bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Recent 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

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

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

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

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

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

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

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

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.
Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry
Authors: Biegala Isabelle C. , Not Fabrice , Vaulot Daniel , Simon Nathalie
Published: September 2003

Picoeukaryotes (cells of <3 μm in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-μm-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.
History of the ADP/ATP-Translocase-Encoding Gene, a Parasitism Gene Transferred from a
Chlamydiales
Ancestor to Plants 1 Billion Years Ago
Authors: Greub Gilbert , Raoult Didier
Published: September 2003

Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene,
tlc
, is found only in
Rickettsiales
,
Chlamydiales
, and plant and alga plastids. We demonstrate the presence of
tlc
in
Parachlamydia acanthamoebae.
This gene shares more similarity with the
tlc
1 gene of
Chlamydiaceae
and the
tlc
of plant and alga plastids than with the
tlc
2 gene of
Chlamydiaceae
. Phylogenetic analysis, including all other
tlc
homologs found in GenBank, showed that
tlc
was duplicated in a
Chlamydiales
ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by
tlc
. Plant and alga plastids acquired
tlc
approximately when
Parachlamydiaceae
and
Chlamydiaceae
diverged, at the eucaryotic radiation time, ca. 1 billion years ago.
Plasmid Transfer from
Pseudomonas putida
to the Indigenous Bacteria on Alfalfa Sprouts: Characterization, Direct Quantification, and In Situ Location of Transconjugant Cells
Authors: Mølbak Lars , Licht Tine Rask , Kvist Thomas , Kroer Niels , Andersen Sigrid Rita
Published: September 2003

The transfer of the plasmids pJKJ5 and TOL (pWWO) from
Pseudomonas putida
to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 × 10
−4
and 2.0 × 10
−6
transconjugant cells per donor cell for pKJK5::
gfp
and TOL::
gfp
, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera
Erwinia
and
Paenibacillus
were gradually replaced by
Pseudomonas
on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera
Pseudomonas
and
Erwinia.
The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from
P. putida
in the absence of selective pressure that could favor the presence of the investigated plasmids.
Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms
Authors: Lawrence J. R. , Swerhone G. D. W. , Leppard G. G. , Araki T. , Zhang X. , West M. M. , Hitchcock A. P.
Published: September 2003

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.
Foreshore Sand as a Source of
Escherichia coli
in Nearshore Water of a Lake Michigan Beach
Authors: Whitman Richard L. , Nevers Meredith B.
Published: September 2003

Swimming advisories due to excessive
Escherichia coli
concentrations are common at 63rd Street Beach, Chicago, Ill. An intensive study was undertaken to characterize the source and fate of
E. coli
in beach water and sand at the beach. From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples,
n
= 1,747; sand samples,
n
= 858); hydrometeorological conditions and bird and bather distributions were also recorded.
E. coli
concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth. Gull contributions to
E. coli
densities in sand and water were most apparent on the day following gull activity in a given area.
E. coli
recolonized newly placed foreshore sand within 2 weeks. Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that
E. coli
may be able to sustain population density in temperate beach sand during summer months without external inputs. This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of
E. coli
to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input.
Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient
Authors: Feris Kevin , Ramsey Philip , Frazar Chris , Moore Johnnie N. , Gannon James E. , Holben William E.
Published: September 2003

The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to α-, β-, and γ-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.
Characterization of a Theta-Type Plasmid from
Lactobacillus sakei
: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli
Authors: Alpert Carl-Alfred , Crutz-Le Coq Anne-Marie , Malleret Christine , Zagorec Monique
Published: September 2003

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from
Lactobacillus sakei
RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative
ori
segment containing 11- and 22-bp repeats and a
repA
gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an
Escherichia coli
replicon to produce the shuttle vector pRV566 and was observed to be functional in
L
.
sakei
for plasmid replication. The
L
.
sakei
replicon alone could not support replication in
E
.
coli
. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in
L
.
sakei
. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as
Lactobacillus curvatus
,
Lactobacillus casei
, and
Lactobacillus plantarum
, in addition to
L
.
sakei
, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.
A Gene Cluster for Chlorate Metabolism in
Ideonella dechloratans
Authors: Thorell Helena Danielsson , Stenklo Katarina , Karlsson Jan , Nilsson Thomas
Published: September 2003

