K , Van Gool A , Vanderleyden J
Published: January 2020

The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.
Ecology of Vibrio mimicus in aquatic environments
Authors: Chowdhury M A , Yamanaka H , Miyoshi S , Aziz K M , Shinoda S
Published: January 2020

An environmental study was done to examine the prevalence of Vibrio mimicus in some aquatic environments of Dhaka, Bangladesh, and of Okayama, Japan. Water samples from Dhaka environments and water and plankton samples from Okayama environments were quantitatively as well as qualitatively analyzed throughout the seasons for V. mimicus. The organism was isolated from Bangladesh environments throughout the year, whereas it was not isolated in Okayama when the water temperature fell below 10 degrees C. Samples with as many as 9.0 x 10(2) CFU of V. mimicus per 100 ml of water in Dhaka and 1.5 x 10(4) CFU of V. mimicus per 100 ml of water in Okayama were detected during the study period. V. mimicus was not found in any environment with an average salinity of 10% or more. Brackish environments with an average salinity of 4% were observed to be the optimal natural condition for the pathogen. Using the API 20E system with the conventional test methods, we observed variations in biochemical properties within the V. mimicus species. This study reveals the inefficacy of the API 20E system to identify a significant percentage of V. mimicus. Therefore, in addition to the API 20E system, a salt tolerance test and a string test are recommended for identification of this species. Susceptibility testing of strains isolated from Okayama environments showed higher resistance to ampicillin and susceptibility to trimethoprim-sulfamethoxazole when compared with environmental isolates of V. mimicus from Bangladesh.
Induction of
Lipomyces starkeyi
Dextranase
Authors: Koenig David W. , Day Donal F.
Published: January 2020

Lipomyces starkeyi
ATCC 20825 is a derepressed mutant derived from
L. starkeyi
ATCC 12659. It requires the presence of an inducer before it produces dextranase. This study was undertaken to determine the most efficient, commercially feasible method for inducing this enzyme. The following compounds induced dextranase synthesis: 1-
O
-β-methyl-glucopyranoside, 1-
O
-α-methyl-glucopyranoside, dextran, isomaltopentose, isomaltotetraose, isomaltotriose, and isomaltose. 1-
O
-β-Methyl-glucopyranoside was found to be a gratuitous inducer. Early in the growth phase, cells produced higher specific levels of enzyme than they did in late log phase. The length of exposure of the yeast cells to the inducer also affected the amount of dextranase produced. The maximum amount of enzyme was produced after 12 h of exposure to the inducer. The saturation concentration was the same for all inducers tested, i.e., approximately 1 mg of inducer for every 2 � 10
8
cells.
Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum
Authors: Bates E E , Gilbert H J , Hazlewood G P , Huckle J , Laurie J I , Mann S P
Published: January 2020

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.
Isolation of Aeromonas spp. from water by using anaerobic incubation
Authors: Cunliffe D A , Adcock P
Published: January 2020

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.
DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry)
Authors: Bloom R A , Mullin B C , Tate R L
Published: January 2020

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.
Molecular cloning and expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli
Authors: Liaw H J , Srinivasan V R
Published: January 2020

A 2.1-kilobase fragment obtained by restriction enzyme HindIII digestion of Erwinia sp. genomic DNA was cloned into plasmid pUC19 and introduced into Escherichia coli by transformation. The transformants with diphenyl ether cleaving activity (Dpe+) were selected on agar plates with a specially designed medium (LTFN) containing 4-nitrodiphenyl ether. The positive clones showed a clear zone around the colonies. Analysis of mutants obtained by transposon mini-Mu dI(lacZ Kmr) mutagenesis indicated the coding region of the gene (dpe) and the utilization of a lacZ promoter of pUC19 for transcription of dpe. Clones with dpe in the opposite orientation in pUC19 were not expressed, confirming the need for a lacZ promoter. Utilization of a lacZ promoter in pUC19 was further confirmed by the observation that the degradation of 4-nitrodiphenyl ether was enhanced in the presence of isopropyl-beta-D-thiogalactoside. Expression of dpe was also found in pDPE7321, generated from cloning this gene into another plasmid, pSP73. Analysis of the plasmid-encoded proteins by the maxicell technique showed a polypeptide of 21,000 molecular weight as the product of dpe.
Sodium lactate delays toxin production by Clostridium botulinum in cook-in-bag turkey products
Authors: Maas M R , Glass K A , Doyle M P
Published: January 2020

