bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, Characterization, and Substrate Specificities of Multiple Xylanases from
Streptomyces
sp. Strain B-12-2
Authors: Elegir Graziano , Szakács George , Jeffries Thomas W.
Published: January 2020

The endoxylanase complex from
Streptomyces
sp. strain B-12-2 was purified and characterized. The organism forms five distinct xylanases in the absence of significant cellulase activity when grown on oat spelt xylan. This is the largest number of endoxylanases yet reported for a streptomycete. On the basis of their physiochemical characteristics, they can be divided into two groups: the first group (xyl 1a and xyl 1b) consists of low-molecular-mass (26.4 and 23.8 kDa, respectively) neutral- to high-pI (6.5 and 8.3, respectively) endoxylanases. Group 1 endoxylanases are unable to hydrolyze aryl-β-
d
-cellobioside, have low levels of activity against xylotetraose (X
4
) and limited activity against xylopentaose, produce little or no xylose, and form products having a higher degree of polymerization with complex substrates. These enzymes apparently carry out transglycosylation. The second group (xyl 2, xyl 3, and xyl 4) consists of high-molecular-mass (36.2, 36.2, and 40.5 kDa, respectively), low-pI (5.4, 5.0, and 4.8, respectively) xylanases. Group 2 endoxylanases are able to hydrolyze aryl-β-
d
-cellobioside, show higher levels of activity against X
4
, and hydrolyze xylopentaose completely with the formation of xylobiose and xylotriose plus limited amounts of X
4
and xylose. The enzymes display intergroup synergism when acting on kraft pulp. Despite intragroup similarities, each enzyme exhibited a unique action pattern and physiochemical characteristic. xyl 2 was highly glycosylated, and xyl 1b (but no other enzyme) was completely inhibited by
p
-hydroxymercuribenzoate.
Kinetics and Modeling of Lactic Acid Production by
Lactobacillus plantarum
Authors: Passos Frederico V. , Fleming Henry P. , Ollis David F. , Felder Richard M. , McFeeters R. F.
Published: January 2020

An unstructured model was developed to describe bacterial growth, substrate utilization, and lactic acid production by
Lactobacillus plantarum
in cucumber juice. Significant lactic acid production occurred during growth, as well as stationary phases. The percentage of acid produced after growth ceased was a function of the medium composition. Up to 51% of the lactic acid was produced after growth ceased when NaCl was not present in the medium, whereas not more than 18% of the total lactic acid was produced after the growth ceased in presence of NaCl, probably because of an increase in the cell death rate. An equation relating the specific death rate and NaCl concentration was developed. With the kinetic model proposed by R. Luedeking and E. L. Piret (J. Biochem. Microbiol. Technol. Eng. 1:393-412, 1958) for lactic acid production rate, the growth-associated and non-growth-associated coefficients were determined as 51.9 (±4.2) mmol/g of cells and 7.2 (±0.9) mmol/g of cells h
-1
respectively. The model was demonstrated for batch growth of
L. plantarum
in cucumber juice. Mathematical simulations were used to predict the influence of variations in death rate, proton concentration when growth ceased, and buffer capacity of the juice on the overall fermentation process.
Tn
5
Insertion Mutants of
Pseudomonas fluorescens
Defective in Adhesion to Soil and Seeds
Authors: DeFlaun Mary F. , Marshall Bonnie M. , Kulle E.-Peter , Levy Stuart B.
Published: January 2020

