bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Genetic Properties of the Primary Endosymbionts of Mealybugs Differ from Those of Other Endosymbionts of Plant Sap-Sucking Insects
Authors: Baumann Linda , Thao MyLo Ly , Hess Justin M. , Johnson Marshall W. , Baumann Paul 
Published: July 2002

ABSTRACT

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae), like aphids and psyllids, are plant sap-sucking insects that have an obligate association with prokaryotic endosymbionts that are acquired through vertical, maternal transmission. We sequenced two fragments of the genome of
Tremblaya princeps,
the endosymbiont of mealybugs, which is a member of the β subdivision of the
Proteobacteria
. Each of the fragments (35 and 30 kb) contains a copy of 16S-23S-5S rRNA genes. A total of 37 open reading frames were detected, which corresponded to putative rRNA proteins, chaperones, and enzymes of branched-chain amino acid biosynthesis, DNA replication, protein translation, and RNA synthesis. The genome of
T. princeps
has a number of properties that distinguish it from the genomes of
Buchnera aphidicola
and
Carsonella ruddii,
the endosymbionts of aphids and psyllids, respectively. Among these properties are a high G+C content (57.1 mol%), the same G+C content in intergenic spaces and structural genes, and similar G+C contents of the genes encoding highly and poorly conserved proteins. The high G+C content has a substantial effect on protein composition; about one-third of the residues consist of four amino acids with high-G+C-content codons. Sequence analysis of DNA fragments containing the rRNA operon and adjacent regions from endosymbionts of several mealybug species suggested that there was a single duplication of the rRNA operon and the adjacent genes in an ancestor of the present
T. princeps.
Subsequently, in one mealybug lineage
rpS15,
one of the duplicated genes, was retained, while in another lineage it decayed. These results extend the diversity of the types of endosymbiotic associations found in plant sap-sucking insects.
Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology
Authors: Koizumi Yoshikazu , Kelly John J. , Nakagawa Tatsunori , Urakawa Hidetoshi , El-Fantroussi Saïd , Al-Muzaini Saleh , Fukui Manabu , Urushigawa Yoshikuni , Stahl David A. 
Published: July 2002

ABSTRACT

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family
Desulfobacteriaceae
. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.
Molecular Characterization of the S-Layer Gene,sbpA, ofBacillus sphaericusCCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen
Authors: Ilk Nicola , Völlenkle Christine , Egelseer Eva M. , Breitwieser Andreas , Sleytr Uwe B. , Sára Margit 
Published: July 2002

ABSTRACTThe nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA ofBacillus sphaericusCCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entiresbpAsequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encodingStrep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility ofStrep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.
Monocyclic Aromatic Hydrocarbon Degradation by
Rhodococcus
sp. Strain DK17
Authors: Kim Dockyu , Kim Young-Soo , Kim Seong-Ki , Kim Si Wouk , Zylstra Gerben J. , Kim Young Min , Kim Eungbin 
Published: July 2002

ABSTRACT

Rhodococcus
sp. strain DK17 was isolated from soil and analyzed for the ability to grow on
o-
xylene as the sole carbon and energy source. Although DK17 cannot grow on
m
- and
p
-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on
o
-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of
o
-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on
o
-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a
meta
-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an
ortho
-cleavage pathway which is specifically induced by benzene but not by
o
-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from
o
-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.
Heterologous Expression of the
Lactococcus lactis
Bacteriocin, Nisin, in a Dairy
Enterococcus
Strain
Authors: Li Haiping , O'Sullivan Daniel J. 
Published: July 2002

ABSTRACT

The bacteriocin nisin is produced only by some strains of
Lactococcus lactis
, and to date production in other lactic acid bacteria has not been achieved.
Enterococcus
sp. strain N12β is a nisin-immune transconjugant obtained from a nisin-producing donor (
L. lactis
ATCC 11454) and a dairy recipient (
Enterococcus
sp. strain S12β), but it does not produce nisin. In this study, using PCR amplification, we confirmed that the whole nisin operon is likely present in
Enterococcus
sp. strain N12β. Northern hybridization of total RNA from strain N12β with a
nisA
probe and the results of reverse transcriptase PCR showed the lack of
nisA
transcription in this strain. However,
nisA
transcription was partially restored in strain N12β upon growth in the presence of exogenous nisin, and the
nisA
transcription signal was intensified after an increase in the external nisin level. Furthermore, bioassays showed that active nisin was produced in a dose-dependent fashion by strain N12β following induction by exogenous nisin. These results indicated that expression of the nisin genes in
Enterococcus
sp. strain N12β depended on autoinduction via signal transduction. However, the amount of external inducing signal required was significantly greater than the amount needed for autoinduction in
L. lactis
.
Improving the Pathogenicity of a Nematode-Trapping Fungus by Genetic Engineering of a Subtilisin with Nematotoxic Activity
Authors: Åhman Johan , Johansson Tomas , Olsson Maja , Punt Peter J. , van den Hondel Cees A. M. J. J. , Tunlid Anders 
Published: July 2002