Comminuted raw turkey, containing 1.4% sodium chloride, 0.3% sodium phosphate, and 0 (control), 2.0, 2.5, 3.0, or 3.5% sodium lactate, was inoculated with a 10-strain mixture of proteolytic type A and B Clostridium botulinum spores. The inoculated turkey was vacuum packaged and cooked by immersion in heated water to an internal temperature of 71.1 degrees C. Samples were incubated at 27 degrees C for up to 10 days. Five samples per treatment were examined for botulinal toxin at specific intervals. Sodium lactate exhibited an antibotulinal effect which was concentration dependent. Processed turkey containing 0, 2.0, 2.5, 3.0, or 3.5% sodium lactate was toxic after 3, 4 to 5, 4 to 6, 7 or 7 to 8 days, respectively. Subsequent studies with a broth medium revealed that lactate, not the sodium ion, was the principal factor in delaying botulinal-toxin formation.
Disinfection of model indicator organisms in a drinking water pilot plant by using PEROXONE
Authors: Wolfe R L , Stewart M H , Liang S , McGuire M J
Published: January 2020

PEROXONE is an advanced oxidation process generated by combining ozone and hydrogen peroxide. This process stimulates the production of hydroxyl radicals, which have been shown to be superior to ozone for the destruction of some organic contaminants. In this study, pilot-scale experiments were conducted to evaluate the microbicidal effectiveness of PEROXONE and ozone against three model indicator groups. Escherichia coli and MS2 coliphage were seeded into the influent to the preozonation contactors of a pilot plant simulating conventional water treatment and were exposed to four ozone dosages (0.5, 1.0, 2.0, and 4.0 mg/liter), four hydrogen peroxide/ozone (H2O2/O3) weight ratios (0, 0.3, 0.5, and 0.8), and four contact times (4, 5, 12, and 16 min) in two source waters--Colorado River water and state project water--of different quality. The removal of heterotrophic plate count bacteria was also monitored. Results of the study indicated that the microbicidal activity of PEROXONE was greatly affected by the applied ozone dose, H2O2/O3 ratio, contact time, source water quality, and type of microorganism tested. At contact times of 5 min or less, ozone alone was a more potent bactericide than PEROXONE at all H2O2/O3 ratios tested. However, this decrease in the bactericidal potency of PEROXONE was dramatic only as the H2O2/O3 ratio was increased from 0.5 to 0.8. The fact that the bactericidal activity of PEROXONE generally decreased with increasing H2O2/O3 ratios was thought to be related to the lower ozone residuals produced. The viricidal activity of PEROXONE and ozone was comparable at all of the H2O2/O3 ratios. Heterotrophic plate count bacteria were the most resistant group of organisms. Greater inactivation of E. coli and MS2 was observed in Colorado River water than in state project water and appeared to result from differences in the turbidity and alkalinity of the two waters. Regardless of source water, greater than 4.5 log10 of E. coli and MS2 was inactivated at an applied ozone dosage of 2.0 mg/liter (and a 4-min contact time) when the H2O2/O3 ratio was less than or equal to 0.5. Comparative disinfection experiments indicated that free chlorine was the most potent bactericidal agent, followed (in descending order of effectiveness) by ozone, PEROXONE, and chloramines. These results indicate that the PEROXONE process must be optimized for each source water to achieve microbicidal effectiveness.
Synergism in Degradation and Utilization of Intact Forage Cellulose, Hemicellulose, and Pectin by Three Pure Cultures of Ruminal Bacteria
Authors: Osborne J. M. , Dehority B. A.
Published: January 2020

