bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans
Authors: Rowland S S , Speedie M K , Pogell B M
Published: January 2020

A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.
Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures
Authors: Evans P J , Mang D T , Young L Y
Published: January 2020

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.
Fermentation of Inulin by
Clostridium thermosuccinogenes
sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats
Authors: Drent Wim J. , Lahpor Greetje A. , Wiegant Wim M. , Gottschal Jan C.
Published: January 2020

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H
2
, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species,
Clostridium thermosuccinogenes
sp. nov., with a G+C content of 35.9 mol%.
Organization and nucleotide sequences of two lactococcal bacteriocin operons
Authors: van Belkum M J , Hayema B J , Jeeninga R E , Kok J , Venema G
Published: January 2020

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.
Characteristics of the adhesive determinants of Lactobacillus fermentum 104
Authors: Henriksson A , Szewzyk R , Conway P L
Published: January 2020

The adhesion of Lactobacillus fermentum 104-R and the variant strain 104-S to porcine gastric squamous epithelium was investigated. An epithelium-specific adhesion was detected for strain 104-S; however, strain 104-R expressed enhanced adhesion capacity to the control surfaces of polystyrene and bovine serum albumin. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. The adhesion pattern of bacterial cells in buffers of pH values ranging from 2 to 7 was determined. The adhesion of strain 104-S to epithelium was greater in a buffer with a higher pH value. On the other hand, adhesion of strain 104-R to the epithelium was rather unaffected by a change in pH. To the control surfaces of polystyrene or bovine serum albumin, the adhesion of both strains was greatest at pH 2 to 4. Treatment of strain 104-S with metaperiodate did not affect the adhesion to epithelium or polystyrene; however, protease treatment dramatically decreased the adhesion of both strains, thus suggesting that the determinants responsible for the adhesion were proteinaceous. Carbohydrates may be partially involved in the adhesion of 104-R because metaperiodate-treated cells adhered more poorly than control, iodate-treated cells. The adhesion-promoting components are most probably tightly bound to the cell wall, because washing with low-pH buffer (pH 1.2) or sodium dodecyl sulfate had no major effect on the adhesion.
Microbial transformation of zearalenone to a zearalenone sulfate
Authors: el-Sharkaway S H , Selim M I , Afifi M S , Halaweish F T
Published: January 2020

The conversion of zearalenone by various microorganisms was studied. A new polar metabolite was formed in addition to alpha- and beta-zearalenols. The structure of the new metabolite was determined as zearalenone-4-O-sulfate conjugate on the basis of enzymatic and acid hydrolysis, followed by mass spectrometry, nuclear magnetic resonance, and infrared spectroscopic analysis. The results obtained demonstrate that Rhizopus arrhizus catalyzes sulfation of zearalenone at the C-4 hydroxyl group.
Species-specific detection of Listeria monocytogenes by DNA amplification
Authors: Deneer H G , Boychuk I
Published: January 2020

The polymerase chain reaction was used to detect and specifically identify Listeria monocytogenes. A 174-bp region of the listeriolysin O gene was shown to be specifically amplified in L. monocytogenes but not in other species of Listeria or in a number of other gram-positive and gram-negative organisms. Less than 50 organisms could routinely be detected by a procedure involving two rounds of 35 amplification cycles each and without the need for subsequent hybridization with labeled probes.
Continuous Production of Long-Side-Chain Poly-β-Hydroxyalkanoates by
Pseudomonas oleovorans
Authors: Ramsay Bruce A. , Saracovan Ilie , Ramsay Juliana A. , Marchessault Robert H.
Published: January 2020

Shake flask experiments showed that
Pseudomonas oleovorans
began to be growth inhibited at 4.65 g of sodium octanoate liter
-1
, with total inhibition at 6 g liter
-1
. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter
-1
in the feed, the maximum specific growth rate was 0.51 h
-1
, and the maximum specific rate of poly-β-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein
-1
h
-1
at a dilution rate (
D
) of 0.25 h
-1
. When the specific growth rate (μ) was <0.3 h
-1
, the PHA composition was relatively constant with a C
4
/C
6
/C
8
/C
10
ratio of 0.1:1.7:20.7:1.0. At μ > 0.3 h
-1
, a decrease in the percentage of C
8
with a concomitant increase in C
10
monomers as μ increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At
D
= 0.24 h
-1
and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate
-1
consumed, the yield of PHA was 0.085 g of PHA g of octanoate
-1
used, and 7.9 g of octanoate was consumed for each gram of NH
4
+
supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass
-1
h
-1
. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.
Biodegradation of Degradable Plastic Polyethylene by
Phanerochaete
and
Streptomyces
Species
Authors: Lee Byungtae , Pometto Anthony L. , Fratzke Alfred , Bailey Theodore B.
Published: January 2020

