bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria
Authors: Wu W M , Hickey R F , Zeikus J G
Published: January 2020

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.
Measurement of Acetate Concentrations in Marine Pore Waters by Using an Enzymatic Approach
Authors: King Gary M.
Published: January 2020

Acetate concentrations in marine and freshwater matrices were measured by an enzymatic technique which coupled the synthesis of acetyl coenzyme A to AMP production. The resulting AMP was assayed by a sensitive and relatively rapid high-pressure liquid chromatography method, using an aqueous, isocratic mobile phase for elution. The method was insensitive to the presence of seawater salts and required no sample prepurification or distillation. Propionate caused a minor, but statistically insignificant, interference when equimolar with acetate; butyrate caused no interference, even at relatively high concentrations. Detection limits for acetate were approximately 100 nM with a precision of about 5%. Pore waters from two intertidal sediments contained approximately 1 to 12 μM acetate; the concentrations were linearly but inversely correlated with porewater sulfate.
Isolation and Characterization of a Competition-Defective
Bradyrhizobium japonicum
Mutant
Authors: Bhagwat Arvind A. , Tully Raymond E. , Keister Donald L.
Published: January 2020

Tn
5
mutagenesis was coupled with a competition assay to isolate mutants of
Bradyrhizobium japonicum
defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn
5
insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.
Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples
Authors: Bej A K , Mahbubani M H , Dicesare J L , Atlas R M
Published: January 2020

To detect low levels of microorganisms in environmental samples by using polymerase chain reaction (PCR)-gene probe detection, samples were concentrated by filtration. Fluoropore (Millipore Corp.) filters were compatible with PCR DNA amplification, whereas various other filters including nitrocellulose and cellulose acetate filters inhibited PCR amplification. By concentrating cells on Fluoropore filters and releasing the DNA by freeze-thaw cycling, PCR DNA amplification could be performed without removing the filter. Concentration with Fluoropore FHLP and FGLP filters permitted the detection of single cells of microorganisms in 100-ml samples by PCR-gene probes.
1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon
Authors: Tong I T , Liao H H , Cameron D C
Published: January 2020

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.
α-Glucuronidase and Other Hemicellulase Activities of
Fibrobacter succinogenes
S85 Grown on Crystalline Cellulose or Ball-Milled Barley Straw
Authors: Smith D. C. , Forsberg C. W.
Published: January 2020

Fibrobacter succinogenes
produces an α-glucuronidase which cleaves 4-
O
-methyl-α-
d
-glucuronic acid from birch wood 4-
O
-methyl-α-
d
-glucuronoxylan. Very low levels of α-glucuronidase activity were detected in extracellular enzyme preparations of
F. succinogenes
on birch wood xylan substrate. The release of 4-
O
-methyl-α-
d
-glucuronic acid was enhanced when the birch wood xylan substrate was predigested by either a purified
Schizophyllum commune
xylanase or a cloned
F. succinogenes
S85 xylanase. These data suggest that the α-glucuronidase is unable to cleave 4-
O
-methyl-α-
d
-glucuronic acid from intact xylan but can act on unique low-molecular-weight glucuronoxylan fragments created by the cloned
F. succinogenes
xylanase. The cloned xylanase presumably must account for a small proportion of the indigenous xylanase activity of
F. succinogenes
cultures, since this xylanase source does not support high glucuronidase activity. The α-glucuronidase and associated hemicellulolytic enzymes exhibited higher activities in culture fluid from cells grown on ball-milled barley straw than in that of cellulose-grown cells. The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on barley straw was more complex than that of culture filtrates from cells grown on cellulose. These data demonstrate that
F. succinogenes
produces an α-glucuronidase with an exacting substrate specificity which enables extensive cleavage of glucuronic acid residues from xylan as a consequence of synergistic xylanase action.
Effect of gene amplification on mercuric ion reduction activity of Escherichia coli
Authors: Philippidis G P , Malmberg L H , Hu W S , Schottel J L
Published: January 2020