Pure cultures of ruminal bacteria characterized as using only a single forage polysaccharide (
Fibrobacter succinogenes
A3c, cellulolytic;
Bacteroides ruminicola
H2b, hemicellulolytic;
Lachnospira multiparus
D15d, pectinolytic) were inoculated separately and in all possible combinations into fermentation tubes containing orchard grass as the sole substrate. Fermentations were run to completion, and then cultures were analyzed for digestion of cellulose plus degradation and utilization of hemicellulose and pectin. Addition of the noncellulolytic organisms, in any combination, to the cellulolytic organism
F. succinogenes
had little effect on overall cellulose utilization.
F. succinogenes
degraded but could not utilize hemicellulose; however, when it was combined with
B. ruminicola
, total utilization of hemicellulose increased markedly over that by
B. ruminicola
alone.
L. multiparus
was inactive in hemicellulose digestion, alone or in any combination. Although unable to degrade and utilize purified pectin,
B. ruminicola
degraded and utilized considerable quantities of the forage pectin. In contrast,
L. multiparus
was very active against purified pectin, but had extremely limited ability to degrade and utilize pectin from the intact forage. Both degradation and utilization of forage pectin increased when
F. succinogenes
was combined with
B. ruminicola.
Sequential addition of two cultures, allowing one to complete its fermentation before adding the second, was used to study synergism between cultures on forage pectin digestion. In general, synergistic effects did not appear to be related to a particular sequence of utilization. The ability of
F. succinogenes
to degrade and
B. ruminicola
to degrade and utilize forage pectin contradicts both previous and present data obtained with purified pectin. Thus, isolation and characterization of ruminal bacteria on purified substrates may be misleading with regard to their role in the overall ruminal fermentation.
Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis
Authors: Crissman J W , Causey S C , Thorne L , Pollock T J
Published: January 2020

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.
Intracellular Location and O
2
Sensitivity of Uptake Hydrogenase in
Azospirillum
spp
Authors: Fu Changlin , Knowles Roger
Published: January 2020

Uptake hydrogenase activity of
Azospirillum brasilense
in vitro (cell-free extract) was very much more sensitive to O
2
than was that of
A. amazonense
, and the O
2
pressure optima for uptake hydrogenase activities were 0.01 and 0.4 to 3 kPa for
A. brasilense
and
A. amazonense
, respectively. The addition of superoxide dismutase did not increase uptake hydrogenase activity of
A. brasilense
either in vivo or in vitro. The O
2
uptake rates of
A. brasilense
and
A. amazonense
were nearly the same. Inhibition of
A. brasilense
O
2
-dependent uptake hydrogenase activity by O
2
was highly reversible under the conditions tested. O
2
also markedly inhibited in vitro methylene blue-dependent uptake hydrogenase activity of
A. brasilense
, and this inhibition was highly reversible. It is concluded that the difference in O
2
tolerance of the uptake hydrogenases is not due to a difference in respiratory protection in the two species and may be due to inherent differences in the two enzymes. For the three species,
A. brasilense
,
A. amazonense
, and
A. lipoferum
, almost all the recovered methylene blue-dependent uptake hydrogenase activity was associated with the membrane fraction.
Influence of diet and monensin on development of anaerobic fungi in the rumen, duodenum, cecum, and feces of cows
Authors: Grenet E , Fonty G , Jamot J , Bonnemoy F
Published: January 2020

Three cows with fistulated rumens, duodenums, and ceca were fed five different diets: lucerne hay, lucerne hay plus whey (40:60), lucerne hay plus beets (50:50), corn silage plus monensin (40 ppm [40 g/kg] of dry matter intake), and lucerne hay plus monensin (80 ppm of dry matter intake). The fungal population was observed in the rumen, duodenum, cecum, and rectum and varied with diet; it was most abundant with lucerne hay alone and with corn silage plus monensin. The proportion of particles colonized by fungi in the duodenum, the cecum, and feces was measured by microscopic observation and varied from 5 to 50%, depending on the diet. The further sporangia attached to the plant particles were from the rumen, the more likely they were to be devoid of spores. Results confirmed the influence of diet on the development of the ruminal fungal population and showed that monensin does not eliminate these microorganisms. They also confirmed the presence of anaerobic fungi in the ruminant intestine. It is likely that anaerobic fungi leave the rumen attached to plant particles. However, large colonies of nonrhizoidal-type fungi were observed in cecum samples and in feces; at these sites, environmental conditions are perhaps more favorable for this type of fungus than they are in the rumen.
Physiological and Morphological Modifications in Immobilized
Gibberella fujikuroi
Mycelia
Authors: Saucedo José Edmundo Nava , Barbotin Jean-Noël , Thomas Daniel
Published: January 2020