The ability of lignin-degrading microorganisms to attack degradable plastics was investigated in pure shake flask culture studies. The degradable plastic used in this study was produced commercially by using the Archer-Daniels-Midland POLYCLEAN masterbatch and contained pro-oxidant and 6% starch. The known lignin-degrading bacteria
Streptomyces viridosporus
T7A,
S. badius
252, and
S. setonii
75Vi2 and fungus
Phanerochaete chrysosporium
were used. Pro-oxidant activity was accelerated by placing a sheet of plastic into a drying oven at 70°C under atmospheric pressure and air for 0, 4, 8, 12, 16, or 20 days. The effect of 2-, 4-, and 8-week longwave UV irradiation at 365 nm on plastic biodegradability was also investigated. For shake flask cultures, plastics were chemically disinfected and incubated-shaken at 125 rpm at 37°C in 0.6% yeast extract medium (pH 7.1) for
Streptomyces
spp. and at 30°C for the fungus in 3% malt extract medium (pH 4.5) for 4 weeks along with an uninoculated control for each treatment. Weight loss data were inconclusive because of cell mass accumulation. For almost every 70°C heat-treated film, the
Streptomyces
spp. demonstrated a further reduction in percent elongation and polyethylene molecular weight average when compared with the corresponding uninoculated control. Significant (
P
< 0.05) reductions were demonstrated for the 4- and 8-day heat-treated films by all three bacteria. Heat-treated films incubated with
P. chrysosporium
consistently demonstrated higher percent elongation and molecular weight average than the corresponding uninoculated controls, but were lower than the corresponding zero controls (heat-treated films without 4-week incubation). The 2- and 4-week UV-treated films showed the greatest biodegradation by all three bacteria. Virtually no degradation by the fungus was observed. To our knowledge, this is the first report demonstrating bacterial degradation of these oxidized polyethylenes in pure culture.
Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"
Authors: Lüthi E , Jasmat N B , Grayling R A , Love D R , Bergquist P L
Published: January 2020

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.
Methodological Modifications for Accurate and Efficient Determination of Contaminant Biodegradation in Unsaturated Calcareous Soils
Authors: Watwood Mary E. , White Carleton S. , Dahm Clifford N.
Published: January 2020

Many techniques for quantifying microbial biodegradation of
14
C-labeled compounds use soil-water slurries and trap mineralization-derived
14
CO
2
in solution wells suspended within the incubation flasks. These methods are not satisfactory for studies of arid-region soils that are highly calcareous and unsaturated because (i) slurries do not simulate unsaturated conditions and (ii) the amount of CO
2
released from calcareous soils exceeds the capacity of the suspended well. This report describes simple, inexpensive methodological modifications for quantifying microbial degradation of [
14
C]benzene and 1,2-dichloro[U-
14
C]ethane in calcareous soils under unsaturated conditions. Soils at 50% water holding capacity were incubated with labeled contaminants for periods up to 10 weeks, followed by acidification of the soil and trapping of the evolved CO
2
in a separate container of 2 N NaOH. The CO
2
was transferred from the incubation flask to the trap solution by a gas transfer shunt containing activated charcoal to remove any volatilized labeled organics. The amount of
14
CO
2
in the trap solution was measured by scintillation counting (disintegrations per minute). The method was tested by using two regional unamended surface soils, a sandy aridisol and a clay-rich riparian soil. The results demonstrated that both [
14
C]benzene and 1,2-dichloro[U-
14
C]ethane were mineralized to release substantial amounts of
14
CO
2
within 10 weeks. Levels of mineralization varied with contaminant type, soil type, and aeration status (anaerobic vs. aerobic); no significant degradation was observed in abiotic control samples. Methodological refinements of this technique resulted in total
14
CO
2
recovery efficiency of approximately 90%.
Pathway for Biodegradation of
p
-Nitrophenol in a
Moraxella
sp
Authors: Spain Jim C. , Gibson David T.
Published: January 2020