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.
Cloning and Detection of Chromosomal and Extrachromosomal DNA from Mycoplasmalike Organisms That Cause Yellow Dwarf Disease of Rice
Authors: Nakashima Kazuo , Kato Shosuke , Iwanami Setsuo , Murata Nobuo
Published: January 2020

DNA was extracted from rice plants infected with mycoplasmalike organisms (MLOs) causing yellow dwarf disease. DNA of the causal agent was separated from the host DNA by repeated bisbenzimide-CsCl equilibrium density gradient centrifugations. MLO DNA cut by
Hin
dIII was ligated into plasmid Bluescript II and cloned in
Escherichia coli
NM522. The DNA inserts were labeled with peroxidase and employed as probes in hybridization. Southern analysis revealed that the insert in pRYD-12 consisted of one, presumably chromosomal, piece of MLO DNA, whereas the insert in pRYD-19, another recombinant plasmid, consisted of one, presumably extrachromosomal, piece of MLO DNA. Cloned DNA probes were successfully applied in dot blot hybridization for the detection of rice yellow dwarf disease MLOs in rice plants and in an insect vector, the green rice leafhopper (
Nephotettix cincticeps
).
19
F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of
Nectria haematococca
Authors: Parisot Denise , Malet-Martino Marie C. , Martino Robert , Crasnier Philippe
Published: January 2020

19
F nuclear magnetic resonance spectroscopy was used to study the metabolism of 5-fluorouracil in four strains of
Nectria haematococca
which displayed similar sensitivities to growth inhibition by this compound but differed in their pigmentation. The major metabolites, 5-fluorouridine and α-fluoro-β-alanine, were excreted into the medium by all four strains. The classical ribofluoronucleotides (5-fluorouridine-5′-monophosphate, -diphosphate, and -triphosphate) and α-fluoro-β-alanine were identified in the acid-soluble fraction of perchloric acid extracts of mycelia. Two hydrolysis products of 5-fluorouracil incorporated into RNA were found in the acid-insoluble pool. They were unambiguously assigned to 5-fluorouridine-2′-monophosphate and 3′-monophosphate with specific hydrolysis reactions on isolated RNA. The lack of fluorodeoxyribonucleotides and the fact that the four strains incorporated similar amounts of fluororibonucleotides into their RNAs strongly suggest an RNA-directed mechanism of cytotoxicity for 5-fluorouracil. The heavily pigmented wild type differed from the three low-pigmented strains in its low uptake of 5-fluorouracil and, consequently, in its reduced biosynthesis of 5-fluorouridine and α-fluoro-β-alanine. At present, it is not clear whether this change in 5-fluorouracil metabolism is a side effect of pigment production or results from another event.
Inhibition of bacterial and phytoplanktonic metabolic activity in the lower River Rhine by ditallowdimethylammonium chloride
Authors: Tubbing D M , Admiraal W
Published: January 2020

The effects of a quaternary ammonium compound, ditallowdimethylammonium chloride (DTDMAC), on natural populations of bacteria and phytoplankton from the lower River Rhine were examined to estimate their sensitivity to the discharges of cationic surfactants in the river basin. In short-term experiments, significant decreases in the growth rate of bacterioplankton and in the photosynthetic rate of phytoplankton were observed at a nominal concentration of 0.03 to 0.1 mg of DTDMAC liter-1. Nitrification was measured with an ion-selective electrode and by the rate of acid production in ammonium-spiked river water and was found to be only sensitive to the addition of concentrations higher than 1 mg of DTDMAC liter-1. This does not support an earlier suggestion that ammonium-oxidizing bacteria are specifically sensitive to quaternary ammonium compounds. The effect of DTDMAC on thymidine incorporation was shown to depend strongly on the concentration of suspended material, which varied with the sampling date. This effect was also quantified in experimental manipulations with Rhine water. Calculations on the partitioning of DTDMAC between water and suspended matter confirmed the role of suspended solids and showed that an increase of the dissolved DTDMAC concentration in Rhine water by circa 0.01 mg liter-1 leads to a slight inhibition of the growth of heterotrophic bacteria. It is concluded that a total concentration of circa 0.01 mg of DTDMAC liter-1 measured in the River Rhine is likely to have biological consequences.
Immunocytochemical localization of nitrogenase in bacteria symbiotically associated with Azolla spp
Authors: Lindblad P , Bergman B , Nierzwicki-Bauer S A
Published: January 2020

