bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydroxycinnamate (
hca
) Catabolic Genes from
Acinetobacter
sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters
Authors: Parke Donna , Ornston L. Nicholas 
Published: September 2003

ABSTRACT

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium
Acinetobacter
sp. strain ADP1. Genes for protocatechuate catabolism are central to the
dca-pca-qui-pob-hca
chromosomal island, for which gene designations corresponding to catabolic function are
dca
(dicarboxylic acid),
pca
(protocatechuate),
qui
(quinate),
pob
(
p
-hydroxybenzoate), and
hca
(hydroxycinnamate).
Acinetobacter hcaC
had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate,
p
-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from
pobS
to an
Xba
I site 5.3 kb beyond
hcaC
was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the
hca
region of the chromosome. pZR8200 enabled
Escherichia coli
to convert
p
-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated
hcaA
,
hcaB
,
hcaK
,
hcaR
, and ORF1. An
Acinetobacter
strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with
p
-coumarate. A chromosomal fusion of
lacZ
to the
hcaE
gene was used to monitor expression of the
hcaABCDE
promoter. LacZ was induced over 100-fold by growth in the presence of caffeate,
p
-coumarate, or ferulate. The protein deduced to be encoded by
hcaR
shares 28% identity with the aligned
E. coli
repressor, MarR. A knockout of
hcaR
produced a constitutive phenotype, as assessed in the
hcaE
::
lacZ
-Km
r
genetic background, revealing HcaR to be a repressor as well. Expression of
hcaE
::
lac
Z in strains with knockouts in
hcaA
,
hcaB
, or
hcaC
revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the
hcaABCDE
genes by HcaR.
The Metabolic Pathway of 4-Aminophenol in
Burkholderia
sp. Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents
Authors: Takenaka Shinji , Okugawa Susumu , Kadowaki Maho , Murakami Shuichiro , Aoki Kenji 
Published: September 2003

ABSTRACT

Burkholderia
sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with

K
m

and
V
max
values of 9.6 μM and 6.8 μmol min
−1
mg of protein
−1
, respectively.
Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria
Authors: Ekstrom Eileen B. , Morel François M. M. , Benoit Janina M. 
Published: September 2003

ABSTRACT

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain,
Desulfovibrio desulfuricans
LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two
Desulfobacter
strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B
12
in some and perhaps many incomplete-oxidizing SRB strains.
Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System ofStreptococcus thermophilus
Authors: Cochu Armelle , Vadeboncoeur Christian , Moineau Sylvain , Frenette Michel 
Published: September 2003

ABSTRACTIn most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains ofStreptococcus thermophilusdo not or poorly metabolize glucose, compelling evidence suggests thatS. thermophiluspossesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. Theptsoperon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kbptsHmRNA and a 2.3-kbptsHImRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded bymanL,manM,manN, andmanO, respectively, which make up themanoperon. Themanoperon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABManas well as membrane fragments containing IICManand IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of theS. thermophilusPTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that inS. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.
Exposure of Sink Drain Microcosms to Triclosan: Population Dynamics and Antimicrobial Susceptibility
Authors: McBain Andrew J. , Bartolo Robert G. , Catrenich Carl E. , Charbonneau Duane , Ledder Ruth G. , Price Bradford B. , Gilbert Peter 
Published: September 2003

ABSTRACTRecent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca.10 log10CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, andAlcaligenesspp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related toAchromobacter xylosoxidansbecame clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.
Effects of Current Velocity on the Nascent Architecture of Stream Microbial Biofilms
Authors: Battin Tom J. , Kaplan Louis A. , Newbold J. Denis , Cheng Xianhao , Hansen Claude 
Published: September 2003

ABSTRACT

Current velocity affected the architecture and dynamics of natural, multiphyla, and cross-trophic level biofilms from a forested piedmont stream. We monitored the development and activity of biofilms in streamside flumes operated under two flow regimes (slow [0.065 m s
−1
] and fast [0.23 m s
−1
]) by combined confocal laser scanning microscopy with cryosectioning to observe biofilm structure and composition. Biofilm growth started as bacterial microcolonies embedded in extracellular polymeric substances and transformed into ripple-like structures and ultimately conspicuous quasihexagonal networks. These structures were particularly pronounced in biofilms grown under slow current velocities and were characterized by the prominence of pennate diatoms oriented along their long axes to form the hexagons. Microstructural heterogeneity was dynamic, and biofilms that developed under slower velocities were thicker and had larger surface sinuosity and higher areal densities than their counterparts exposed to higher velocities. Surface sinuosity and biofilm fragmentation increased with thickness, and these changes likely reduced resistance to the mass transfer of solutes from the water column into the biofilms. Nevertheless, estimates of dissolved organic carbon uptake and microbial growth suggested that internal cycling of carbon was more important in thick biofilms grown in slow flow conditions. High-pressure liquid chromatography-pulsed amperometric detection analyses of exopolysaccharides documented a temporal shift in monosaccharide composition as the glucose levels decreased and the levels of rhamnose, galactose, mannose, xylose, and arabinose increased. We attribute this change in chemical composition to the accumulation of diatoms and increased incorporation of detrital particles in mature biofilms.
Colonization of Flax Roots and Early Physiological Responses of Flax Cells Inoculated with Pathogenic and Nonpathogenic Strains of
Fusarium oxysporum
Authors: Olivain Chantal , Trouvelot Sophie , Binet Marie-Noëlle , Cordier Christelle , Pugin Alain , Alabouvette Claude 
Published: September 2003

