bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from
Sphingomonas
sp. Strain AO1
Authors: Sasaki Miho , Akahira Ayako , Oshiman Ko-ichi , Tsuchido Tetsuaki , Matsumura Yoshinobu 
Published: December 2005

ABSTRACT

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation
16:
449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by
Sphingomonas
sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450
bisd
), ferredoxin (Fd
bisd
), and ferredoxin reductase (Red
bisd
). We demonstrated that P450
bisd
and Fd
bisd
are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd
bisd
revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450
bisd
, in the presence of Fd
bisd
, Red
bisd
, and NADH, was able to convert BPA. The

K
m

and
k
cat
values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min
−1
, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450
bisd
might exhibit strict specificity. Two BPA degradation products of the P450
bisd
system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.
Microarray-Based Detection and Typing of the Rhizobium Nodulation Gene
nodC
: Potential of DNA Arrays To Diagnose Biological Functions of Interest
Authors: Bontemps Cyril , Golfier Geoffroy , Gris-Liebe Carine , Carrere Sébastien , Talini Luc , Boivin-Masson Catherine 
Published: December 2005

ABSTRACT

Environmental screening of bacteria for the presence of genes of interest is a challenging problem, due to the high variability of the nucleotide sequence of a given gene between species. Here, we tackle this general issue using a particularly well-suited model system that consists of the nodulation gene
nodC
, which is shared by phylogenetically distant rhizobia. 41mer and 50mer oligonucleotides featuring the nucleotide diversity of two highly conserved regions of the NodC protein were spotted on glass slides and cross hybridized with the radioactive-labeled target genomic DNA under low-stringency conditions. Statistical analysis of the hybridization patterns allowed the detection of known, as well as new,
nodC
sequences and classified the rhizobial strains accordingly. The microarray was successfully used to type the
nodC
gene directly from legume nodules, thus eliminating the need of cultivation of the endosymbiont. This approach could be extended to a panel of diagnostic genes and constitute a powerful tool for studying the distribution of genes of interest in the environment, as well as for bacteria identification.
Physiology and Phylogeny of Green Sulfur Bacteria Forming a Monospecific Phototrophic Assemblage at a Depth of 100 Meters in the Black Sea
Authors: Manske Ann K. , Glaeser Jens , Kuypers Marcel M. M. , Overmann Jörg 
Published: December 2005

ABSTRACTThe biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophylle(BChle) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter−1. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChle. Only traces of BChle(8 ng liter−1) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m−2s−1at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of theChlorobiaceaeand was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m−2s−1for photosynthetic14CO2fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.
Heterologous Production of Antimicrobial Peptides in
Propionibacterium freudenreichii
Authors: Brede Dag Anders , Faye Therese , Stierli Melanie Patricia , Dasen Gottfried , Theiler Anita , Nes Ingolf F. , Meile Leo , Holo Helge 
Published: December 2005

ABSTRACT

Heterologous bacteriocin production in
Propionibacterium freudenreichii
is described. We developed an efficient system for DNA shuttling between
Escherichia coli
and
P. freudenreichii
using vector pAMT1. It is based on the
P. freudenreichii
rolling-circle replicating plasmid pLME108 and carries the
cml
(A)/
cmx
(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (
pctA
) into pAMT1 under the control of the constitutive promoter (P
4
) yielded bacteriocin in amounts equal to those of the wild-type producer
Propionibacterium thoenii
419. The
P. freudenreichii
clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The
pamA
gene from
P. thoenii
419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in
P. freudenreichii
, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the
pamA
signal peptide and PAMP were fused in frame. The promoter region of
pamA
was identified by fusion of putative promoter fragments to the coding sequence of the
pctA
gene. The P
4
and P
pamp
promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.
Sequential Reductive Dechlorination of
meta
-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different
Chloroflexi
Phylotypes
Authors: Fagervold Sonja K. , Watts Joy E. M. , May Harold D. , Sowers Kevin R. 
Published: December 2005

ABSTRACT

Three species within a deeply branching cluster of the
Chloroflexi
are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the
Chloroflexi
including
Dehalococcoides
spp. and the
o
-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the
para-
flanked
meta
position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the
Dehalococcoides
group. This same species reductively dechlorinated the
para
- and
ortho-
flanked
meta
-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the
o
-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the
Chloroflexi
exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of
Chloroflexi
with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.
Diversity of Carotenoid Synthesis Gene Clusters from Environmental
Enterobacteriaceae
Strains
Authors: Sedkova Natalia , Tao Luan , Rouvière Pierre E. , Cheng Qiong 
Published: December 2005