Tn
5
insertion mutants of a soil isolate,
Pseudomonas fluorescens
Pf0-1, were selected for decreased ability to adhere to quartz sand in a column assay. Three adhesion-deficient mutants that differed in the location of the Tn
5
insertion in the chromosome were isolated and compared with the wild-type strain. One mutant, Pf0-5, was described previously as an adhesion-defective, nonmobile, flagellumless mutant (M. F. DeFlaun, A. S. Tanzer, A. L. McAteer, B. Marshall, and S. B. Levy, Appl. Environ. Microbiol. 56:112-119, 1990). Another insertion mutant, Pf0-10, was also missing flagella and the 34-kDa outer membrane protein that was absent in Pf0-5 but present in the wild-type strain. The third mutant (Pf0-15) had increased amounts of this 34-kDa outer membrane protein and more flagella than the wild-type strain. These mutants also displayed decreased ability to adhere to sterile and natural (live) soil and to a variety of plant seeds. In kinetics studies, the wild-type strain showed an initial rapid binding to seeds followed by a later slow phase of binding. The mutant strains were defective in the initial stages of attachment but did show the later slow binding. The findings indicate that the same mutations that affect binding to sand and soil also affect adhesion to plant seeds.
Accumulation of selenium in a model freshwater microbial food web
Authors: Sanders R W , Gilmour C C
Published: January 2020

The transfer of selenium between bacteria and the ciliated protozoan, Paramecium putrinum, was examined in laboratory cultures. The population growth of the ciliate was not inhibited in the presence of the highest concentrations of dissolved selenite or selenate tested (10(3) micrograms liter-1). Experiments with radioactive 75selenite or 75selenate indicated that accumulation of selenium by ciliates through time was low when feeding and metabolism were reduced by incubating at 0 degrees C. However, selenium accumulated in ciliate biomass during incubation with dissolved 75Se and bacteria at 24 degrees C and also when bacteria prelabeled with 75Se were offered as food in the absence of dissolved selenium. When 75Se-labeled bacterial food was diluted by the addition of nonradioactive bacteria, the amount of selenite and selenate in ciliates decreased over time, indicating depuration by the ciliates. In longer-term (> 5-day) fed-batch incubations with 75selenite-labeled bacteria, the selenium concentration in ciliates equilibrated at approximately 1.4 micrograms of Se g (dry weight)-1. The selenium content of ciliates was similar to that of their bacterial food on a dry-weight basis. These data indicate that selenium uptake by this ciliate occurred primarily during feeding and that biomagnification of selenium did not occur in this simple food chain.
Liquid Flow in Biofilm Systems
Authors: Stoodley Paul , deBeer Dirk , Lewandowski Zbigniew
Published: January 2020

A model biofilm consisting of
Pseudomonas aeruginosa, Pseudomonas fluorescens
, and
Klebsiella pneumoniae
was developed to study the relationships between structural heterogeneity and hydrodynamics. Local fluid velocity in the biofilm system was measured by a noninvasive method of particle image velocimetry, using confocal scanning laser microscopy. Velocity profiles were measured in conduit and porous medium reactors in the presence and absence of biofilm. Liquid flow was observed within biofilm channels; simultaneous imaging of the biofilm allowed the liquid velocity to be related to the physical structure of the biofilm.
Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water
Authors: Fayer R
Published: January 2020

Cryptosporidium parvum oocysts suspended in 0.5 ml of distilled water were pipetted into plastic vials which were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 60 to 100 degrees C. At each temperature setting four vials containing C. parvum oocysts were placed into wells and held for 15 s before time was recorded as zero, and then pairs of vials were removed 1 and 5 min later. Upon removal, all vials were immediately cooled on crushed ice. Also, at each temperature interval one vial containing 0.5 ml of distilled water was placed in a well and a digital thermometer was used to record the actual water temperature at 30-s intervals. Heated oocyst suspensions as well as unheated control suspensions were orally inoculated by gavage into 7- to 10-day-old BALB/c mouse pups to test for infectivity. At 96 h after inoculation the ileum, cecum, and colon from each mouse were removed and prepared for histology. Tissue sections were examined microscopically. Developmental-stage C. parvum was found in all three gut segments from all mice that received oocysts in unheated water and in water that reached temperatures of 54.4, 59.9, and 67.5 degrees C at 1 min when vials were removed from the heat source. C. parvum was also found in the ileum of one of six mice that received oocysts in water that reached a temperature of 59.7 degrees C at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Standing Stock, Activity, and Carbon Production during Formation and Growth of Sea Ice in the Weddell Sea, Antarctica
Authors: Grossmann Sönnke , Dieckmann Gerhard S.
Published: January 2020

