bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant
Cellulomonas fimi
β-1,4-Glucanases
Authors: Stålbrand Henrik , Mansfield Shawn D. , Saddler John N. , Kilburn Douglas G. , Warren R. Antony J. , Gilkes Neil R. 
Published: December 2019

ABSTRACT

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium
Cellulomonas fimi
were purified from
Escherichia coli
cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.
Abundance in Sewage of Bacteriophages That InfectEscherichia coliO157:H7 and That Carry the Shiga Toxin 2 Gene
Authors: Muniesa Maite , Jofre Juan 
Published: December 2019

ABSTRACTShiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such asEscherichia coliO157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negativeE. coliO157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious forE. coliO157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infectingE. coliO157:H7.
Cloning of
Phanerochaete chrysosporium leu2
by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs
Authors: Schick Zapanta Laura , Hattori Takefumi , Rzetskaya Magarita , Tien Ming 
Published: December 2019

ABSTRACT

A
Phanerochaete chrysosporium
cDNA library was constructed in an expression vector that allows expression in both
Escherichia coli
and
Saccharomyces cerevisiae
. This expression vector, λYES, contains the
lacZ
promoter for expression in
E. coli
and the
GAL1
promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from
P. chrysosporium
was characterized further. The genomic clone (g
leu2
) was subsequently isolated and was used successfully as a selectable marker to transform
P. chrysosporium
auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous
P. chrysosporium
transformation procedures. The method described here allows other genes to be isolated from
P. chrysosporium
for use as selectable markers.
Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization
Authors: Llobet-Brossa Enric , Rosselló-Mora Ramon , Amann Rudolf 
Published: December 2019

ABSTRACT

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the
Cytophaga-Flavobacterium
cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.
Plasposons: Modular Self-Cloning Minitransposon Derivatives for Rapid Genetic Analysis of Gram-Negative Bacterial Genomes
Authors: Dennis Jonathan J. , Zylstra Gerben J. 
Published: December 2019

ABSTRACT

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon’s site of insertion has been developed. The basic plasposon, named Tn
Mod
, consists of the Tn
5
inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn
5
transposase, in combination with the flexibility afforded by the modular arrangement of Tn
Mod
, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.
Use of Luciferase Genes as Biosensors To Study Bacterial Physiology in the Digestive Tract
Authors: Corthier G. , Delorme C. , Ehrlich S. D. , Renault P. 
Published: December 2019

ABSTRACT

A method based on the use of the bacterial luciferase genes was developed in order to investigate
Lactococcus lactis
gene expression in the mouse digestive tract. Germfree mice were monoassociated with different strains containing transcriptional fusions of promoters with the luciferase genes. Our results demonstrate that this method is readily applicable to the study of promoter strength and physiology of bacteria in the digestive tract.
Importance of Extracellular Polymeric Substances fromThiobacillus ferrooxidansfor Bioleaching
Authors: Gehrke Tilman , Telegdi Judit , Thierry Dominique , Sand Wolfgang 
Published: December 2019

ABSTRACTLeaching bacteria such asThiobacillus ferrooxidansattach to pyrite or sulfur by means of extracellular polymeric substances (EPS) (lipopolysaccharides). The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrochemical interaction with the negatively charged pyrite surface. EPS from sulfur cells possess increased hydrophobic properties and do not attach to pyrite, indicating adaptability to the substrate or substratum.
Gene Transfer by Transduction in the Marine Environment
Authors: Jiang Sunny C. , Paul John H. 
Published: December 2019

ABSTRACT

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn
5
and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (
npt
II) gene in Tn
5
. The transduction frequencies ranged from 1.33 × 10
−7
to 5.13 × 10
−9
transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the
npt
II probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10
−8
to 3.7 × 10
−8
transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10
14
transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.
A Glutathione
S
-Transferase with Activity towards
cis
-1,2-Dichloroepoxyethane Is Involved in Isoprene Utilization by
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Kingma Jaap , van den Wijngaard Arjan J. , Janssen Dick B. 
Published: December 2019