Constraints created by immobilization conditions modified the physiological behavior and morphological characteristics of
Gibberella fujikuroi
mycelia in comparison with their development in free-cell conditions.
G. fujikuroi
mycelia were immobilized in different support matrices (polyurethane, carrageenan, and alginate) and showed a variety of reactions in response to the different microenvironmental factors encountered during and after immobilization. The best support with respect to gibberellic acid yield and biocatalyst stability was found to be an alginate with a high degree of polymerization. The most visible effects of immobilization included changes in growth development, morphological appearance, metabolite production, mycelial pigmentation, mycelial viability under starvation conditions, and induction of resting forms when previously immobilized mycelia were subcultured.
Survey of metal tolerance in moderately halophilic eubacteria
Authors: Nieto J J , Fernández-Castillo R , Márquez M C , Ventosa A , Quesada E , Ruiz-Berraquero F
Published: January 2020

The tolerance patterns, expressed as MICs, for 250 moderately halophilic eubacteria to 10 heavy metals were surveyed by using an agar dilution method. The moderate halophiles tested included 12 culture collection strains and fresh isolates representative of Deleya halophila (37 strains), Acinetobacter sp. (24 strains), Flavobacterium sp. (28 strains), and 149 moderately halophilic gram-positive cocci included in the genera Marinococcus, Sporosarcina, Micrococcus, and Staphylococcus. On the basis of the MICs, the collection strains showed, overall, similar responses to silver, cobalt, mercury, nickel, lead, and zinc. All were sensitive to silver, mercury, and zinc and tolerant of lead. The response to arsenate, cadmium, chromium, and copper was very heterogeneous. The metal susceptibility levels of the 238 freshly isolated strains were, in general, very heterogeneous among the four taxonomic groups as well as within the strains included in each group. The highest toxicities were found with mercury, silver, and zinc, while arsenate showed the lowest activity. All these strains were tolerant of nickel, lead, and chromium and sensitive to silver and mercury. Acinetobacter sp. strains were the most heavy-metal tolerant, with the majority of them showing tolerance of eight different metal ions. In contrast, Flavobacterium sp. strains were the most metal sensitive. The influence of salinity and yeast extract concentrations of the culture medium on the toxicity of the heavy metals tested for some representative strains was also studied. Lowering the salinity, in general, led to enhanced sensitivity to cadmium and, in some cases, to cobalt and copper. However, increasing the salinity resulted in only a slight decrease in the cadmium, copper, and nickel toxicities.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolism of Ferulic Acid by
Paecilomyces variotii
and
Pestalotia palmarum
Authors: Rahouti Mohammed , Seigle-Murandi Françoise , Steiman Régine , Eriksson Karl-Erik
Published: January 2020

Ferulic acid metabolism was studied in cultures of two micromycetes producing different amounts of phenol oxidases. In cultures of the low phenol oxidase producer
Paecilomyces variotii
, ferulic acid was decarboxylated to 4-vinylguaiacol, which was converted to vanillin and then either oxidized to vanillic acid or reduced to vanillyl alcohol. Vanillic acid underwent simultaneously an oxidative decarboxylation to methoxyhydroquinone and a nonoxidative decarboxylation to guaiacol. Methoxyhydroquinone and guaiacol were demethylated to yield hydroxyquinol and catechol, respectively. Catechol was hydroxylated to pyrogallol. Degradation of ferulic acid by
Paecilomyces variotii
proceeded mainly via methoxyhydroquinone. The high phenol oxidase producer
Pestalotia palmarum
catabolized ferulic acid via 4-vinylguaiacol, vanillin, vanillyl alcohol, vanillic acid, and methoxyhydroquinone. However, the main reactions observed with this fungus involved polymerization reactions.
Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells
Authors: Brand B , Boos W
Published: January 2020