In situ immunogold labeling and transmission electron microscopy were used to detect nitrogenase in bacteria (bactobionts) symbiotically associated with leaf cavities of Azolla caroliniana and Azolla filiculoides. In A. caroliniana, the Fe protein of the nitrogenase complex was detected in a subset of the distinct bactobiont types present in leaf cavities of all ages. Similar results were obtained for the bactobionts of A. filiculoides with antisera against both the Fe and MoFe subunits of nitrogenase.
Use of Bioluminescence Markers To Detect
Pseudomonas
spp. in the Rhizosphere
Authors: de Weger Letty A. , Dunbar Paul , Mahafee Walter F. , Lugtenberg Ben J. J. , Sayler Gary S.
Published: January 2020

The use of bioluminescence as a sensitive marker for detection of
Pseudomonas
spp. in the rhizosphere was investigated. Continuous expression of the
luxCDABE
genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible
luxCDABE
construct or a constitutive
luxAB
construct (coding only for the luciferase) was introduced into the
Pseudomonas
cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase,
n
-decyl aldehyde, respectively. These
Pseudomonas
cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10
3
to 10
4
CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems.
Diglyceride Kinase Activity in Cell Extracts of
Rhizobium meliloti
: Evidence for a Diglyceride Cycle during Cyclic β-1,2-Glucan Biosynthesis
Authors: Hunt William P. , Gore Richard S. , Miller Karen J.
Published: January 2020

In this article, we provide evidence for the presence of diglyceride kinase activity in cell extracts of
Rhizobium meliloti
1021. Characterization of the rhizobial enzyme revealed that it shares many properties with the diglyceride kinase of
Escherichia coli
. A possible role for this enzyme during cyclic β-1,2-glucan biosynthesis is discussed.
Lactobacilli and azoreductase activity in the murine cecum
Authors: McConnell M A , Tannock G W
Published: January 2020

Azoreductase activity in the ceca of mice lacking lactobacilli as members of the normal microflora (reconstituted-lactobacillus-free [RLF] mice) was compared with that of RLF mice whose gastrointestinal tracts were colonized by strains of Lactobacillus delbrueckii and Lactobacillus fermentum. Azoreductase activity was 31% lower in the ceca of mice colonized by lactobacilli.
Improved Technique for Identification and Enumeration of Methanogenic Bacterial Colonies on Roll Tubes by Epifluorescence Microscopy
Authors: Kataoka Naoaki , Tokiwa Yutaka , Takeda Kiyoshi
Published: January 2020

Methanogenic fluorescent colonies can be clearly identified on roll tubes by using an epifluorescence microscope equipped with a × 2 objective. Methanogenic and nonmethanogenic colonies could be counted in roll tubes prepared from methanogenic enrichment cultures. Late-developing colonies appearing after 25 days of incubation were mainly methanogenic.
Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus
Authors: Benbadis L , Garel J R , Hartley D L
Published: January 2020

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.
Lipid Content and Cryotolerance of Bakers' Yeast in Frozen Doughs
Authors: Gélinas Pierre , Fiset Gisèle , Willemot Claude , Goulet Jacques
Published: January 2020

The relationship between lipid content and tolerance to freezing at −50°C was studied in
Saccharomyces cerevisiae
grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness).
Biolistic transformation of a procaryote, Bacillus megaterium
Authors: Shark K B , Smith F D , Harpending P R , Rasmussen J L , Sanford J C
Published: January 2020

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.
Rapid Immunocapture of
Pseudomonas putida
Cells from Lake Water by Using Bacterial Flagella
Authors: Morgan J. Alun W. , Winstanley Craig , Pickup Roger W. , Saunders Jon R.
Published: January 2020