ABSTRACT

Rhodococcus
sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene,
cis
-1,2-dichloroethene to
cis
-1,2-dichloroepoxyethane, and
trans
-1,2-dichloroethene to
trans
-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded
cis
-1,2-dichloroepoxyethane and
trans
-1,2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of
cis
-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3,4-epoxy-3-methyl-1-butene, epoxypropane,
cis
-1,2-dichloroepoxyethane, and
trans
-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1,2-epoxyhexane was detected in cell extracts of cells exposed to 1,2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH
S
-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.
Acyl Transfer Activity of an Amidase from
Rhodococcus
sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids
Authors: Fournand David , Bigey Frederic , Arnaud Alain 
Published: December 2019

ABSTRACT

The enantioselective amidase from
Rhodococcus
sp. strain R312 was produced in
Escherichia coli
and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the
K

m
amide

values (e.g.,
K

m
amide

= 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (
k
cat
) were obtained with linear aliphatic amides (e.g.,
k
cat
= 333 s
−1
with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g.,
K

m

NH
2
OH

= 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be
l
-enantioselective towards α-hydroxy- and α-aminoamides.
Detection ofEhrlichia risticii, the Agent of Potomac Horse Fever, in Freshwater Stream Snails (Pleuroceridae:Jugaspp.) from Northern California
Authors: Barlough Jeffrey E. , Reubel Gerhard H. , Madigan John E. , Vredevoe Larisa K. , Miller Paul E. , Rikihisa Yasuko 
Published: December 2019

ABSTRACTEhrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae:Jugaspp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic. Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA,groESLheat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembledEhrlichia risticii, the causative agent of PHF. The minimum percentage ofJugaspp. harboring the organism in the population studied was 3.5% (2 of 57 snails). No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site. These data suggest that pleurocerid stream snails may play a role in the life cycle ofE. risticiiin northern California.
Strain IMB-1, a Novel Bacterium for the Removal of Methyl Bromide in Fumigated Agricultural Soils
Authors: Connell Hancock Tracy L. , Costello Andria M. , Lidstrom Mary E. , Oremland Ronald S. 
Published: December 2019

ABSTRACT

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class
Proteobacteria
and is closely related to members of the genus
Rhizobium
. The ability of strain IMB-1 to oxidize MeBr to CO
2
is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.
Spatial and Temporal Deposition of
Hyphomonas
Strain VP-6 Capsules Involved in Biofilm Formation
Authors: Langille Stephen E. , Weiner Ronald M. 
Published: December 2019

ABSTRACT

Hyphomonas
strain VP-6 is a prosthecate bacterium isolated from the Guayamas vent region and is a member of a genus of primary and common colonizers of marine surfaces. It adheres to solid substrata as a first step in biofilm formation. Fine-structure microscopy and the use of specific stains and lectins reveal that it synthesizes two different extracellular polymeric substances (EPS). One is a temporally synthesized, polar holdfast EPS, and the other is a capsular EPS that is present during the complete life cycle and surrounds the entire cell, including the prosthecum. The timing and location of
Hyphomonas
strain VP-6 EPS elaboration correlate with adhesion to surfaces, suggesting that the EPS serves not only as the biofilm matrix but also as a primary adhesin. The temporality and polarity of VP-6 EPS expression substantially differ from those properties of
Hyphomonas
strain MHS-3 EPS expression.
Volatile Compounds Originating from Mixed Microbial Cultures on Building Materials under Various Humidity Conditions
Authors: Korpi Anne , Pasanen Anna-Liisa , Pasanen Pertti 
Published: December 2019

