bluuebunny/crossref_metadata_2025 huggingface dataset:
Root Nodule
Bradyrhizobium
spp. Harbor
tfdA
α and
cadA
, Homologous with Genes Encoding 2,4-Dichlorophenoxyacetic Acid-Degrading Proteins
Authors: Itoh Kazuhito , Tashiro Yoshiko , Uobe Kazuko , Kamagata Yoichi , Suyama Kousuke , Yamamoto Hiroki
Published: April 2004

The distribution of
tfdA
α and
cadA
, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading
Bradyrhizobium
sp. isolated from pristine environments, was examined by PCR and Southern hybridization in several
Bradyrhizobium
strains including type strains of
Bradyrhizobium japonicum
USDA110 and
Bradyrhizobium elkanii
USDA94, in phylogenetically closely related
Agromonas oligotrophica
and
Rhodopseudomonas palustris
, and in 2,4-D-degrading
Sphingomonas
strains. All strains showed positive signals for
tfdA
α, and its phylogenetic tree was congruent with that of 16S rRNA genes in α-
Proteobacteria
, indicating evolution of
tfdA
α without horizontal gene transfer. The nucleotide sequence identities between
tfdA
α and canonical
tfdA
in β- and γ-
Proteobacteria
were 46 to 57%, and the deduced amino acid sequence of TfdAα revealed conserved residues characteristic of the active site of α-ketoglutarate-dependent dioxygenases. On the other hand,
cadA
showed limited distribution in 2,4-D-degrading
Bradyrhizobium
sp. and
Sphingomonas
sp. and some strains of non-2,4-D-degrading
B. elkanii.
The
cadA
genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between
Bradyrhizobium
sp. and
Sphingomonas
sp., indicating the incongruency of
cadA
with 16S rRNA genes. The nucleotide sequence identities between
cadA
and
tftA
of 2,4,5-trichlorophenoxyacetate-degrading
Burkholderia cepacia
AC1100 were 46 to 53%. Although all root nodule
Bradyrhizobium
strains were unable to degrade 2,4-D, three strains carrying
cadA
homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of
cadA
homologs in the cleavage of the aryl ether linkage. Based on codon usage patterns and GC content, it was suggested that the
cadA
genes of 2,4-D-degrading and nondegrading
Bradyrhizobium
spp. have different origins and that the genes would be obtained in the former through horizontal gene transfer.
Gas Exchange in the Filamentous Cyanobacterium
Nostoc punctiforme
Strain ATCC 29133 and ItsHydrogenase-Deficient Mutant StrainNHM5
Authors: Lindberg Pia , Lindblad Peter , Cournac Laurent
Published: April 2004

Nostoc
punctiforme
ATCC 29133 is a nitrogen-fixing, heterocystous
cyanobacterium of symbiotic origin. During nitrogen fixation, it
produces molecular hydrogen (H
2
), which is recaptured by an
uptake hydrogenase. Gas exchange in cultures of
N.
punctiforme
ATCC 29133 and its hydrogenase-free mutant strain
NHM5 was studied. Exchange of O
2
, CO
2
,
N
2
, and H
2
was followed simultaneously with a
mass spectrometer in cultures grown under nitrogen-fixing conditions.
Isotopic tracing was used to separate evolution and uptake of
CO
2
and O
2
. The amount of H
2
produced
per molecule of N
2
fixed was found to vary with light
conditions, high light giving a greater increase in H
2
production than N
2
fixation. The ratio under low light and
high light was approximately 1.4 and 6.1 molecules of H
2
produced per molecule of N
2
fixed, respectively. Incubation
under high light for a longer time, until the culture was depleted of
CO
2
, caused a decrease in the nitrogen fixation rate. At the
same time, hydrogen production in the hydrogenase-deficient strain was
increased from an initial rate of approximately 6 μmol (mg of
chlorophyll
a
)
−1
h
−1
to
9 μmol (mg of chlorophyll
a
)
−1
h
−1
after about 50 min. A light-stimulated
hydrogen-deuterium exchange activity stemming from the nitrogenase was
observed in the two strains. The present findings are important for
understanding this nitrogenase-based system, aiming at photobiological
hydrogen production, as we have identified the conditions under which
the energy flow through the nitrogenase can be directed towards
hydrogen production rather than nitrogen
fixation.
Cloning, Deletion, and Characterization of PadR, the Transcriptional Repressor of the Phenolic Acid Decarboxylase-EncodingpadAGene ofLactobacillus plantarum
Authors: Gury Jérôme , Barthelmebs Lise , Tran Ngoc Phuong , Diviès Charles , Cavin Jean-François
Published: April 2004