Chlorate reductase has been isolated from the chlorate-respiring bacterium
Ideonella dechloratans
, and the genes encoding the enzyme have been sequenced. The enzyme is composed of three different subunits and contains molybdopterin, iron, probably in iron-sulfur clusters, and heme
b
. The genes (
clr
) encoding chlorate reductase are arranged as
clrABDC
, where
clrA
,
clrB
, and
clrC
encode the subunits and
clrD
encodes a specific chaperone. Judging from the subunit composition, cofactor content, and sequence comparisons, chlorate reductase belongs to class II of the dimethyl sulfoxide reductase family. The
clr
genes are preceded by a novel insertion sequence (transposase gene surrounded by inverted repeats), denoted ISIde1. Further upstream, we find the previously characterized gene for chlorite dismutase (
cld
), oriented in the opposite direction. Chlorate metabolism in
I. dechloratans
starts with the reduction of chlorate, which is followed by the decomposition of the resulting chlorite to chloride and molecular oxygen. The present work reveals that the genes encoding the enzymes catalyzing both these reactions are in close proximity.
Wide Distribution of a Novel
pmoA
-Like Gene Copy among Type II Methanotrophs, and Its Expression in
Methylocystis
Strain SC2
Authors: Tchawa Yimga Merlin , Dunfield Peter F. , Ricke Peter , Heyer Jürgen , Liesack Werner
Published: September 2003

Experiments were conducted to determine if a novel
pmoA
-like gene (
pmoA2
) recently discovered in the methane-oxidizing bacterium
Methylocystis
strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of
pmoA1
and
pmoA2
. By using this primer combination, we identified
pmoA2
in a wide range of type II MOB of the
Methylosinus-Methylocystis
group. However, screening by PCR and by Southern hybridization using a newly developed
pmoA2
-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor
pmoA2
. No
pmoA2
was detected in five type I MOB tested:
Methylococcus capsulatus
strain Bath,
Methylocaldum
strain E10A,
Methylobacter luteus
,
Methylomicrobium album
, and
Methylomonas
strain D1a. In comparative sequence analyses, all
pmoA2
-like sequences formed a coherent cluster clearly distinct from
pmoA1
sequences of type I and type II MOB, and from
amoA
sequences of the
Nitrosomonas
-
Nitrosospira
group. Phylogenetic analysis using the paml model suggested that
pmoA2
is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of
Methylocystis
strain SC2 for gene expression of
pmoA
. A strong signal was observed for
pmoA1
in Northern hybridization, while the results obtained for
pmoA2
were ambiguous. However, reverse transcription-PCR confirmed that
pmoA2
was expressed, albeit at lower level than
pmoA1
. This provided experimental evidence that the gene product of
pmoA2
may be a functionally active enzyme.
Isolation and Identification of Actinobacteria from Surface-Sterilized Wheat Roots
Authors: Coombs Justin T. , Franco Christopher M. M.
Published: September 2003

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (
Triticum aestivum
L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including
Streptomyces
,
Microbispora
,
Micromonospora
, and
Nocardiodes
spp. Many of the
Streptomyces
spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to
Streptomyces
spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to
Streptomyces caviscabies
and
S. setonii
. None of these isolates, nor the
S. caviscabies
and
S. setonii
type strains, were found to carry the
nec1
pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.
Bacterial Diversity and Sulfur Cycling in a Mesophilic Sulfide-Rich Spring
Authors: Elshahed Mostafa S. , Senko John M. , Najar Fares Z. , Kenton Stephen M. , Roe Bruce A. , Dewers Thomas A. , Spear John R. , Krumholz Lee R.
Published: September 2003