Monoclonal antibodies to
Pseudomonas putida
Paw340 cells were produced. In an enzyme-linked immunosorbent assay (ELISA) against whole bacterial cells, a hybridoma cell line termed MLV1 produced a monoclonal antibody that reacted with
P. putida
Paw340 but showed no cross-reaction with 100 medical isolates and 150 aquatic isolates. By ELISA, immunogold electron microscopy, and Western blot (immunoblot) analysis, MLV1 antibody was found to react with purified bacterial flagella. The surfaces of magnetic polystyrene beads were coated with MLV1 antibody. By mixing MLV1 antibody-coated beads with lake water samples containing the target
P. putida
host, bead-cell complexes which could be recovered by attraction towards a magnet were formed. Prevention of nonspecific attachment of cells to the beads required the incorporation of detergents in the isolation protocol. These detergents affected colony-forming ability; however, the cells remained intact for direct detection. When reisolated by standard cultural methods, approximately 20% of the initial target population was recovered. Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched. This method may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.
Genetic construction of nisin-producing Lactococcus lactis subsp. cremoris and analysis of a rapid method for conjugation
Authors: Broadbent J R , Kondo J K
Published: January 2020

Conjugation was used to construct nisin-producing Lactococcus lactis subsp. cremoris strains. Recipients were obtained by electroporation of L. lactis subsp. cremoris strains with the drug resistance plasmid pGK13 or pGB301. A method, direct-plate conjugation, was developed in which donor and recipient cells were concentrated and then combined directly on selective media. This method facilitated transfer of the nisin-sucrose (Nip+ Suc+) phenotype from the donor strain, L. lactis subsp. lactis 11454, to three L. lactis subsp. cremoris recipient strains. Nip+ Suc+ L. lactis subsp. cremoris transconjugants were obtained at frequencies which ranged from 10(-7) to 10(-8) per donor CFU. DNA-DNA hybridization to transconjugant DNAs, performed with an oligonucleotide probe synthesized to detect the nisin precursor gene, showed that this gene was transferred during conjugation but was not associated with detectable plasmid DNA. Further investigation indicated that L. lactis subsp. cremoris Nip+ Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. Results suggested that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for application as mixed and multiple starter systems. Additionally, the direct-plate conjugation method required less time than filter or milk agar matings and may also be useful for investigations of conjugal mechanisms in these organisms.
Gut microflora of vervet and samango monkeys in relation to diet
Authors: Bruorton M R , Davis C L , Perrin M R
Published: January 2020

The microflora in the gastrointestinal tracts of wild vervet and samango monkeys (Cercopithecus aethiops and C. mitis, respectively) were studied, using fermentation acid analysis, electron microscopy, and culturing methods. The diets of the two species of monkey differ considerably, with that of the samango including a greater proportion of cellulose-rich leaf material, and this is reflected in the microflora. Volatile fatty acid measurements along the gut of both species showed that these end products of bacterial metabolism were concentrated in the cecum and colon. Electron microscopy indicated that morphologically similar bacteria were present in the cecum and colon of both species, but the samango possessed a distinct stomach microflora. Bacteria in the lumina of the four main regions of the gut of the monkeys (stomach, small intestine, cecum, and colon) were plated on a number of anaerobic media (Mann, Rogosa, and Sharp; clostridial basal; and complex media). The cecum and colon were found to contain higher numbers of microbes per gram (wet weight) of gut content than the stomach and small intestine. Microbial isolates were able to catabolize carboxymethyl cellulose and other polymers. This may aid the monkeys, particularly samangos, in the digestion of fibrous dietary components such as leaves.
Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods
Authors: Bej A K , Mahbubani M H , Atlas R M
Published: January 2020