ABSTRACT

We examined growth of mixed microbial cultures (13 fungal species and one actinomycete species) and production of volatile compounds (VOCs) in typical building materials in outside walls, separating walls, and bathroom floors at various relative humidities (RHs) of air. Air samples from incubation chambers were adsorbed on Tenax TA and dinitrophenylhydrazine cartridges and were analyzed by thermal desorption-gas chromatography and high-performance liquid chromatography, respectively. Metabolic activity was measured by determining CO
2
production, and microbial concentrations were determined by a dilution plate method. At 80 to 82% RH, CO
2
production did not indicate that microbial activity occurred, and only 10% of the spores germinated, while slight increases in the concentrations of some VOCs were detected. All of the parameters showed that microbial activity occurred at 90 to 99% RH. The microbiological analyses revealed weak microbial growth even under drying conditions (32 to 33% RH). The main VOCs produced on the building materials studied were 3-methyl-1-butanol, 1-pentanol, 1-hexanol, and 1-octen-3-ol. In some cases fungal growth decreased aldehyde emissions. We found that various VOCs accompany microbial activity but that no single VOC is a reliable indicator of biocontamination in building materials.
Effect of Tungstate on Nitrate Reduction by the Hyperthermophilic Archaeon
Pyrobaculum aerophilum
Authors: Afshar Sepideh , Kim Christopher , Monbouquette Harold G. , Schröder Imke 
Published: December 2019

ABSTRACT

Pyrobaculum aerophilum
, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that
P. aerophilum
requires the metal oxyanion WO
4
2−
for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 μM MoO
4
2−
did not replace WO
4
2−
for the growth of
P. aerophilum
. However, cell growth was completely inhibited by the addition of 100 μM MoO
4
2−
to the culture medium. At lower tungstate concentrations (0.3 μM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO
4
2−
concentrations (<0.7 μM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO
4
2−
levels of 0.7 μM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO
4
2−
levels (0.3 μM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in
P. aerophilum
occurs in the presence of high concentrations of tungstate.
Randomly Amplified Polymorphic DNA Analysis of Starved and Viable but Nonculturable
Vibrio vulnificus
Cells
Authors: Warner Jennifer M. , Oliver James D. 
Published: December 2019

ABSTRACT

Vibrio vulnificus
is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism’s natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of
V. vulnificus
strains grown in rich media and has been applied to starved and VBNC cells of
V. vulnificus
in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify
V. vulnificus
DNA in these states.
Comparison of C
18
-Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of
Mycobacterium bovis
in Bovine Milk Samples and Analysis of Samples by PCR
Authors: Cornejo Brandon J. , Sahagún-Ruiz Alfredo , Suárez-Güemes Francisco , Thornton Charles G. , Ficht Thomas A. , Adams L. Garry 
Published: December 2019

ABSTRACT

The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence of
Mycobacterium bovis
by PCR. Detection by a C
18
-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS
6110
-based PCR was 94.1 and 58.8%, respectively (
P
< 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis.
Isolation ofMycobacterium paratuberculosisfrom Milk by Immunomagnetic Separation
Authors: Grant Irene R. , Ball Hywel J. , Rowe Michael T. 
Published: December 2019

ABSTRACTAn immunomagnetic separation (IMS) technique was developed to facilitate selective isolation ofMycobacterium paratuberculosiscells from milk. Rabbit polyclonal antibodies against radiation-killed intactM. paratuberculosiscells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosisIgG-coated beads (IMB) reacted strongly with laboratory strains ofM. paratuberculosisas determined by slide agglutination, and microscopic examination confirmed thatM. paratuberculosiscells attached to the IMB. The IMB were found to have a maximum binding capacity of 104to 105CFU ofM. paratuberculosis. Studies showed that IMS selectively recoveredM. paratuberculosisfrom inoculated milk containing as few as 10 CFU ofM. paratuberculosisper ml when 10 μl of IMB (ca. 106beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline–0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation ofM. paratuberculosisfrom a milk sample relies on chemical decontamination, followed by culturing on Herrold’s egg yolk medium, which must be incubated at 37°C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection ofM. paratuberculosisin milk when it is used in conjunction with end point detection methods, such as IS900PCR or an enzyme-linked immunosorbent assay.