An artesian sulfide- and sulfur-rich spring in southwestern Oklahoma is shown to sustain an extremely rich and diverse microbial community. Laboratory incubations and autoradiography studies indicated that active sulfur cycling is occurring in the abundant microbial mats at Zodletone spring. Anoxygenic phototrophic bacteria oxidize sulfide to sulfate, which is reduced by sulfate-reducing bacterial populations. The microbial community at Zodletone spring was analyzed by cloning and sequencing 16S rRNA genes. A large fraction (83%) of the microbial mat clones belong to sulfur- and sulfate-reducing lineages within δ-
Proteobacteria
, purple sulfur γ
-Proteobacteria
, ε
-Proteobacteria
,
Chloroflexi
, and filamentous
Cyanobacteria
of the order
Oscillatoria
as well as a novel group within γ
-Proteobacteria
. The 16S clone library constructed from hydrocarbon-exposed sediments at the source of the spring had a higher diversity than the mat clone library (Shannon-Weiner index of 3.84 compared to 2.95 for the mat), with a higher percentage of clones belonging to nonphototrophic lineages (e.g.,
Cytophaga
,
Spirochaetes
,
Planctomycetes
,
Firmicutes
, and
Verrucomicrobiae
). Many of these clones were closely related to clones retrieved from hydrocarbon-contaminated environments and anaerobic hydrocarbon-degrading enrichments. In addition, 18 of the source clones did not cluster with any of the previously described microbial divisions. These 18 clones, together with previously published or database-deposited related sequences retrieved from a wide variety of environments, could be clustered into at least four novel candidate divisions. The sulfate-reducing community at Zodletone spring was characterized by cloning and sequencing a 1.9-kb fragment of the dissimilatory sulfite reductase (DSR) gene. DSR clones belonged to the
Desulfococcus
-
Desulfosarcina
-
Desulfonema
group,
Desulfobacter
group, and
Desulfovibrio
group as well as to a deeply branched group in the DSR tree with no representatives from cultures. Overall, this work expands the division-level diversity of the bacterial domain and highlights the complexity of microbial communities involved in sulfur cycling in mesophilic microbial mats.
Purification and Properties of a Feruloyl Esterase Involved in Lignocellulose Degradation byAureobasidium pullulans
Authors: Rumbold Karl , Biely Peter , Mastihubová Maria , Gudelj Marinka , Gübitz Georg , Robra Karl-Heinz , Prior Bernard A.
Published: September 2003

The lignocellulolytic fungusAureobasidium pullulansNRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methylp-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-Oand 5-Oarabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside,O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, andp-nitrophenyl-acetate but reveals only low activity onp-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methylp-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.
Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated σ
54
-Dependent Promoter in
Acinetobacter
sp. Strain ADP1
Authors: Jones Rheinallt M. , Williams Peter A.
Published: September 2003

The
areR
gene in
Acinetobacter
sp. strain ADP1 regulates the expression of the
areCBA
genes, which determine growth on benzyl alkanoates. AreR is a member of the NtrC/XylR family of regulatory proteins as determined by sequence homology. Seventy-nine bases upstream of the start of transcription is a region carrying two overlapping inverted repeat (IR) sequences that we predict to be the AreR binding site, also known as the upstream activator site (UAS). IR1 is a near-perfect (16 of 17 bp) repeat separated by 1 bp, and IR2 consists of 9- and 7-bp perfect repeats with a 3-bp gap, with the central bases of the two arms of the repeat separated by 44 and 22 bp. We report here a method for site-directed mutagenesis of chromosomal genes in ADP1 in which linear fragments generated by overlap extension PCR are used to transform ADP1 via its natural transformation system and recombinants are selected by a marker exchange-eviction strategy with a newly created
sacB
-Km cassette. This method was used to generate 38 strains with designed mutations in the putative UAS upstream of
areCBA.
The effects of the mutations on
areCBA
expression were measured by enzyme assays of benzyl alcohol dehydrogenase (AreB) and by reporter gene assays of
lacZ
inserted into
areA
. Substitutions or deletions in IR1 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of IR2, than when they were in its outer regions. By contrast, substitutions in the right arm of IR2 resulted in mutants with relatively high expression levels compared to that of the wild type. Effects of deletions in the right arm of IR2 were very dependent upon the length of the deletion, with 3- or 5-bp deletions reducing expression by >90% whereas an 11-bp deletion in the same area reduced the expression levels by only 50%, suggesting that alterations in the distance and the orientation of the UAS relative to the −24, −12 σ
54
promoter are critical.
Conversion of 2-Fluoromuconate tocis-Dienelactone by Purified Enzymes ofRhodococcus opacus1cp
Authors: Solyanikova Inna P. , Moiseeva Olga V. , Boeren Sjef , Boersma Marelle G. , Kolomytseva Marina P. , Vervoort Jacques , Rietjens Ivonne M. C. M. , Golovleva Ludmila A. , van Berkel Willem J. H.
Published: September 2003