A
Moraxella
strain grew on
p
-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on
p
-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl,
p
-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of
p
-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD
+
, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from
p
-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of
p
-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.
Analysis of Free Amino Acids in Microbially Colonized Sandstone by Precolumn Phenyl Isothiocyanate Derivatization and High-Performance Liquid Chromatography
Authors: Siebert Jörg , Palmer Robert J. , Hirsch Peter
Published: January 2020

A fast, sensitive method for extraction and analysis of soluble free amino acids from microbially colonized sandstone is described. After precolumn phenyl isothiocyanate (PITC) derivatization, the PITC-amino acids were identified and quantified by reversed-phase high-performance liquid chromatography. This kind of analysis could be used to elucidate the role and function of amino acids in the nutrition of epi- and endolithic microorganisms active in biological weathering processes.
Mortality of fecal bacteria in seawater
Authors: Garcia-Lara J , Menon P , Servais P , Billen G
Published: January 2020

We propose a method for determining the mortality rate for allochthonous bacteria released in aquatic environments without interference due to the loss of culturability in specific culture media. This method consists of following the disappearance of radioactivity from the trichloroacetic acid-insoluble fraction in water samples to which [3H]thymidine-prelabeled allochthonous bacteria have been added. In coastal seawater, we found that the actual rate of disappearance of fecal bacteria was 1 order of magnitude lower than the rate of loss of culturability on specific media. Minor adaptation of the procedure may facilitate assessment of the effect of protozoan grazing and bacteriophage lysis on the overall bacterial mortality rate.
Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage
Authors: Walter T M , Aronson A I
Published: January 2020

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.
Trajectory of aerosol droplets from a sprayed bacterial suspension
Authors: Lighthart B , Shaffer B T , Marthi B , Ganio L
Published: January 2020

Simulated droplet trajectories of a polydispersed microbial aerosol in a laminar air flow regimen were compared with observed dispersal patterns of aerosolized Bacillus subtilis subsp. niger spores in quasilaminar airflow. Simulated dispersal patterns could be explained in terms of initial droplet sizes and whether the droplets evaporated to residual aeroplanktonic size before settling to the ground. For droplets that evaporated prior to settling out, a vertical downwind size fractionation is predicted in which the microbial residue of the smallest droplets settles the least, and is found in the airstream at about sprayer height, and progressively larger droplet residues settle to progressively lower heights. Observations of spore particle size distributions downwind from a spray source support the simulation. Droplet and particle size distributions near the source had three size fractions: one containing large, presumably nonevaporated droplets of greater than or equal to 7 microns in diameter, and two smaller fractions, with diameters of 2 to 3 microns (probably the residue of droplets containing more than one spore) and 1 to 2 microns (probably the residue from single-spore droplets). As predicted by the simulation, the aerosol settled and progressed downwind, with the number of small droplets and particles increasing in proportion to the height and distance downwind.
Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid
Authors: Bej A K , DiCesare J L , Haff L , Atlas R M
Published: January 2020

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.
Production and characterization of antibodies against nivalenol tetraacetate
Authors: Wang C R , Chu F S
Published: January 2020

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.
Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells
Authors: Alvarez-Cohen L , McCarty P L
Published: January 2020

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.
Modeling of bacterial growth as a function of temperature
Authors: Zwietering M H , de Koos J T , Hasenack B E , de Witt J C , van't Riet K
Published: January 2020

The temperature of chilled foods is a very important variable for microbial safety in a production and distribution chain. To predict the number of organisms as a function of temperature and time, it is essential to model the lag time, specific growth rate, and asymptote (growth yield) as a function of temperature. The objective of this research was to determine the suitability and usefulness of different models, either available from the literature or newly developed. The models were compared by using an F test, by which the lack of fit of the models was compared with the measuring error. From the results, a hyperbolic model was selected for the description of the lag time as a function of temperature. Modified forms of the Ratkowsky model were selected as the most suitable model for both the growth rate and the asymptote as a function of temperature. The selected models could be used to predict experimentally determined numbers of organisms as a function of temperature and time.
16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA
Authors: Weller R , Weller J W , Ward D M
Published: January 2020