Bacterial response to formation and growth of sea ice was investigated during autumn in the northeastern Weddell Sea. Changes in standing stock, activity, and carbon production of bacteria were determined in successive stages of ice development. During initial ice formation, concentrations of bacterial cells, in the order of 1 × 10
8
to 3 × 10
8
liter
-1
, were not enhanced within the ice matrix. This suggests that physical enrichment of bacteria by ice crystals is not effective. Due to low concentrations of phytoplankton in the water column during freezing, incorporation of bacteria into newly formed ice via attachment to algal cells or aggregates was not recorded in this study. As soon as the ice had formed, the general metabolic activity of bacterial populations was strongly suppressed. Furthermore, the ratio of [
3
H]leucine incorporation into proteins to [
3
H]thymidine incorporation into DNA changed during ice growth. In thick pack ice, bacterial activity recovered and growth rates up to 0.6 day
-1
indicated actively dividing populations. However, biomass-specific utilization of organic compounds remained lower than in open water. Bacterial concentrations of up to 2.8 × 10
9
cells liter
-1
along with considerably enlarged cell volumes accumulated within thick pack ice, suggesting reduced mortality rates of bacteria within the small brine pores. In the course of ice development, bacterial carbon production increased from about 0.01 to 0.4 μg of C liter
-1
h
-1
. In thick ice, bacterial secondary production exceeded primary production of microalgae.
Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass
Authors: McEldowney S
Published: January 2020

The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined. Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal. The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment. The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells. The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment. Cells exposed to Cd prior to attachment showed increased adhesion. Zinc-treated cells did not. There was a positive correlation between adhesion and Cd concentration in the attachment solution. No such relationship existed for Zn. P. fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls. Zinc pretreatment resulted in decreased desorption. Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1. The presence of Cd or Zn during detachment had little effect on desorption. The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface. In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases.
Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene
Authors: Fujii T , Nagasawa N , Iwamatsu A , Bogaki T , Tamai Y , Hamachi M
Published: January 2020

The ATF1 gene, which encodes alcohol acetyltransferase (AATase), was cloned from Saccharomyces cerevisiae and brewery lager yeast (Saccharomyces uvarum). The nucleotide sequence of the ATF1 gene isolated from S. cerevisiae was determined. The structural gene consists of a 1,575-bp open reading frame that encodes 525 amino acids with a calculated molecular weight of 61,059. Although the yeast AATase is considered a membrane-bound enzyme, the results of a hydrophobicity analysis suggested that this gene product does not have a membrane-spanning region that is significantly hydrophobic. A Southern analysis of the yeast genomes in which the ATF1 gene was used as a probe revealed that S. cerevisiae has one ATF1 gene, while brewery lager yeast has one ATF1 gene and another, homologous gene (Lg-ATF1). Transformants carrying multiple copies of the ATF1 gene or the Lg-ATF1 gene exhibited high AATase activity in static cultures and produced greater concentrations of acetate esters than the control.
Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats
Authors: Fries M R , Zhou J , Chee-Sanford J , Tiedje J M
Published: January 2020

Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS)
Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H
2
O
2
Production by
Pleurotus eryngii
Authors: Guillén Francisco , Evans Christine S.
Published: January 2020