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Strøm, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.
Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater
Authors: Fiksdal L , Pommepuy M , Derrien A , Cormier M
Published: January 2020

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.
Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor
Authors: Chalmers J J , Kim E , Telford J N , Wong E Y , Tacon W C , Shuler M L , Wilson D B
Published: January 2020

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.
Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting
Saccharomyces cerevisiae
Cells
Authors: Senac Thomas , Hahn-Hägerdal Bärbel
Published: January 2020

Glucose and xylulose fermentation and product formation by
Saccharomyces cerevisiae
were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter
−1
, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass
−1
h
−1
for glucose and 0.39 mmol g of initial biomass
−1
h
−1
for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a
Torula
yeast and
Candida utilis
. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by
S. cerevisiae.
Esterase Activities of
Fibrobacter succinogenes
subsp.
succinogenes
S85
Authors: McDermid Kevin P. , MacKenzie C. Roger , Forsberg Cecil W.
Published: January 2020

Cells of the anaerobic ruminal bacterium
Fibrobacter succinogenes
subsp.
succinogenes
S85 (formerly
Bacteroides succinogenes
) exhibit arylesterase activity. When cells were grown on cellulose, it was found that 69% of the total esterase activity was extracellular while 65% was nonsedimentable upon centrifugation of the culture supernatant at 100,000 ×
g
. Disruption of the cells by various different methods failed to increase the esterase activity, indicating that the substrate was fully accessible to esterase enzymes in intact cells. During growth of cells with either glucose or cellulose as the sole carbon source, the increase in acetylesterase activity corresponded to an increase in cell density, suggesting constitutive production. The enzyme(s) hydrolyzed α-naphthyl,
p
-nitrophenyl, and 4-methylumbelliferyl derivatives of acetic acid; xylose tetraacetate; glucose pentaacetate; acetylxylan; and a polymer composed of ferulic acid, arabinose, and xylose in molar proportions of 1:1.1:2.2 (FAX). These data demonstrate the presence of an acetylxylan esterase and a ferulic acid esterase. The cleavage of FAX also documents the presence of an α-
l
-arabinofuranosidase.
Transduction of linked chromosomal genes between Pseudomonas aeruginosa strains during incubation in situ in a freshwater habitat
Authors: Saye D J , Ogunseitan O A , Sayler G S , Miller R V
Published: January 2020

Both transduction of single chromosomal loci and cotransduction of closely linked loci were observed between lysogenic and nonlysogenic strains of Pseudomonas aeruginosa in a freshwater habitat. Transductants were recovered at frequencies of 10(-6) to 10(-5) transductants per CFU. Transductants of lysogenized strains were recovered 10- to 100-fold more frequently than were transductants of nonlysogenic parents. Lysogens are thus capable of introducing phages which mediate generalized transduction into the natural microbial community and serving as recipients of transduced DNA. It would appear that lysogeny has the potential of increasing the size and flexibility of the gene pool available to natural populations of bacteria. The ability to generate and select new genetic combinations through phage-mediated exchange can be significant in the face of a continually changing environment and may contribute to the apparent fitness of the lysogenic state in natural ecosystems.
Toxicity of protease-resistant domains from the delta-endotoxin of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus and Aedes aegypti bioassays
Authors: Pfannenstiel M A , Cray W C , Couche G A , Nickerson K W
Published: January 2020

The mosquitocidal glycoprotein endotoxin of Bacillus thuringiensis subsp. israelensis was digested with chymotrypsin to yield protease-resistant domains which were then separated from smaller protease digestion products by high-performance liquid chromatography. Once purified, the domains no longer bound wheat germ agglutinin, a lectin which binds N-acetylglucosamine (GlcNAc) and GlcNAc oligomers. Purified protease-resistant domains were as toxic for Culex quinquefasciatus larvae as intact solubilized toxin. In separate experiments, the toxicity of chymotrypsin-digested endotoxin for Aedes aegypti larvae was reduced fivefold or more. A model is presented in which GlcNAc-containing oligosaccharides are required for toxicity for A. aegypti larvae but not C. quinquefasciatus larvae.
A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica
Authors: Kapperud G , Dommarsnes K , Skurnik M , Hornes E
Published: January 2020