ABSTRACT

Eight
Enterobacteriaceae
strains that produce zeaxanthin and derivatives of this compound were isolated from a variety of environmental samples. Phylogenetic analysis showed that these strains grouped with different clusters of
Erwinia
type strains. Four strains representing the phylogenetic diversity were chosen for further characterization, which revealed their genetic diversity as well as their biochemical diversity. The carotenoid synthesis gene clusters cloned from the four strains had three different gene organizations. Two of the gene clusters, those from strains DC416 and DC260, had the classical organization
crtEXYIBZ
; the gene cluster from DC413 had the rare organization
crtE-idi-XYIBZ
; and the gene cluster from DC404 had the unique organization
crtE-idi-YIBZ
. Besides the diversity in genetic organization, these genes also exhibited considerable sequence diversity. On average, they exhibited 60 to 70% identity with each other, as well as with the corresponding genes of the
Pantoea
type strains. The four different clusters were individually expressed in
Escherichia coli
, and the two
idi
-containing clusters gave more than fivefold-higher carotenoid titers than the two clusters lacking
idi
. Expression of the
crtEYIB
genes with and without
idi
confirmed the effect of increasing carotenoid titer by the type II
idi
gene linked with the carotenoid synthesis gene clusters.
Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland
Authors: Metje Martina , Frenzel Peter 
Published: December 2005

ABSTRACT

The effects of temperature on rates and pathways of CH
4
production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH
4
and CO
2
over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH
3
F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH
4
· g [dry weight]
−1
· day
−1
), but the activity was relatively high even at 4°C (0.25 μmol CH
4
· g [dry weight]
−1
· day
−1
). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order
Methanobacteriales
, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H
2
sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.
Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis inSaccharomyces cerevisiae
Authors: Leonard Effendi , Yan Yajun , Lim Kok Hong , Koffas Mattheos A. G. 
Published: December 2005

ABSTRACTFlavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing inSaccharomyces cerevisiaea four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain whenp-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.
Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from X
anthomonas campestris
pv. vesicatoria
Authors: Basim Huseyin , Minsavage Gerald V. , Stall Robert E. , Wang Jaw-Fen , Shanker Savita , Jones Jeffrey B. 
Published: December 2005

ABSTRACT

We characterized the copper resistance genes in strain XvP26 of
Xanthomonas campestris
pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in
Xanthomonas
and
Pseudomonas
strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in
Pseudomonas syringae
pv. tomato and
Escherichia coli
, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in
E. coli
and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in
Sinorhizobium meliloti
, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant
X. campestris
pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in
X. campestris
pv. vesicatoria.
Bleeding Sap and Old Wood Are the Two Main Sources of Contamination of Merging Organs of Vine Plants byXylophilus ampelinus, the Causal Agent of Bacterial Necrosis
Authors: Grall S. , Roulland C. , Guillaumès J. , Manceau C. 
Published: December 2005

ABSTRACTThe spatial distribution of vine plants contaminated byXylophilus ampelinus, the agent responsible for bacterial necrosis, was studied over a 5-year period within two vineyards in the Cognac area. Both vineyards were planted withVitis viniferacv. Ugni blanc but were different in age and agronomic location. The emission ofX. ampelinusin contaminated bleeding sap was observed during vine sprouting. Contaminated bleeding sap is an important source of inoculum for external contamination due to the high susceptibility of young merging shoots to the pathogen.X. ampelinusemission by bleeding sap was not affected by the age of the plants or the location of the vineyards. However, its emission was irregular with time, and it varied between two fruit canes from individual plants and between plants as well as between years. Moreover, the two vineyards appeared to be entirely contaminated. Consequently, the behavior of the pathogen is not predictable. The distribution of the pathogen inside vine plant organs was analyzed through the four growing seasons. The old wood was contaminated throughout the year and constituted a stock inoculum for endophytic contamination of crude sap during the winter and the spring. Despite the fact that most of the young green shoots were contaminated in May,X.ampelinuswas not found in green shoots in June and September, refuting the hypothesis of an epiphytic life of the pathogen under natural conditions. Although all plants were entirely contaminated in both vineyards, symptoms were rare and were observed on different plants each year.
Molecular Characterization ofClostridium perfringensIsolates from Humans with Sporadic Diarrhea: Evidence for Transcriptional Regulation of the Beta2-Toxin-Encoding Gene
Authors: Harrison Ben , Raju Deepa , Garmory Helen S. , Brett Moira M. , Titball Richard W. , Sarker Mahfuzur R. 
Published: December 2005

ABSTRACTClostridium perfringenstype A food poisoning is caused byC. perfringensisolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused byC. perfringensplasmidcpeisolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14cpe+C. perfringensfecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified ascpe+type A, and 12 of the 14cpe+isolates carry theircpegene on the plasmid and 2 carry it on the chromosome. Interestingly,cpb2is present in only 12 plasmidcpeisolates; 11 isolates carrycpeandcpb2on different plasmids, butcpeandcpb2are located on the same plasmid in one isolate.C. perfringensenterotoxin is produced by all 14cpe+SD isolates. However, only 10 of the 12cpe+/cpb2+SD isolates produced CPB2, with significant variation in amounts. The levels ofcpb2mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypicalcpb2was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression ofcpb2mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94cpb2, which overexpressedcpb2mRNA, restored CPB2 production in F4969 (acpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin inC. perfringens-associated SD.
Visualization and Detection of Infectious Coxsackievirus Replication Using a Combined Cell Culture-Molecular Beacon Assay
Authors: Wang Aijun , Salazar Andre M. , Yates Marylynn V. , Mulchandani Ashok , Chen Wilfred 
Published: December 2005

ABSTRACT
Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5′ noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.