The existence of a redox cycle leading to the production of hydrogen peroxide (H
2
O
2
) in the white rot fungus
Pleurotus eryngii
has been confirmed by incubations of 10-day-old mycelium with veratryl (3,4-dimethoxybenzyl) and anisyl (4-methoxybenzyl) compounds (alcohols, aldehydes, and acids). Veratraldehyde and anisaldehyde were reduced by aryl-alcohol dehydrogenase to their corresponding alcohols, which were oxidized by aryl-alcohol oxidase, producing H
2
O
2
. Veratric and anisic acids were incorporated into the cycle after their reduction, which was catalyzed by aryl-aldehyde dehydrogenase. With the use of different initial concentrations of either veratryl alcohol, veratraldehyde, or veratric acid (0.5 to 4.0 mM), around 94% of veratraldehyde and 3% of veratryl alcohol (compared with initial concentrations) and trace amounts of veratric acid were found when equilibrium between reductive and oxidative activities had been reached, regardless of the initial compound used. At concentrations higher than 1 mM, veratric acid was not transformed, and at 1.0 mM, it produced a negative effect on the activities of aryl-alcohol oxidase and both dehydrogenases. H
2
O
2
levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H
2
O
2
-reducing system kept these levels steady. On the other hand, the concomitant production of the three above-mentioned enzymes during the active growth phase of the fungus was demonstrated. Finally, the possibility that anisaldehyde is the metabolite produced by
P. eryngii
for the maintenance of this redox cycle is discussed.
Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens
Authors: Dong Xiuzhu , Plugge Caroline M. , Stams Alfons J. M.
Published: January 2020

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO
2
in cocultures with the formate- and hydrogen-utilizing methanogens
Methanospirillum hungatei
and
Methanobacterium formicicum.
Propionate oxidation did not occur in cocultures with two
Methanobrevibacter
strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the
Methanobrevibacter
strains, and organisms which are able to convert formate into H
2
plus CO
2
(
Desulfovibrio
strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with
Methanospirillum hungatei
and the aceticlastic
Methanothrix soehngenii
, propionate was degraded faster than in biculture with
Methanospirillum hungatei
, indicating that low acetate concentrations are favorable for propionate oxidation as well.
Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits
Authors: Roberts Rodney G. , Reymond Stephen T.
Published: January 2020

Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruit-handling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of
Botrytis cinerea, Penicillium expansum, Mucor piriformis
, and
Cryptosporiopsis perennans
demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5 μg · ml
-1
. Longer exposure times were necessary to achieve similar spore mortalities with 1 μg · ml
-1
. Of the fungi tested,
B. cinerea
and
P. expansum
were the least sensitive to ClO
2
. In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0- to 18.0-μg · ml
-1
ClO
2
foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed.
Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO
Authors: Jarausch W , Saillard C , Dosba F , Bové J M
Published: January 2020

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle.
Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products
Authors: Bereswill S , Bugert P , Völksch B , Ullrich M , Bender C L , Geider K
Published: January 2020

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin. In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P. syringae. The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65-kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)
Variable colonization of chickens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs
Authors: Schoeni J L , Doyle M P
Published: January 2020

Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen.
Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification
Authors: Puig M , Jofre J , Lucena F , Allard A , Wadell G , Girones R
Published: January 2020

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.
Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model
Authors: Lees D N , Henshilwood K , Doré W J
Published: January 2020

The application of the PCR to complex samples is hindered by amplification inhibitors. We describe a reverse transcription-PCR-based method capable of inhibitor removal for the detection of enteroviruses in shellfish. Initial virus extraction stages based on a modified polyethylene glycol precipitation technique (G.D. Lewis and T.G. Metcalf, Appl. Environ. Microbiol. 54:1983-1988, 1988) were followed by virus purification with 1,1,2-trichloro,2,2,1-trifluoroethane and concentration by ultrafiltration. A guanidine isothiocyanate-glass powder extraction system was utilized for sample lysis, RNase protection, and nucleic acid purification. Removal of PCR inhibitors and method sensitivity were quantified in shellfish (oysters and mussels) seeded with poliovirus. PCR sample tolerance exceeded 4 g for depurated shellfish; however, polluted field samples were more inhibitory. Virus recoveries of 31% for oyster extracts and 17% for mussel extracts and nucleic acid extraction reverse transcription-PCR detection limits down to 1 PFU yielded an overall sensitivity limit of < 10 PFU of poliovirus in up to 5 g of shellfish. PCR-positive results were obtained from a variety of polluted field samples naturally contaminated with human enteroviruses. The methods developed for virus recovery and PCR inhibitor removal should be equally applicable to detection of other RNA viruses such as hepatitis A virus, Norwalk virus, and other small round-structured viruses in shellfish.
Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artificially contaminated milk samples
Authors: Cooray K J , Nishibori T , Xiong H , Matsuyama T , Fujita M , Mitsuyama M
Published: January 2020