Methods using polymerase chain reaction (PCR) and gene probes to detect viable Legionella pneumophila were investigated with cells exposed to biocide or elevated temperature. Exposure to hypochlorite caused viable nonculturable cells to form. Culturable and viable nonculturable cells showed positive PCR amplification, whereas nonviable cells did not. Viable cells were also specifically detected with mip mRNA as the target, reverse transcription (to form cDNA), and PCR amplification. After exposure to elevated temperature, only viable culturable cells were detected, which corresponded with positive PCR amplification.
Improved method for coliform verification
Authors: Diehl J D
Published: January 2020

Modification of a method for coliform verification presented in Standard Methods for the Examination of Water and Wastewater is described. Modification of the method, which is based on beta-galactosidase production, involves incorporation of a lactose operon inducer in medium upon which presumptive coliform isolates are cultured prior to beta-galactosidase assay.
Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase
Authors: Pogell B M , Rowland S S , Steinmann K E , Speedie M K , Hoskin F C
Published: January 2020

Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate. Studies with wild-type and recombinant strains of S. lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S. lividans of other significant hydrolytic enzymatic activity towards soman.
High-Molecular-Weight Amylase Activities from Bacteria Degrading Starch-Plastic Films
Authors: Burgess-Cassler Anthony , Imam Syed H. , Gould J. Michael
Published: January 2020

Amylases having unusually high molecular weights (
M
r
, >150,000) were found in culture supernatants of an environmentally derived microbial mixed culture selected for its ability to utilize starch-containing plastic films as sole carbon sources. The mixed culture produced amylases active at pHs 5.5 and 8.0.
Rate of Pyrite Bioleaching by
Thiobacillus ferrooxidans
: Results of an Interlaboratory Comparison
Authors: Olson Gregory J.
Published: January 2020

Ten laboratories participated in an interlaboratory comparison of determination of bioleaching rates of a pyrite reference material. A standardized procedure and a single strain of
Thiobacillus ferrooxidans
were used in this study. The mean rate of bioleaching of the pyrite reference material was 12.4 mg of Fe per liter per h, with a coefficient of variation (percent relative standard deviation) of 32% as determined by eight laboratories. These results show the precision among laboratories of the determination of rates of pyrite bioleaching when a standard test procedure and reference material are used.
Accumulation of Trehalose and Sucrose in Cyanobacteria Exposed to Matric Water Stress
Authors: Hershkovitz Nitzan , Oren Aharon , Cohen Yehuda
Published: January 2020

The drought-resistant cyanobacteria
Phormidium autumnale
, strain LPP
4
, and a
Chroococcidiopsis
sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 μg of trehalose per μg of chlorophyll in
P. autumnale
, 6.9 μg of sucrose per μg of chlorophyll in LPP
4
, and 4.1 μg of sucrose and 3.2 μg of trehalose per μg of chlorophyll in the
Chroococcidiopsis
sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (
Plectonema boryanum
and
Synechococcus
strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.
Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination
Authors: Abouzied M M , Azcona J I , Braselton W E , Pestka J J
Published: January 2020

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.
Use of a Plasmid DNA Probe To Monitor Populations of
Bacillus pumilus
Inoculant Strains in Hay
Authors: Hendrick Carol A. , Smiley Brenda K. , Shelley Terrence H. , Tomes Nancy J.
Published: January 2020

We are evaluating naturally occurring isolates of
Bacillus pumilus
for use as microbial hay preservatives. Seven isolates of
B. pumilus
from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of
B. pumilus
and other
Bacillus
spp. were probed with
32
P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb
Hin
dIII-
Pst
I fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.
Use of Ultrafiltration To Isolate Viruses from Seawater Which Are Pathogens of Marine Phytoplankton
Authors: Suttle Curtis A. , Chan Amy M. , Cottrell Matthew T.
Published: January 2020

