bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biophysical Characterization of Fungal Phytases (
myo
-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance
Authors: Wyss Markus , Pasamontes Luis , Friedlein Arno , Rémy Roland , Tessier Michel , Kronenberger Alexandra , Middendorf Anke , Lehmann Martin , Schnoebelen Line , Röthlisberger Urs , Kusznir Eric , Wahl Guido , Müller Francis , Lahm Hans-Werner , Vogel Kurt , van Loon Adolphus P. G. M. 
Published: December 2019

ABSTRACT

Phytases (
myo
-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus fumigatus
,
Emericella nidulans
,
Myceliophthora thermophila
, and
Talaromyces thermophilus
, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from
Escherichia coli
. All of the phytases examined are monomeric proteins. While
E. coli
phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in
A. niger
, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of
A. fumigatus
and
E. nidulans
phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.
Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class
Proteobacteria
in Compost and Composted Materials
Authors: Kowalchuk George A. , Naoumenko Zinaida S. , Derikx Piet J. L. , Felske Andreas , Stephen John R. , Arkhipchenko Irina A. 
Published: December 2019

ABSTRACT

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class
Proteobacteria
in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera
Nitrosospira
and
Nitrosomonas
. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active.
amoA
, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.
Attachment of the Yeast
Rhodosporidium toruloides
Is Mediated by Adhesives Localized at Sites of Bud Cell Development
Authors: Buck James W. , Andrews John H. 
Published: December 2019

ABSTRACT

The basidiomycetous yeast
Rhodosporidium toruloides
(anamorph,
Rhodotorula glutinis
) is a common phylloplane epiphyte with biocontrol potential. To understand how
R. toruloides
adheres to plant surfaces, we obtained nonadherent fungal mutants after chemical mutagenesis with methane-sulfonic acid ethyl ester. Sixteen attachment-minus (Att
−
) mutants were identified by three methods: (i) screening capsule-minus colonies for loss of adhesive ability; (ii) enrichment for mutants unable to attach to polystyrene; and (iii) selection for reduced fluorescence of fluorescein isothiocyanate-concanavalin A (Con A)-stained cells by fluorescence-activated cell sorting. None of the 16 mutants attached to polystyrene or barley leaves. The lectin Con A eliminated adhesion in all of the wild-type isolates tested. Hapten competition assays indicated that Con A bound to mannose residues on the cell surface. Adhesion of wild-type
R. toruloides
was transient; nonadhesive cells subsequently became adhesive, with bud development. All Att
−
mutants and nonattaching wild-type cells lacked polar regions that stained intensely with fluorescein isothiocyanate-Con A and India ink. Lectin, enzyme, and chemical treatments showed that the polar regions consisted of alkali-soluble materials, including mannose residues. Tunicamycin treatment reduced wild-type adhesion, indicating that the mannose residues could be associated with glycoproteins. We concluded that compounds, including mannose residues, that are localized at sites of bud development mediate adhesion of
R. toruloides
to both polystyrene and barley leaf surfaces.
Use of the Integration Elements Encoded by the Temperate Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy Transcriptional Fusions in
Lactococcus lactis
Authors: Brøndsted Lone , Hammer Karin 
Published: December 2019

ABSTRACT

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the
orf1
gene (presumably encoding a promoter), and the
attP
region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of
Lactococcus lactis
subsp.
cremoris
(B. Christiansen, L. Brøndsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164–5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the
orf1
gene is separated from the
attP
region, the Orf1 protein is able to promote site-specific integration of an
attP
-carrying plasmid into the
attB
site on the
L. lactis
subsp.
cremoris
chromosome. Furthermore, the first detailed deletion analysis of an
attP
region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the
attP
region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of
L. lactis
subsp.
cremoris
when the
orf1
gene is donated in
trans
. The functional 56-bp
attP
region of TP901-1 is substantially smaller than minimal
attP
regions identified for other phages. Based on the deletion analysis, several repeats located within the
attP
region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (
attP
and
orf1
) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in
L. lactis
was constructed. Two promoter-reporter integration vectors containing the reporter gene
gusA
or
lacLM
, encoding β-glucuronidase or β-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of
L. lactis
subsp.
cremoris
MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site
attB
used by bacteriophage TP901-1.
Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources
Authors: Chu Kung-Hui , Alvarez-Cohen Lisa 
Published: December 2019

ABSTRACT
In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.
Phylogenetic Differences between Particle-Associated and Planktonic Ammonia-Oxidizing Bacteria of the β Subdivision of the Class
Proteobacteria
in the Northwestern Mediterranean Sea
Authors: Phillips Carol J. , Smith Zena , Embley T. Martin , Prosser James I. 
Published: December 2019