The inhibitory effect of milk in the PCR detection of Listeria monocytogenes could be overcome by washing the contaminated milk sample with phosphate-buffered saline and concentrating the bacteria to 1/10 of the original volume. In order to avoid a possible failure in the detection of virulent L. monocytogenes, a one-step procedure which enabled demonstration of three virulence-associated genes, prfA, hlyA, and plcB, simultaneously in a single PCR mixture was developed.
Identification of
Aeromonas
Species Isolated from Freshwater Fish with the Microplate Hybridization Method
Authors: Sugita Haruo , Nakamura Tomoyoshi , Tanaka Katsunao , Deguchi Yoshiaki
Published: January 2020

Aeromonas
isolates were obtained from the intestinal tracts of six species of cultured freshwater fish and identified on the basis of their genotypic and phenotypic characters. The microplate hybridization method could differentiate type strains of
Aeromonas
species and related bacteria. DNA-DNA hybridization analysis showed that 65 aeromonad isolates were 72 to 100% related with either
Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas sobria
, or
Aeromonas veronii.
As many as 48% of the genotypically identified
A. caviae, A. hydrophila
, and
A. sobria
isolates differed from the type strains of corresponding species in one to three phenotypic characters. These results strongly suggest that not all aeromonad isolates from freshwater fish could be identified correctly on the basis of only the phenotypic characters, indicating the usefulness of the microplate hybridization method for the identification of aeromonads.
Controlled expression and structural organization of a Lactococcus lactis bacteriophage lysin encoded by two overlapping genes
Authors: Shearman C A , Jury K L , Gasson M J
Published: January 2020

The phi vML3 bacteriophage lysin is specific for lactococci and could be used to promote enzyme release during cheese manufacture. The level of lysin expression from the cloned gene using its own upstream sequences is very low. Expression in Escherichia coli by using a synthetic hybrid lysin gene and a series of BAL 31 deletions of the original cloned DNA fragment suggested that the start of the gene had previously been incorrectly assigned. Reevaluation of homology between the lysin and Bacillus subtilis PZA protein 15 led to the identification of a new potential ribosome binding site (RBS). A 0.72-kb PCR-generated fragment including this RBS and the complete lysin gene was expressed and inducibly controlled. The translational start of the lysin gene was identified as an isoleucine codon, and this may lead to a low translation rate. During the analysis of the BAL 31 deletion fragments, two proteins of 20 and 8 kDa were shown to be expressed from the originally defined lysin gene. The DNA sequence has a second open reading frame with a good RBS and two potential start methionines. The smaller lysin protein was isolated, and the N terminus was sequenced, confirming that one methionine codon acted as the start of a second gene. The larger lysin protein has homology with lysozymes. The smaller lysin protein has some features resembling those of a holin. The possible roles of these two proteins in lysis of lactococci are discussed.
Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy
Authors: Mehmann B , Brunner I , Braus G H
Published: January 2020

DNA sequences of single-copy genes coding for chitin synthases (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase; EC 2.4.1.16) were used to characterize ectomycorrhizal fungi. Degenerate primers deduced from short, completely conserved amino acid stretches flanking a region of about 200 amino acids of zymogenic chitin synthases allowed the amplification of DNA fragments of several members of this gene family. Different DNA band patterns were obtained from basidiomycetes because of variation in the number and length of amplified fragments. Cloning and sequencing of the most prominent DNA fragments revealed that these differences were due to various introns at conserved positions. The presence of introns in basidiomycetous fungi therefore has a potential use in identification of genera by analyzing PCR-generated DNA fragment patterns. Analyses of the nucleotide sequences of cloned fragments revealed variations in nucleotide sequences from 4 to 45%. By comparison of the deduced amino acid sequences, the majority of the DNA fragments were identified as members of genes for chitin synthase class II. The deduced amino acid sequences from species of the same genus differed only in one amino acid residue, whereas identity between the amino acid sequences of ascomycetous and basidiomycetous fungi within the same taxonomic class was found to be approximately 43 to 66%. Phylogenetic analysis of the amino acid sequence of class II chitin synthase-encoding gene fragments by using parsimony confirmed the current taxonomic groupings. In addition, our data revealed a fourth class of putative zymogenic chitin synthesis.
Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters
Authors: Lang A L , Tsai Y L , Mayer C L , Patton K C , Palmer C J
Published: January 2020