Viruses may be major structuring elements of phytoplankton communities and hence important regulators of nutrient and energy fluxes in aquatic environments. In order to ascertain whether viruses are potentially important in dictating phytoplankton community structure, it is essential to determine the extent to which representative phytoplankton taxa are susceptible to viral infection. We used a spiral ultrafiltration cartridge (30,000-molecular-weight cutoff) to concentrate viruses from seawater at efficiencies approaching 100%. Natural virus communities were concentrated from stations in the Gulf of Mexico, a barrier island pass, and a hypersaline lagoon (Laguna Madre) and added to cultures of potential phytoplankton hosts. By following changes in in vivo fluorescence over time, it was possible to isolate several viruses that were pathogens to a variety of marine phytoplankton, including a prasinophyte (
Micromonas pusilla
), a pennate diatom (likely a
Navicula
sp.), a centric diatom (of unknown taxa), and a chroococcoid cyanobacterium (a
Synechococcus
sp.). As well, we observed changes in fluorescence in cultures of a cryptophyte (a
Rhodomonas
sp.) and a chlorophyte (
Nannochloropsis oculata
) which were consistent with the presence of viral pathogens. Although pathogens were isolated from all stations, all the pathogens were not isolated from every station. Filterability studies on the viruses infecting
M. pusilla
and the
Navicula
sp. showed that the viruses were consistently infective after filtration through polycarbonate and glass-fiber filters but were affected by most other filter types. Establishment of phytoplankton-pathogen systems will be important in elucidating the effect that viruses have on primary producers in aquatic systems.
Immunological Cross-Reactivities of Adenosine-5′-Phosphosulfate Reductases from Sulfate-Reducing and Sulfide-Oxidizing Bacteria
Authors: Odom J. Martin , Jessie Karen , Knodel Elinor , Emptage Mark
Published: January 2020

Crude extracts from 14 species of sulfate-reducing bacteria comprising the genera
Desulfovibrio, Desulfotomaculum, Desulfobulbus
, and
Desulfosarcina
and from three species of sulfide-oxidizing bacteria were tested in an enzyme-linked immunosorbent assay with polyclonal antisera to adenosine 5′-phosphosulfate reductase from
Desulfovibrio desulfuricans
G100A. The results showed that extracts from
Desulfovibrio
species were all highly cross-reactive, whereas extracts from the other sulfate-reducing genera showed significantly less cross-reaction. An exception was
Desulfotomaculum orientis
, which responded more like
Desulfovibrio
species than the other
Desulfotomaculum
strains tested. Extracts from colorless or photosynthetic sulfur bacteria were either unreactive or exhibited very low levels of reactivity with the antibodies to the enzyme from sulfate reducers. These results were confirmed by using partially purified enzymes from sulfate reducers and the most cross-reactive sulfide oxidizer,
Thiobacillus denitrificans
. Two types of monoclonal antibodies to adenosine 5′-phosphosulfate reductase were also isolated. One type reacted more variably with the enzymes of the sulfate reducers and poorly with the
Thiobacillus
enzyme, whereas the second reacted strongly with
Desulfovibrio, Desulfotomaculum orientis
, and
Thiobacillus
enzymes.
Characterization of caulobacters isolated from wastewater treatment systems
Authors: MacRae J D , Smit J
Published: January 2020

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.
Identification of plasmid partition function in coryneform bacteria
Authors: Kurusu Y , Satoh Y , Inui M , Kohama K , Kobayashi M , Terasawa M , Yukawa H
Published: January 2020

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.
Impact of Water Column Acidification on Protozoan Bacterivory at the Lake Sediment-Water Interface
Authors: Tremaine Sarah C. , Mills Aaron L.
Published: January 2020

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h
-1
, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (
P
= 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (
P
< 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.
Isolation and Characterization of a Subsurface Bacterium Capable of Growth on Toluene, Naphthalene, and Other Aromatic Compounds
Authors: Fredrickson J. K. , Brockman F. J. , Workman D. J. , Li S. W. , Stevens T. O.
Published: January 2020