ABSTRACT

The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the β subdivision of the class
Proteobacteria
associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to
Nitrosomonas eutropha
, while the sequences obtained from the planktonic samples were related to a novel marine
Nitrosospira
group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as
Nitrosomonas
clones, while 96% of these clones were identified as clones that were related to the marine
Nitrosospira
species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the
Nitrosomonas
group. This is the first indication that
Nitrosomonas
species and
Nitrosospira
species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.
Green Fluorescent Protein as a Reporter To Monitor Gene Expression and Food Colonization by
Aspergillus flavus
Authors: Du Wanglei , Huang Zhengyu , Flaherty Joseph E. , Wells Kevin , Payne Gary A. 
Published: December 2019

ABSTRACT

Transformants of
Aspergillus flavus
containing the
Aequorea victoria gfp
gene fused to a viral promoter or the promoter region and 483 bp of the coding region of
A. flavus aflR
expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants.
Phylogenetic Diversity of Symbiotic Methanogens Living in the Hindgut of the Lower Termite
Reticulitermes speratus
Analyzed by PCR and In Situ Hybridization
Authors: Shinzato Naoya , Matsumoto Tadao , Yamaoka Ikuo , Oshima Tairo , Yamagishi Akihiko 
Published: December 2019

ABSTRACT

A phylogenetic analysis of the sequences of 60 clones of archaeal small-subunit rRNA genes amplified from the termite
Reticulitermes speratus
revealed that most of them (56 clones) clustered in the genus
Methanobrevibacter
. Three clones were classified in the order Thermoplasmales. The
Methanobrevibacter
-related symbionts were detected by in situ hybridization analysis.
Effect of Aromatic Compounds on Cellular Fatty Acid Composition of
Rhodococcus opacus
Authors: Tsitko Irina V. , Zaitsev Gennadi M. , Lobanok Anatoli G. , Salkinoja-Salonen Mirja S. 
Published: December 2019

ABSTRACT

In cells of
Rhodococcus opacus
GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of
trans
-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of
R. opacus
GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of
R. opacus
to lipophilic aromatic compounds.
Involvement of Two Plasmids in the Degradation of Carbaryl by
Arthrobacter
sp. Strain RC100
Authors: Hayatsu Masahito , Hirano Motoko , Nagata Tadahiro 
Published: December 2019

ABSTRACT

A bacterium capable of utilizing carbaryl (1-naphthyl
N
-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as
Arthrobacter
and was designated strain RC100. RC100 hydrolyzes the
N
-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah
−
) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat
−
) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah
−
mutants or Nat
−
mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes.
Purification and Characterization of a Novel Peroxidase from
Geotrichum candidum
Dec 1 Involved in Decolorization of Dyes
Authors: Kim Seong Jun , Shoda Makoto 
Published: December 2019

ABSTRACT

A peroxidase (DyP) involved in the decolorization of dyes and produced by the fungus strain
Geotrichum candidum
Dec 1 was purified. DyP, a glycoprotein, is glycosylated with
N
-acetylglucosamine and mannose (17%) and has a molecular mass of 60 kDa and an isoelectric point (pI) of 3.8. The absorption spectrum of DyP exhibited a Soret band at 406 nm corresponding to a hemoprotein, and its Na
2
S
2
O
4
-reduced form revealed a peak at 556 nm that indicates the presence of a protoheme as its prosthetic group. Nine of the 21 types of dyes that were decolorized by Dec 1 cells were decolorized by DyP; in particular, anthraquinone dyes were highly decolorized. DyP also oxidized 2,6-dimethoxyphenol and guaiacol but not veratryl alcohol. The optimal temperature for DyP activity was 30°C, and DyP activity was stable even after incubation at 50°C for 11 h.
Cell Surface-Associated Lipoteichoic Acid Acts as an Adhesion Factor for Attachment of
Lactobacillus johnsonii
La1 to Human Enterocyte-Like Caco-2 Cells
Authors: Granato Dominique , Perotti Fabienne , Masserey Isabelle , Rouvet Martine , Golliard Mireille , Servin Alain , Brassart Dominique 
Published: December 2019

ABSTRACT

The influence of pH on the adhesion of two
Lactobacillus
strains to Caco-2 human intestinal cells was investigated. One strain,
Lactobacillus johnsonii
La1, was adherent at any pH between 4 and 7. The other one,
L. acidophilus
La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for
L. johnsonii
La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the
L. johnsonii
La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of
L. johnsonii
La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by
L. johnsonii
La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of
L. johnsonii
La1 to human Caco-2 cells involves LTA.
Effects of Salinity and Temperature on Long-Term Survival of the Eel Pathogen
Vibrio vulnificus
Biotype 2 (Serovar E)
Authors: Marco-Noales Ester , Biosca Elena G. , Amaro Carmen 
Published: December 2019

ABSTRACT

Vibrio vulnificus
biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30°C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25°C for microcosms at 0.3 and 0.5% and 12°C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion,
V. vulnificus
biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs.
Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria
Authors: Urbach Ena , Vergin Kevin L. , Giovannoni Stephen J. 
Published: December 2019

ABSTRACT
Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.