Lactobacillus plantarumdisplays a substrate-induciblepadAgene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator ofpadAwas located in thepadAlocus based on its 52% identity with PadR, thepadAgene transcriptional regulator ofPediococcus pentosaceus(L. Barthelmebs, B. Lecomte, C. Diviès, and J.-F. Cavin, J. Bacteriol.182:6724-6731, 2000). Deletion of theL. plantarum padRgene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently orientedpadA. ThepadRgene is cotranscribed with a downstream open reading frame (ORF1), the product of which may belong to a group of universal stress proteins (Usp). ThepadRdeletion mutant overexpressedpadAconstitutively, and thepadApromoter appears to be tightly regulated in this bacterium. Gel mobility shift assays using thepadAgene promoter region and purified PadR expressed inEscherichia coliindicated that operator DNA binding by PadR was not eliminated by addition ofp-coumarate. Gel mobility shift assays using partially purified extracts of native PadR protein from both phenolic acid-induced and noninducedL. plantarumcells demonstrate that inactivation of PadR by phenolic acids requires the integrity ofL. plantarumand mediation by a specific protein absent inE. coli.
Detection of Noroviruses in Tap Water in Japan by Means of a New Method for Concentrating Enteric Viruses in Large Volumes of Freshwater
Authors: Haramoto Eiji , Katayama Hiroyuki , Ohgaki Shinichiro
Published: April 2004

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H
2
SO
4
(pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.
Characterization of
Bacillus
Probiotics Available for Human Use
Authors: Duc Le H. , Hong Huynh A. , Barbosa Teresa M. , Henriques Adriano O. , Cutting Simon M.
Published: April 2004

Bacillus
species (
Bacillus cereus
,
Bacillus clausii
,
Bacillus pumilus
) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three
B. cereus
strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one
B. cereus
strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the
B. pumilus
strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of
B. pumilus
and of a laboratory strain of
B. subtilis
were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of
B. pumilus
and
B. subtilis
induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The
B. pumilus
strain and one
B. cereus
strain (
B. cereus
var. vietnami) were found to produce a bacteriocin-like activity against other
Bacillus
species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three
B. cereus
strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.
Multilocus Sequence Typing of
Listeria monocytogenes
by Use of Hypervariable Genes Reveals Clonal and Recombination Histories of Three Lineages
Authors: Meinersmann Richard J. , Phillips Robert W. , Wiedmann Martin , Berrang Mark E.
Published: April 2004

In an attempt to develop a method to discriminate among isolates of
Listeria monocytogenes
, the sequences of all of the annotated genes from the fully sequenced strain
L. monocytogenes
EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of
L. monocytogenes
ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes (
hisJ
,
cbiE
,
truB
,
ribC
,
comEA
,
purM
,
aroE
,
hisC
, and
addB
) in the 80 to 90% identity group and two genes (
gyrB
and
rnhB
) with approximately 97% identity were selected for multilocus sequence analysis in two sets of
L. monocytogenes
isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of
L. monocytogenes
that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the
L. monocytogenes
isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of
L. monocytogenes
can share habitats, they appear to differ in their histories of horizontal gene transfer.
Identification ofStreptococcus thermophilusCNRZ368 Genes Involved in Defense against Superoxide Stress
Authors: Thibessard Annabelle , Borges Frédéric , Fernandez Annabelle , Gintz Brigitte , Decaris Bernard , Leblond-Bourget Nathalie
Published: April 2004

To better understand the defense mechanism ofStreptococcus thermophilusagainst superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt,ossF, andossG) from those whose action may be generalized to other stressful conditions (mreD,rodA,pbp2b,cpsX, andiscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1in two loci close to each other. More precisely, these two loci are homologous to thesufDandiscUgenes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called thesufregion, was further characterized inS. thermophilusCNRZ368 by sequencing and by construction of ΔsufDandiscU97nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.
Survey of Archaeal Diversity Reveals an Abundance of HalophilicArchaeain a Low-Salt, Sulfide- and Sulfur-Rich Spring
Authors: Elshahed Mostafa S. , Najar Fares Z. , Roe Bruce A. , Oren Aharon , Dewers Thomas A. , Krumholz Lee R.
Published: April 2004