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.
Regulation of the production of extracellular pectinase, cellulase, and protease in the soft rot bacterium Erwinia carotovora subsp. carotovora: evidence that aepH of E. carotovora subsp. carotovora 71 activates gene expression in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli
Authors: Murata H , Chatterjee A , Liu Y , Chatterjee A K
Published: January 2020

The production of pectolytic enzymes (pectate lyase [Pel] and polygalacturonase [Peh]), cellulase (Cel), and protease (Prt) is activated in the soft rot bacterium Erwinia carotovora subsp. carotovora by aepA (activator of extracellular protein production) and celery extract (Y. Liu, H. Murata, A. Chatterjee, and A. K. Chatterjee, Mol. Plant-Microbe Interact. 6:299-308, 1993). We recently isolated a new class of mutants of strain E. carotovora subsp. carotovora 71 which overproduces Pel, Peh, Cel, and Prt. From the overproducing strain AC5034, we identified an activator locus, designated aepH*, which stimulated Pel, Peh, Cel, and Prt production in E. carotovora subsp. carotovora 71 or its derivatives. The nucleotide sequence of the aepH* DNA segment revealed an open reading frame of 141 bp that could encode a small (5.45-kDa) highly basic (pI 11.7) protein of 47 amino acid residues. Analyses of deletions and MudI insertions indicated that the activator function required the 508-bp DNA segment which contains this open reading frame. The wild-type locus, aepH+, is localized within a DNA segment upstream of aepA. An AepH- strain constructed by exchanging aepH+ with aepH*::MudI was deficient in Pel, Peh, Cel, and Prt production; exoenzyme production was restored upon the introduction of a plasmid carrying aepH+ or aepH*. Plasmids carrying either aepH+ or aepH* activated the production of Pel-1, Peh-1, and Cel in Escherichia coli HB101 carrying the cognate genes. The aepH effect in E. coli was due to the activation of transcription, as indicated by assays of pel-1 and peh-1 mRNAs. The aepH+ and aepH* plasmids also stimulated Pel, Peh, Cel, and Prt production in other wild-type E. carotovora subsp. carotovora strains as well as in E. carotovora subsp. atroseptica. Although the stimulatory effect was generally more pronounced with aepH* than with aepH+, the extent of activation in the wild-type strains depended upon the bacterial strain and the growth medium. Southern blot hybridization revealed the presence of aepH homologs in E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, and provided physical evidence for linkage between aepA and aepH homologs in genomes of these bacteria. We conclude that aepH-mediated activation of exoprotein gene expression is a feature common to most strains of E. carotovora.
Dynamics and Distribution of Cyanophages and Their Effect on Marine
Synechococcus
spp
Authors: Suttle Curtis A. , Chan Amy M.
Published: January 2020

Cyanophages infecting marine
Synechococcus
cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting
Synechococcus
strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 × 10
5
ml
-1
near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and
Synechococcus
concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 × 10
3
ml
-1
). When
Synechococcus
concentrations exceeded ca. 10
3
ml
-1
, cyanophage concentrations increased markedly (ca. 10
2
to > 10
5
ml
-1
), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity
d
(per day), as a function of solar irradiance
I
(millimoles of quanta per square meter per second), were used to develop a relationship (
d
= 0.2610
I
- 0.00718;
r
2
= 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of
Synechococcus
cells would be infected daily by viruses. Calculations based on contact rates between
Synechococcus
cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most
Synechococcus
cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of
Synechococcus
cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the
Synechococcus
population on a daily basis.
PCR-based preparation of 23S rRNA-targeted group-specific polynucleotide probes
Authors: Ludwig W , Dorn S , Springer N , Kirchhof G , Schleifer K H
Published: January 2020