Cloning and analysis of cDNAs synthesized from rRNAs is one approach to assess the species composition of natural microbial communities. In some earlier attempts to synthesize cDNA from 16S rRNA (16S rcDNA) from the Octopus Spring cyanobacterial mat, a dominance of short 16S rcDNAs was observed, which appear to have originated only from certain organisms. Priming of cDNA synthesis from small ribosomal subunit RNA with random deoxyhexanucleotides can retrieve longer sequences, more suitable for phylogenetic analysis. Here we report the retrieval of 16S rRNA sequences from three formerly uncultured community members. One sequence type, which was retrieved three times from a total of five sequences analyzed, can be placed in the cyanobacterial phylum. A second sequence type is related to 16S rRNAs from green nonsulfur bacteria. The third sequence type may represent a novel phylogenetic type.
Intestinal carriage of Yersinia pseudotuberculosis by wild birds and mammals in Japan
Authors: Fukushima H , Gomyoda M
Published: January 2020

Fecal specimens were obtained from wild birds and mammals in the eastern part of Shimane Prefecture, Japan, an area where serotype 1b, 2b, 3, and 4b strains of Yersinia pseudotuberculosis were found to be prevalent in humans. Each of 869 animals, including 259 wild birds and 610 wild mammals, was screened for yersiniae. A total of 37 strains of Y. pseudotuberculosis were isolated from 34 (5.6%) mammals, including 23 raccoon dogs (Nyctereutes procyonoides), eight deer (Cervus nippon), two hares (Lepus brachyurus), and one marten (Martes melampus), and from two (0.8%) birds, including one eastern spot-billed duck (Anas poecilorhyncha) and one wigeon (Anas penelope). The Y. pseudotuberculosis isolates collected belonged to virulence plasmid-positive (serotypes 1b, 2b, 3, 4b, and 6) and virulence plasmid-negative (serotype 5a) strains, the most predominant serotype being 4b. The close relationship between the regional distributions of Y. pseudotuberculosis in wild animals and humans suggests that wild animals are an important source of infection.
Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli
Authors: Heipieper H J , Keweloh H , Rehm H J
Published: January 2020

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.
Development and application of new positively charged filters for recovery of bacteriophages from water
Authors: Borrego J J , Cornax R , Preston D R , Farrah S R , McElhaney B , Bitton G
Published: January 2020

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.
Mold flora and aflatoxin contamination of stored and cooked samples of pearl millet in the Paharia tribal belt of Santhal paragana, Bihar, India
Authors: Mishra N K , Daradhiyar S K
Published: January 2020

Stored and cooked samples of pearl millet (Pennesetum typhoides), which is regularly consumed as food by the Paharia tribe in the hilly regions of Santhal Pargana, Bihar State, India, that were harvested in January 1989 were analyzed for mold flora, natural occurrence of Aspergillus flavus and A. parasiticus, and incidence and levels of aflatoxin B1. Of the 22 fungal species isolated, A. flavus and A. parasiticus were the predominant species (63.8%) during the rainy season, followed by other species of Aspergillus, Penicillium, Fusarium, Rhizopus, Helminthosporium, and Curvularia. Screening of 169 A. flavus and A. parasiticus strains showed that 59 of them were toxigenic, producing various combinations of aflatoxins B1, B2, G1, and G2. The amounts of aflatoxin B1 ranged between 4 and 30 mg/100 ml of liquid medium. Analysis of stored and cooked samples also revealed a high incidence and alarming levels of naturally produced aflatoxin B1. Forty-nine of 75 stored and 16 of 38 cooked samples contained various combinations of aflatoxins. The levels of aflatoxin B1 ranged between 17 and 2,110 ppb in stored samples and 18 and 549 ppb in cooked samples. The correlation of insect damage with A. flavus and A. parasiticus incidence and quantity of aflatoxin B1 was found to be insignificant.
Aerobic mineralization of vinyl chloride by a bacterium of the order Actinomycetales
Authors: Phelps T J , Malachowsky K , Schram R M , White D C
Published: January 2020