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdomEuryarchaeota, whileCrenarchaeotarepresented less than 10% of clones. Euryarchaeotal clones belonged to the ordersMethanomicrobiales,Methanosarcinales, andHalobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within theHalobacterialesrepresented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the familyHalobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genusHaloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilicArchaeain such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of theHalobacterialesare not restricted to their typical high-salt habitats, and we propose a role for theHalobacterialesin sulfur reduction in natural ecosystems.
Prevalence of the Genes Encoding Propionicin T1 andProtease-Activated Antimicrobial Peptide and Their Expressionin ClassicalPropionibacteria
Authors: Faye Therese , Brede Dag Anders , Langsrud Thor , Nes Ingolf F. , Holo Helge
Published: April 2004

The
purpose of this study was to investigate the frequency
of production of the bacteriocin propionicin T1 and the
protease-activated antimicrobial peptide (PAMP) and their corresponding
genes in 64 isolates of classical propionibacteria. This study revealed
that these genes are widespread in
Propionibacterium jensenii
and
Propionibacterium thoenii
but absent from the remaining
species of classical propionibacteria that were studied. The
pro-PAMP-encoding gene (
pamA
) was found in 63% of the
P. jensenii
strains and 61% of the
P. thoenii
strains, and all of these strains displayed PAMP activity. The
propionicin T1-encoding gene (
pctA
) was present in 89%
of the
P. thoenii
strains and 54% of the
P.
jensenii
strains. All
P. thoenii
strains containing the
pctA
gene exhibited antimicrobial activity corresponding to
propionicin T1 activity, whereas only 38% of the
pctA
-containing
P. jensenii
strains displayed this
activity. Sequencing of the
pctA
genes revealed the existence
of two allelic variants that differed in a single nucleotide in six
strains of
P. jensenii
; in these strains the glycine at
position 55 of propionicin T1 was replaced by an aspartate residue (A
variant). No strains harboring the A variant showed any antimicrobial
activity against propionicin T1-sensitive bacteria. An open reading
frame (
orf2
) located immediately downstream from the
pctA
gene was absent in three strains containing the G variant
of propionicin T1. Two of these strains showed low antimicrobial
activity, while the third strain showed no antimicrobial activity at
all. The protein encoded by
orf2
showed strong homology to ABC
transporters, and it has been proposed previously that this protein is
involved in the producer immunity against propionicin T1. The limited
antimicrobial activity exhibited by the strains lacking
orf2
further suggests that this putative ABC transporter plays an important
role in propionicin T1
activity.
Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase
Authors: Nanba K. , King G. M. , Dunfield K.
Published: April 2004

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to constructrbcLclone libraries. Phylogenetic analyses showed that therbcLsequences from all isolates clustered with form ICrbcLsequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IArbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to knownrbcLsequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.
Species Diversity of and Toxin Production by
Gibberella fujikuroi
Species Complex Strains Isolated from Native Prairie Grasses in Kansas
Authors: Leslie John F. , Zeller Kurt A. , Logrieco Antonio , Mulè Giuseppina , Moretti Antonio , Ritieni Alberto
Published: April 2004

Fusarium
species from agricultural crops have been well studied with respect to toxin production and genetic diversity, while similar studies of communities from nonagricultural plants are much more limited. We examined 72
Fusarium
isolates from a native North American tallgrass prairie and found that
Gibberella intermedia
(
Fusarium proliferatum
),
Gibberella moniliformis
(
Fusarium verticillioides
), and
Gibberella konza
(
Fusarium konzum
) dominated.
Gibberella thapsina
(
Fusarium thapsinum
) and
Gibberella subglutinans
(
Fusarium subglutinans
) also were recovered, as were seven isolates that could not be assigned to any previously described species on the basis of either morphological or molecular characters. In general, isolates from the prairie grasses produced the same toxins in quantities similar to those produced by isolates of the same species recovered from agricultural hosts. The
G. konza
isolates produce little or no fumonisins (up to 120 μg/g by one strain), and variable but generally low to moderate amounts of beauvericin (4 to 320 μg/g) and fusaproliferin (50 to 540 μg/g). Toxicity to
Artemia salina
larvae within most species was correlated with the concentration of either beauvericin or fusaproliferin produced. Organic isolates from some cultures of
G. moniliformis
were highly toxic towards
A. salina
even though they produced little, if any, beauvericin or fusaproliferin. Thus, additional potentially toxigenic compounds may be synthesized by
G. moniliformis
strains isolated from prairie grasses. The
Fusarium
community from these grasses appears to contain some species not found in surrounding agricultural communities, including some that probably are undescribed, and could be capable of serving as a reservoir for strains of potential agricultural importance.
GC Fractionation Enhances Microbial CommunityDiversity Assessment and Detection of Minority Populations ofBacteria by Denaturing Gradient GelElectrophoresis
Authors: Holben William E. , Feris Kevin P. , Kettunen Anu , Apajalahti Juha H. A.
Published: April 2004