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.
Development and application of oligonucleotide probes for molecular identification of Xenorhabdus species
Authors: Pütz J , Meinert F , Wyss U , Ehlers R U , Stackebrandt E
Published: January 2020

Synthetic deoxyoligonucleotide probes that hybridized against the region at positions 455 through 480 of 16S rRNA were developed for the identification of all five Xenorhabdus species. Sequence variation in the respective rRNA region between two strains of Xenorhabdus luminescens in addition allowed the construction of two strain-specific probes. Of 27 isolates determined to be Xenorhabdus strains by phenotypic characterization, 24 could be assigned to four of the five species. Two strains (HL-1 and HL-2) isolated from a Heterorhabditis sp. and a single strain (D-1.1) isolated from Steinernema affinis showed no hybridization signal with any of the five species-specific probes. With regard to the available species descriptions of nematodes, the results presented here confirm that, except for Steinernema affinis, the individual nematode hosts harbor only a single Xenorhabdus species.
Isoverrucarol production by Fusarium oxysporum CJS-12 isolated from corn
Authors: Kim K H , Lee Y W , Mirocha C J , Pawlosky R J
Published: January 2020

Isoverrucarol (3,15-dihydroxy-12,13-epoxy-trichothec-9-ene) was isolated and purified from wheat cultures of a toxic strain of Fusarium oxysporum CJS-12. The toxin was characterized by thin-layer chromatography, gas chromatography-mass spectrometry, and 1H and 13C nuclear magnetic resonance spectrometry. Isoverrucarol caused toxic effects in rats, including loss of appetite, bodily weakness, severe mucosae of the stomach, and death, when administered orally at 10 and 20 mg/kg of body weight. The toxin also caused a definite dermatitic reaction of epidermis and an edematic-necrotic response of the dermis.
Rubber-Degrading Enzyme from a Bacterial Culture
Authors: Tsuchii Akio , Takeda Kiyoshi
Published: January 2020

Rubber-degrading activity was found in the extracellular culture medium of
Xanthomonas
sp. strain 35Y which was grown on natural rubber latex. Natural rubber in the latex state was degraded by the crude enzyme, and two fractions were separately observed by gel permeation chromatography of the reaction products. One fraction was of higher molecular weight (HMW) with a very wide MW distribution from 10
3
to 10
5
, and the other fraction was of lower molecular weight (LMW) with a MW of a few hundred.
1
H-nuclear magnetic resonance spectra of the partially purified fractions were those expected of
cis
-1,4-polyisoprene mixtures with the structure OHC-CH
2
-(-CH
2
-C(-CH
3
) = CH-CH
2
-)

n

-CH
2
-C(=O)-CH
3
, with average values of
n
of about 113 and 2 for HMW and LMW fractions, respectively. The LMW fraction consisted mostly of one component in gas-liquid chromatography as well as in gel permeation chromatography, and the main component was identified as 12-oxo-4,8-dimethyl trideca-4,8-diene-1-al (acetonyl diprenyl acetoaldehyde, A

l

P
2
A
t
) by
13
C-nuclear magnetic resonance and gas chromatography-mass spectra. Not only the latices of natural and synthetic isoprene rubber, but also some kinds of low-MW polyisoprene compounds of
cis
-1,4 type, were degraded by the crude enzyme. The rubber-degrading reaction was found to be at least partly oxygenase catalyzed from the incorporation of
18
O into A

l

P
2
A
t
under an
18
O
2
atmosphere.
Reductive Dehalogenation of a Nitrogen Heterocyclic Herbicide in Anoxic Aquifer Slurries
Authors: Adrian Neal R. , Suflita Joseph M.
Published: January 2020