A bacterium, designated F199, utilized toluene, naphthalene, dibenzothiophene, salicylate, benzoate,
p
-cresol, and all isomers of xylene as a sole carbon and energy source. This bacterium was isolated from Middendorf sediments, a Cretaceous age formation that underlies the Southeast Coastal Plain in South Carolina, at a depth of approximately 410 m. F199 is a gram-positive, irregular-shaped bacterium that has a varied cell morphology that is dependent on culture medium type and growth stage. F199 required microaerobic conditions (40 to 80 μM O
2
) for growth on hydrocarbons, glucose, acetate, and lactate in mineral salts medium but not for growth on rich media. [
14
C]naphthalene mineralization by F199 was induced by either naphthalene or toulene; however, [
14
C]toluene mineralization by this strain was induced by toluene but not naphthalene. F199 was also found to harbor two plasmids larger than 100 kb. Restricted F199 plasmid and genomic DNA did not hybridize with toluene (pWW0) or naphthalene (NAH7) catabolic plasmid DNA probes. The presence in the Middendorf formation of bacteria with the capacity for degrading a variety of aromatic compounds suggests that indigenous microorganisms may have potential for in situ degradation of organic contaminants.
Influence of Substituents on Reductive Dehalogenation of 3-Chlorobenzoate Analogs
Authors: Dolfing Jan , Tiedje James M.
Published: January 2020

The biochemical effects of aryl substituents on the reductive dechlorination of 3-chlorobenzoate analogs were quantified with (i) a stable 3-chlorobenzoate-grown methanogenic sludge enrichment, (ii)
Desulfomonile tiedjei
DCB-1, isolated from this enrichment and able to catalyze the reductive dechlorination of 3-chlorobenzoate, and (iii) a defined 3-chlorobenzoate-degrading methanogenic consortium with
D. tiedjei
as the key dechlorinating organism. The addition of hydrogen stimulated the dechlorination rate in the consortium. The extent of this stimulation depended on the substituent. The data were evaluated with various sets of substituent constants compiled for the Hammett equation. None of the sets yielded a satisfactory correlation between experimental values and theoretical constants. This suggests that the microbially catalyzed reductive dechlorination of 3-chlorobenzoate cannot be described simply as either a nucleophilic or an electrophilic substitution reaction. Nevertheless, observations that the presence of a
para
-amino or -hydroxy group inhibited the rate of dechlorination suggest that the rate-limiting step in the reductive dechlorination of 3-chlorobenzoate is a nucleophilic attack on the negatively charged π electron cloud around the benzene nucleus.
Pathways and Microbiology of Thiosulfate Transformations and Sulfate Reduction in a Marine Sediment (Kattegat, Denmark)
Authors: Jørgensen Bo Barker , Bak Friedhelm
Published: January 2020

Reductive and oxidative pathways of the sulfur cycle were studied in a marine sediment by parallel radiotracer experiments with
35
SO
4
2-
, H
2
35
S, and
35
S
2
O
3
2-
injected into undisturbed sediment cores. The distributions of viable populations of sulfate- and thiosulfate-reducing bacteria and of thiosulfate-disproportionating bacteria were concurrently determined. Sulfate reduction occurred both in the reducing sediment layers and in oxidized and even oxic surface layers. The population density of sulfate-reducing bacteria was >10
6
cm
-3
in the oxic layer, high enough that it could possibly account for the measured rates of sulfate reduction. The bacterial numbers counted in the reducing sediment layers were 100-fold lower. The dominant sulfate reducers growing on acetate or H
2
were gas-vacuolated motile rods which were previously undescribed. The products of sulfide oxidation, which took place in both oxidized and reduced sediment layers, were 65 to 85% S
2
O
3
2-
and 35 to 15% SO
4
2-
. Thiosulfate was concurrently oxidized to sulfate, reduced to sulfide, and disproportionated to sulfate and sulfide. There was a gradual shift from predominance of oxidation toward predominance of reduction with depth in the sediment. Disproportionation was the most important pathway overall. Thiosulfate disproportionation occurred only as cometabolism in the marine acetate-utilizing sulfate-reducing bacteria, which could not conserve energy for growth from this process alone. Oxidative and reductive cycling of sulfur thus occurred in all sediment layers with an intermediate “thiosulfate shunt” as an important mechanism regulating the electron flow.
In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker
Authors: Dunne W M , Burd E M
Published: January 2020

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.