DNA coding for a variable region within domain III of bacterial 23S rRNA was used as the target for group-specific polynucleotide hybridization probes. The corresponding rDNA was amplified in vitro by the PCR technique in combination with a pair of primers specific for flanking conserved target sites. The amplified fragments were cloned or used directly as probes. RNA probes were generated by in vitro transcription of cloned or amplified rDNA. The probes were labeled by incorporating modified nucleotides during in vitro DNA amplification or in vitro transcription or by random priming. The use of in vitro transcribed single-stranded RNA probes instead of double-stranded DNA probes provided stronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, Alcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.
Polyester Biosynthesis Characteristics of
Pseudomonas citronellolis
Grown on Various Carbon Sources, Including 3-Methyl-Branched Substrates
Authors: Choi Mun Hwan , Yoon Sung Chul
Published: January 2020

Forty-two different carbon sources were tested for the polyester synthesis of a citronellol-utilizing bacterium,
Pseudomonas citronellolis
(ATCC 13674). These included linear C
2
to C
10
monocarboxylic acids, C
3
to C
10
dicarboxylic acids, saccharides, α,ω-diols, hydrocarbons, and 3-methyl-branched substrates such as 3,7-dimethyl-6-octen-1-ol (citronellol), 3-methyl-
n
-valerate, 3-methyl-1-butanol, and 3-methyladipate. Isolated polymers were characterized by gas chromatography, infrared spectroscopy,
1
H- or
13
C-nuclear magnetic resonance spectroscopy,
1
H-
13
C heteronuclear correlation spectroscopy (
1
H-
13
C COSY),
1
H-
1
H homonuclear COSY, and differential scanning calorimetry. Polyesters from nine monocarboxylic acids and two related carbon sources could be metabolically divided into three groups. The first group of C
2
to C
4
carbon sources resulted in copolyesters composed of 61 to 70 mol% 3-hydroxydecanoate, 23 to 33 mol% 3-hydroxyoctanoate, 3.6 to 9.0 mol% 3-hydroxy-5-
cis
-dodecenoate, and 1.8 to 2.6 mol% 3-hydroxy-7-
cis
-tetradecenoate. Carbon sources in group II (C
7
to C
10
) produced copolyesters composed of 3-hydroxyacid monomer units with the same number of carbon atoms as the substrate (major constituent) and monomer units with either two less or two more carbons. Negligible amounts of 3-hydroxy-5-
cis
-dodecenoate and 3-hydroxy-7-
cis
-tetradecenoate were detected in copolyesters from this group. Copolyesters from group III (C
5
and C
6
) had a monomer unit distribution that could be said to be between those of groups I and II. In addition, a novel copolyester, poly(3-hydroxy-7-methyl-6-octenoate-co-3-hydroxy-5-methylhexanoate), was synthesized when grown on citronellol. The
1
H-
13
C heteronuclear COSY spectrum for monomer unit II revealed that both methylene and isopropyl groups, proximately connected in series to a single chiral center, had magnetically diastereotopic natures.
Evidence for natural horizontal transfer of tetQ between bacteria that normally colonize humans and bacteria that normally colonize livestock
Authors: Nikolich M P , Hong G , Shoemaker N B , Salyers A A
Published: January 2020