We studied the metabolic fate of bromacil in anaerobic aquifer slurries held under denitrifying, sulfate-reducing, or methanogenic conditions. Liquid chromatograhy-mass spectrometry of the slurries confirmed that bromacil was debrominated under methanogenic conditions but was not degraded under the other incubation conditions. This finding extends the range of aryl reductive dehalogenation reactions to include nitrogen heterocyclic compounds.
Enhancement of enterovirus infectivity in vitro by pretreating host cell monolayers with the cationic polymer polyethyleneimine
Authors: Preston D R , Bitton G , Farrah S R
Published: January 2020

Laboratory strains of enteroviruses, as well as viruses isolated from raw wastewater, were found to exhibit enhanced infectivity in vitro when BGM cell monolayers were pretreated with the cationic polymer polyethyleneimine (PEI). Viruses were assayed by the cytopathic effect technique and as PFU under methylcellulose and agar overlays with monolayers treated with 0 to 5.0 x 10(-3)% (wt/vol) PEI in phosphate-buffered saline supplemented with 2% fetal bovine serum. Poliovirus type 1 cytopathic effect occurred at an enhanced rate in cells treated with 5.0 x 10(-3)% PEI compared with untreated cells. PEI-treated cells were found to adsorb viruses much more effectively than untreated cells did. When the methylcellulose overlay procedure was used, rates of infectivity were enhanced as follows: poliovirus type 1, 5.5-fold; echovirus type 1, 1.2-fold; echovirus type 5, 5.2-fold; and coxsackievirus type B5, 4.9-fold. Viruses concentrated from raw wastewater showed a 3.8-fold increase in titer when quantitated by the most-probable-number method and a 3.3-fold increase when quantitated as PFU under an agar overlay.
Characterization of a Symbiotic Coculture of
Clostridium thermohydrosulfuricum
YM3 and
Clostridium thermocellum
YM4
Authors: Mori Yutaka
Published: January 2020

Clostridium thermohydrosulfuricum
YM3 and
C. thermocellum
YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported
C. thermohydrosulfuricum
strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in
C. thermocellum
strains: it formed spores rarely (at a frequency of <10
-5
), it required CO
2
and Na
2
CO
3
for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.
Isolation of Legionella longbeachae serogroup 1 from potting mixes
Authors: Steele T W , Lanser J , Sangster N
Published: January 2020

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.
New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water
Authors: Brenner K P , Rankin C C
Published: January 2020

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.
Characterization of Palo Podrido, a Natural Process of Delignification in Wood
Authors: Agosin Eduardo , Blanchette Robert A. , Silva Herman , Lapierre Catherine , Cease Kory R. , Ibach Rebecca E. , Abad André R. , Muga Pedro
Published: January 2020

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of
Eucryphia cordifolia
and
Nothofagus dombeyi
, which have the lowest lignin content and whose lignins have the largest amount of β-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A
Ganoderma
species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the β-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified
N. dombeyi
wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.
Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir
Authors: Paul J H , Jeffrey W H , Cannon J P
Published: January 2020

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.
Fungal transformation of fluoranthene
Authors: Pothuluri J V , Freeman J P , Evans F E , Cerniglia C E
Published: January 2020

The fungus Cunninghamella elegans ATCC 36112 metabolized approximately 80% of the 3-14C-labeled fluoranthene (FA) added within 72 h of incubation. C. elegans metabolized FA to trans-2,3-dihydroxy-2,3-dihydrofluoranthene (trans-2,3-dihydrodiol), 8- and 9-hydroxyfluoranthene trans-2,3-dihydrodiol, 3-fluoranthene-beta-glucopyranoside, and 3-(8-hydroxyfluoranthene)-beta-glucopyranoside. These metabolites were separated by thin-layer and reversed-phase high-performance liquid chromatography and identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques. The major pathway involved hydroxylation to form a glucoside conjugate of 3-hydroxyfluoranthene and a glucoside conjugate of 3,8-dihydroxyfluoranthene which together accounted for 52% of the total ethyl acetate-soluble metabolites. C. elegans initially metabolized FA in the 2,3 position to form fluoranthene trans-2,3-dihydrodiol, which has previously been shown to be a biologically active compound in mammalian and bacterial genotoxicity tests. However, C. elegans formed predominantly glucoside conjugates of the phenolic derivatives of FA, which suggests that this fungus has the potential to detoxify FA.