The present study describes the19F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) fromRhodococcus opacus1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase fromTrichosporon cutaneumor 2-hydroxybiphenyl 3-mono-oxygenase fromPseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone tocis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes inR. opacus1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.
Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes
Authors: De Wildeman Stefaan , Diekert Gabriele , Van Langenhove Herman , Verstraete Willy
Published: September 2003

The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C2groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation ofDesulfitobacterium dichloroeliminansstrain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conservingantimechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.
Biofilm Formation and the Presence of the Intercellular Adhesion Locus
ica
among Staphylococci from Food and Food Processing Environments
Authors: Møretrø Trond , Hermansen Lene , Holck Askild L. , Sidhu Maan S. , Rudi Knut , Langsrud Solveig
Published: September 2003

In clinical staphylococci, the presence of the
ica
genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of
Staphylococcus.
The
icaA
gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of
icaA
was positively correlated with strong biofilm formation. The
icaA
gene was partly sequenced for 22 food-related strains from nine different species of
Staphylococcus
, and their
icaA
genes were found to have DNA similarities to previously sequenced
icaA
genes of 69 to 100%. Northern blot analysis indicated that the expression of the
ica
genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.
Novel Eukaryotes from the Permanently Anoxic Cariaco Basin (Caribbean Sea)
Authors: Stoeck Thorsten , Taylor Gordon T. , Epstein Slava S.
Published: September 2003

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84,inD. Kirchman, ed.,Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75,inR. R. Colwell and D. J. Grimes, ed.,Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84,inD. Kirchman, ed.,Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75,inR. R. Colwell and D. J. Grimes, ed.,Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).
Toward an International Standard for PCR-BasedDetection of Food-Borne Thermotolerant Campylobacters: AssayDevelopment and AnalyticalValidation
Authors: Lübeck P.S. , Wolffs P. , On S. L. W. , Ahrens P. , Rådström P. , Hoorfar J.
Published: September 2003

As
part of a European research project (FOOD-PCR), we developed a
standardized and robust PCR detection assay specific for the three most
frequently reported food-borne pathogenic
Campylobacter
species,
C. jejuni
,
C. coli
, and
C. lari
.
Fifteen published and unpublished PCR primers targeting the 16S rRNA
gene were tested in all possible pairwise combinations, as well as two
published primers targeting the 23S rRNA gene. A panel of 150 strains
including target and nontarget strains was used in an in-house
validation. Only one primer pair, OT1559 plus 18-1, was found to be
selective. The inclusivity and exclusivity were 100 and 97%,
respectively. In an attempt to find a thermostable DNA polymerase more
resistant than
Taq
to PCR inhibitors present in chicken
samples, three DNA polymerases were evaluated. The DNA polymerase
T
th
was not inhibited at a concentration of 2%
(vol/vol) chicken carcass rinse, unlike both
Taq
DNA
polymerase and DyNAzyme. Based on these results, T
th
was
selected as the most suitable enzyme for the assay. The standardized
PCR test described shows potential for use in large-scale screening
programs for food-borne
Campylobacter
species under the assay
conditions
specified.
Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Validation in a Multicenter Collaborative Trial
Authors: Lübeck P. S. , Cook N. , Wagner M. , Fach P. , Hoorfar J.
Published: September 2003

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (
Campylobacter jejuni
,
C. coli
, and
C. lari
) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.