ABSTRACT

Fusarium oxysporum
includes nonpathogenic strains and pathogenic strains that can induce necrosis or tracheomycosis in plants. The objective of this study was to compare the abilities of a pathogenic strain (Foln3) and a nonpathogenic strain (Fo47) to colonize flax roots and to induce early physiological responses in flax cell culture suspensions. Both strains colonized the outer cortex of the root; however, plant defense reactions, i.e., the presence of wall appositions, osmiophilic material, and collapsed cells, were less frequent and less intense in a root colonized by Foln3 than by Fo47. Early physiological responses were measured in flax cell suspensions confronted with germinated microconidia of both strains. Both pathogenic (Foln3) and nonpathogenic strains (Fo47) triggered transient H
2
O
2
production in the first few minutes of the interaction, but the nonpathogenic strain also induced a second burst 3 h postinoculation. Ca
2+
influx was more intense in cells inoculated with Fo47 than in cells inoculated with Foln3. Similarly, alkalinization of the extracellular medium was higher with Fo47 than with Foln3. Inoculation of the fungi into flax cell suspensions induced cell death 10 to 20 h postinoculation, with a higher percentage of dead cells observed with Fo47 than with Foln3 beginning at 14 h. This is the first report showing that early physiological responses of flax cells can be used to distinguish pathogenic and nonpathogenic strains of the soil-borne fungus
F. oxysporum
.
Growth of
Escherichia coli
in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine
Authors: Williams Margaret M. , Braun-Howland Ellen B. 
Published: September 2003

ABSTRACT

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an
Escherichia coli
isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH
2
Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH
2
Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically,
Legionella pneumophila
,
E. coli
, and β and δ proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing
E. coli
within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.
Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-Flow Bioreactor
Authors: Girguis Peter R. , Orphan Victoria J. , Hallam Steven J. , DeLong Edward F. 
Published: September 2003

ABSTRACT
Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.
Identification of Methyl Coenzyme M Reductase A (
mcrA
) Genes Associated with Methane-Oxidizing Archaea
Authors: Hallam Steven J. , Girguis Peter R. , Preston Christina M. , Richardson Paul M. , DeLong Edward F. 
Published: September 2003

ABSTRACT

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (
mcrA
) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel
mcrA
types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of
mcrA
types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and
mcrA
types in a microcosm, phylogenetic congruence between
mcrA
and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2
mcrA
sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with
mcrA
sequences and define a functional genomic link between methanogenic and methanotrophic archaea.
Development and Application of a Monoclonal-Antibody Technique for Counting
Aureococcus anophagefferens
, an Alga Causing Recurrent Brown Tides in the Mid-Atlantic United States
Authors: Caron David A. , Dennett Mark R. , Moran Dawn M. , Schaffner Rebecca A. , Lonsdale Darcy J. , Gobler Christopher J. , Nuzzi Robert , McLean Tim I. 
Published: September 2003

ABSTRACT

A
method was developed for the rapid detection and
enumeration of
Aureococcus anophagefferens
, the cause of
harmful algal blooms called “brown tides” in estuaries
of the Mid-Atlantic United States. The method employs a monoclonal
antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay
format. The MAb obtained exhibits high reactivity with
A.
anophagefferens
and very low cross-reactivities with a
phylogenetically diverse array of other protists and bacteria. Standard
curves are constructed for each 96-well microtiter plate by using known
amounts of a preserved culture of
A. anophagefferens
. This
approach allows estimation of the abundance of the alga in natural
samples. The MAb method was compared to an existing method that employs
polyclonal antibodies and epifluorescence microscopy and to direct
microscopic counts of
A. anophagefferens
in samples with high
abundances of the alga. The MAb method provided increased quantitative
accuracy and greatly reduced sample processing time. A spatial survey
of several Long Island estuaries in May 2000 using this new approach
documented a range of abundances of
A. anophagefferens
in
these bays spanning nearly 3 orders of
magnitude.
Filamentous “
Epsilonproteobacteria
” Dominate Microbial Mats from Sulfidic Cave Springs
Authors: Engel Annette Summers , Lee Natuschka , Porter Megan L. , Stern Libby A. , Bennett Philip C. , Wagner Michael 
Published: September 2003

ABSTRACT

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the “
Epsilonproteobacteria
” and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of “
Epsilonproteobacteria
” in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.
Extremely Acidophilic Protists from Acid Mine Drainage Host
Rickettsiales
-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes
Authors: Baker Brett J. , Hugenholtz Philip , Dawson Scott C. , Banfield Jillian F. 
Published: September 2003

ABSTRACT

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order
Rickettsiales
in the
Alphaproteobacteria
. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist
Acanthamoeba
. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “
Candidatus
Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.