ABSTRACT

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated
PII
is an important pathogenicity factor in the common nematode-trapping fungus
Arthrobotrys oligospora
. The transcript of
PII
was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the
PII
gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the
PII
gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in
Aspergillus niger
) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).
Degradation of Humic Acids by the Litter-Decomposing Basidiomycete
Collybia dryophila
Authors: Steffen Kari Timo , Hatakka Annele , Hofrichter Martin 
Published: July 2002

ABSTRACT

The basidiomycete
Collybia dryophila
K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic
14
C-labeled humic acid (
14
C-HA) prepared from [U-
14
C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn
2+
(200 μM), leading to 75% conversion of LHA and 50% mineralization of
14
C-HA (compared to 60% and 20%, respectively, in the absence of Mn
2+
). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn
2+
-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation,
C. dryophila
released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as
C. dryophila
which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.
Bovine Enteroviruses as Indicators of Fecal Contamination
Authors: Ley Victoria , Higgins James , Fayer Ronald 
Published: July 2002

ABSTRACT
Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.
Identification of Proteins Involved in the Heat Stress Response of
Bacillus cereus
ATCC 14579
Authors: Periago Paula M. , van Schaik Willem , Abee Tjakko , Wouters Jeroen A. 
Published: July 2002

ABSTRACT

To monitor the ability of the food-borne opportunistic pathogen
Bacillus cereus
to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C,
B. cereus
ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of
B. subtilis
. These observations indicate that heat adaptation of
B. cereus
involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic
B. cereus
, which may result in increased survival in foods.
Laccase-Catalyzed Oxidation of Mn
2+
in the Presence of Natural Mn
3+
Chelators as a Novel Source of Extracellular H
2
O
2
Production and Its Impact on Manganese Peroxidase
Authors: Schlosser Dietmar , Höfer Christine 
Published: July 2002

ABSTRACT

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete
Stropharia rugosoannulata
with a molecular mass of approximately 66 kDa oxidized Mn
2+
to Mn
3+
, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn
3+
complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H
2
O
2
formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H
2
O
2
production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn
2+
oxidation and abiotic decomposition of these organic chelators by the resulting Mn
3+
, which leads to formation of superoxide and its subsequent reduction to H
2
O
2
. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn
3+
complexes in assays containing 1 mM Mn
2+
and 100 mM oxalate or malonate, but omitting an additional H
2
O
2
source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn
2+
oxidation in providing H
2
O
2
for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.
Purification and Characterization of an Extracellular Protease from
Penicillium chrysogenum
Pg222 Active against Meat Proteins
Authors: Benito María J. , Rodríguez Mar , Núñez Félix , Asensio Miguel A. , Bermúdez María E. , Córdoba Juan J. 
Published: July 2002

ABSTRACT

An extracellular protease from
Penicillium chrysogenum
(Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and
Aspergillus oryzae
protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from
Penicillium citrinum
. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.
Silencing of the Aspergillopepsin B (
pepB
) Gene of
Aspergillus awamori
by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
Authors: Moralejo Francisco J. , Cardoza Rosa Elena , Gutierrez Santiago , Lombraña Marta , Fierro Francisco , Martín Juan F. 
Published: July 2002

ABSTRACT

Aspergillopepsin B was identified in culture broths of
Aspergillus awamori
by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The
pepB
gene encoding aspergillopepsin B of
A. awamori
was cloned and characterized. It is located in chromosome IV of
A. awamori
, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of
Aspergillus niger
var.
macrosporus
. The
pepB
gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the
pepB
gene in the antisense orientation downstream from the
gpdA
promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the
pepB
gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the
pepB
gene was deleted by double crossover. Two of the selected transformants lacked the endogenous
pepB
gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in
A. awamori
.
New Chromogenic Agar Medium for the Identification ofCandidaspp
Authors: Cooke Venitia M. , Miles R. J. , Price R. G. , Midgley G. , Khamri W. , Richardson A. C. 
Published: July 2002

ABSTRACTA new chromogenic agar medium (Candidadiagnostic agar [CDA]) for differentiation ofCandidaspp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed byCandida albicansandCandida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies ofCandida tropicalisandCandida kefyrwere uniformly pink, and colonies of otherCandidaspp., includingCandida glabrataandCandida parapsilosis, were white. CDA was evaluated by using 115 test strains ofCandidaspp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies ofC. albicanswere not distinguished from colonies ofC. dubliniensis. However, for the group containingC. albicansplusC. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containingC. tropicalisplusC. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiateC. tropicalisandC. kefyrstrains but did differentiate members of a broader group (C. tropicalis,C. kefyr,Candida lusitaniaeplusCandida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity ofCandidadetection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.