Though numerous studies have shown that gene transfer occurs between distantly related bacterial genera under laboratory conditions, the frequency and breadth of horizontal transfer events in nature remain unknown. Previous evidence for natural intergeneric transfers came from studies of genes in human pathogens, bacteria that colonize the same host. We present evidence that natural transfer of a tetracycline resistance gene, tetQ, has occurred between bacterial genera that normally colonize different hosts. A DNA sequence comparative approach was taken to examine the extent of horizontal tetQ dissemination between species of Bacteroides, the predominant genus of the human colonic microflora, and between species of Bacteroides and of the distantly related genus Prevotella, a predominant genus of the microflora of the rumens and intestinal tracts of farm animals. Virtually identical tetQ sequences were found in a number of isolate pairs differing in taxonomy and geographic origin, indicating that extensive natural gene transmission has occurred. Among the exchange events indicated by the evidence was the very recent transfer of an allele of tetQ usually found in Prevotella spp. to a Bacteroides fragilis strain.
Oxidative Dissolution of Arsenopyrite by Mesophilic and Moderately Thermophilic Acidophiles
Authors: Tuovinen Olli H. , Bhatti Tariq M. , Bigham Jerry M. , Hallberg Kevin B. , Garcia Oswaldo , Lindström E. Börje
Published: January 2020

The purpose of this work was to determine solution- and solid-phase changes associated with the oxidative leaching of arsenopyrite (FeAsS) by
Thiobacillus ferrooxidans
and a moderately thermoacidophilic mixed culture. Jarosite [KFe
3
(SO
4
)
2
(OH)
6
], elemental sulfur (S
0
), and amorphous ferric arsenate were detected by X-ray diffraction as solid-phase products. The oxidation was not a strongly acid-producing reaction and was accompanied by a relatively low redox level. The X-ray diffraction lines of jarosite increased considerably when ferrous sulfate was used as an additional substrate for
T. ferroxidans.
A moderately thermoacidophilic mixed culture oxidized arsenopyrite faster at 45°C than did
T. ferroxidans
at 22°C, and the oxidation was accompanied by a nearly stoichiometric release of Fe and As. The redox potential was initially low but subsequently increased during arsenopyrite oxidation by the thermoacidophiles. Jarosite, S
0
, and amorphous ferric arsenate were also formed under these conditions.
Regiospecific and stereoselective hydroxylation of 1-indanone and 2-indanone by naphthalene dioxygenase and toluene dioxygenase
Authors: Resnick S M , Torok D S , Lee K , Brand J M , Gibson D T
Published: January 2020

The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterial strains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purified enzyme components. Pseudomonas sp. strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixture of 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%). The (R)-3-hydroxy-1-indanone was formed in 62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic. The same cells also formed 2-hydroxy-1-indanone from 2-indanone. Purified NDO components oxidized 1-indanone and 2-indanone to the same products produced by strain 9816/11. P. putida F39/D cells, expressing TDO, oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone efficiently. Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90% ee (R:S, 5:95) and failed to oxidize 1-indanone. Oxidation of 1- and 2-indanone in the presence of [18O]oxygen indicated that the hydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation) rather than by the dioxygenation of enol tautomers of the ketone substrates. As alternatives to chemical synthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-1-indanone as the major products from 1-indanone and 2-indanone, respectively.
Filamentous Fungi with High Cytosolic Phospholipid Transfer Activity in the Presence of Exogenous Phospholipid
Authors: Record Eric , Lesage Laurence , Cahagnier Bernard , Marion Didier , Asther Marcel
Published: January 2020

The phospholipid transfer activity of cell extracts from 15 filamentous fungus strains grown on a medium containing phospholipids as the carbon source was measured by a fluorescence assay. This assay was based on the transfer of pyrene-labeled phosphatidylcholines forming the donor vesicles to acceptor vesicles composed of egg phosphatidylcholines. The highest phosphatidylcholine transfer activity was obtained with cell extracts from
Aspergillus oryzae
. The presence of exogenous phospholipids in the culture medium of
A. oryzae
was shown to increase markedly the activity of phospholipid transfer as well as the pool of exocellular proteins during the primary phase of growth. Modifications in the biochemical marker activities of cellular organelles were observed: succinate dehydrogenase, a mitochondrial marker; inosine diphosphatase, a Golgi system marker; and cytochrome
c
oxidoreductase, an endoplasmic reticulum marker, were increased 7.3-, 2-, and 22-fold, respectively, when
A. oryzae
was grown in the presence of phospholipids.