A gram-positive branched bacterium isolated from a trichloroethylene-degrading consortium mineralized vinyl chloride in growing cultures and cell suspensions. Greater than 67% of the [1,2-14C]vinyl chloride was mineralized to carbon dioxide, with approximately 10% of the radioactivity appearing in cell biomass and another 10% appearing in 14C-aqueous-phase products.
Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized
Pseudomonas syringae
Authors: Buttner Mark P. , Stetzenbach Linda D.
Published: January 2020

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released
Pseudomonas syringae
. The upper limits of the impactor samplers were exceeded at a spray concentration of 10
7
CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.
Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene
Authors: van der Laan J C , Gerritse G , Mulleners L J , van der Hoek R A , Quax W J
Published: January 2020

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.
A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations
Authors: Kleman G L , Chalmers J J , Luli G W , Strohl W R
Published: January 2020

A combined predictive and feedback control algorithm based on measurements of the concentration of glucose on-line has been developed to control fed-batch fermentations of Escherichia coli. The predictive control algorithm was based on the on-line calculation of glucose demand by the culture and plotting a linear regression to the next datum point to obtain a predicted glucose demand. This provided a predictive "coarse" control for the glucose-based nutrient feed. A direct feedback control using a proportional controller, based on glucose measurements every 2 min, fine-tuned the feed rate. These combined control schemes were used to maintain glucose concentrations in fed-batch fermentations as tight as 0.49 +/- 0.04 g/liter during growth of E. coli to high cell densities.
Detection of Giardia cysts with a cDNA probe and applications to water samples
Authors: Abbaszadegan M , Gerba C P , Rose J B
Published: January 2020

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.
Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris
Authors: Salama M , Sandine W , Giovannoni S
Published: January 2020

Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.
Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus
Authors: Slos P , Bourquin J C , Lemoine Y , Mercenier A
Published: January 2020

A promoter probe vector, pTG244, was constructed with the aim of isolating transcription initiation signals from Streptococcus thermophilus (Streptococcus salivarius subsp. thermophilus). pTG244 is based on the Escherichia coli-streptococcus shuttle vector pTG222, into which the promoterless chloramphenicol acetyltransferase gene of Bacillus pumilus (cat-86) was cloned. Random Sau3A fragments from the S. thermophilus A054 chromosomal DNA were cloned upstream of the cat-86 gene by using E. coli as the host. The pool of recombinant plasmids were introduced into S. thermophilus and Lactococcus lactis subsp. lactis in order to search for promoter activity in these hosts. For S. thermophilus, it was necessary to first select erythromycin-resistant transformants and then to screen for chloramphenicol resistance among these. Direct selection of chloramphenicol-resistant clones was, however, possible in L. lactis subsp. lactis. Six fragments exhibiting promoter activity were characterized in S. thermophilus by measuring the levels of cat-86 transcription and/or chloramphenicol acetyltransferase specific activity. Three of the promoter-carrying fragments were sequenced. The 5' ends of their corresponding mRNAs were determined by S1 mapping and shown to correspond to a purine residue in all cases. Upstream from these potential transcription start points, sequences homologous to the E. coli sigma 70 and the Bacillus subtilis vegetative sigma 43 (or sigma A) consensus promoters were identified.
Comparison of Representative Strains of Infectious Hematopoietic Necrosis Virus by Serological Neutralization and Cross-Protection Assays
Authors: Engelking H. Mark , Harry Jo Beth , Leong Jo-Ann C.
Published: January 2020

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.
Characterization of the Heat Shock Response in
Lactococcus lactis
subsp.
lactis
Authors: Whitaker Richard D. , Batt Carl A.
Published: January 2020

The heat shock response in
Lactococcus lactis
subsp.
lactis
was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of
L. lactis
subsp.
lactis
, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10
3
; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with
35
S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding
Escherichia coli
proteins. Therefore, we conclude that
L. lactis
subsp.
lactis
displays a heat shock response similar to that observed in other mesophilic bacteria.
Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by
Pseudomonas
Strains
Authors: Sander Peter , Wittich Rolf-Michael , Fortnagel Peter , Wilkes Heinz , Francke Wittko
Published: January 2020

Two
Pseudomonas
sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary
cis
-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD
+
-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent
ortho
cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.