Effectively
and accurately assessing total microbial community diversity is one of
the primary challenges in modern microbial ecology. This is
particularly true with regard to the detection and characterization of
unculturable populations and those present only in low abundance. We
report a novel strategy, GC fractionation combined with denaturing
gradient gel electrophoresis (GC-DGGE), which combines mechanistically
different community analysis approaches to enhance assessment of
microbial community diversity and detection of minority populations of
microbes. This approach employs GC fractionation as an initial step to
reduce the complexity of the community in each fraction. This reduced
complexity facilitates subsequent detection of diversity in individual
fractions. DGGE analysis of individual fractions revealed bands that
were undetected or only poorly represented when total bacterial
community DNA was analyzed. Also, directed cloning and sequencing of
individual bands from DGGE lanes corresponding to individual
G+C fractions allowed detection of numerous phylotypes that
were not recovered using a traditional random cloning and sequencing
approach.
Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by
Lactobacillus curvatus
Strain LTH 1174, Originating from Fermented Sausage
Authors: Verluyten Jurgen , Messens Winy , De Vuyst Luc
Published: April 2004

Lactobacillus curvatus
LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of
L. curvatus
LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (

k
B

) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a
w
-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (

X
B

) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in

X
B

, an increase in

k
B

, and hence an increase in the maximum attainable bacteriocin activity.
Characterization of Azo Reduction Activity in a Novel Ascomycete Yeast Strain
Authors: Ramalho Patrícia A. , Cardoso M. Helena , Cavaco-Paulo A. , Ramalho M. Teresa
Published: April 2004

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species,Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity ofI. occidentalisis still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of theN,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, andN,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.
Pulsed Electric Field Alters Molecular Chaperone Expression and Sensitizes
Listeria monocytogenes
to Heat
Authors: Lado Beatrice H. , Bomser Joshua A. , Dunne C. Patrick , Yousef Ahmed E.
Published: April 2004

Pulsed electric field (PEF)-resistant and PEF-sensitive
Listeria monocytogenes
strains were sublethally treated with electric pulses at 15 kV/cm for 29 μs and held at 25°C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 μs) and heat (55°C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of
L. monocytogenes
Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25°C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized
L. monocytogenes
to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.
Molecular Basis for Anaerobic Growth of
Saccharomyces cerevisiae
on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis
Authors: Sonderegger Marco , Jeppsson Marie , Hahn-Hägerdal Bärbel , Sauer Uwe
Published: April 2004

Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a
Saccharomyces cerevisiae
strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.
Seasonal Dynamics of Shallow-Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient
Authors: Feris Kevin P. , Ramsey Philip W. , Frazar Chris , Rillig Matthias , Moore Johnnie N. , Gannon James E. , Holben William E.
Published: April 2004

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (
R
2
= 0.02,
P
= 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (
R
2
= 0.58,
P
< 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the α, β, and γ-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.
Oxygen-Controlled Bacterial Growth in the Sponge
Suberites domuncula
: toward a Molecular Understanding of the Symbiotic Relationships between Sponge and Bacteria
Authors: Müller Werner E. G. , Grebenjuk Vladislav A. , Thakur Narsinh L. , Thakur Archana N. , Batel Renato , Krasko Anatoli , Müller Isabel M. , Breter Hans J.
Published: April 2004

Sponges (phylum Porifera), known to be the richest producers among the metazoans of bioactive secondary metabolites, are assumed to live in a symbiotic relationship with microorganisms, especially bacteria. Until now, the molecular basis of the mutual symbiosis, the exchange of metabolites for the benefit of the other partner, has not been understood. We show with the demosponge
Suberites domuncula
as a model that the sponge expresses under optimal aeration conditions the enzyme tyrosinase, which synthesizes diphenols from monophenolic compounds. The cDNA isolated was used as a probe to determine the steady-state level of gene expression. The gene expression level parallels the level of specific activity in sponge tissue, indicating that without aeration the tyrosinase level drops drastically; this effect is reversible. The SB2 bacterium isolated from the sponge surface grew well in M9 minimal salt medium supplemented with the dihydroxylated aromatic compound protocatechuate; this carbon source supported growth more than did glucose. From the SB2 bacterium the protocatechuate gene cluster was cloned and sequenced. This cluster comprises all genes coding for enzymes involved in the conversion of protocatechuate to acetyl coenzyme A. Expression is strongly induced if the bacteria are cultivated on M9-protocatechuate medium; the genes
pcaQ
(encoding the putative transcriptional activator of the
pca
operon) and
pcaDC
were used for quantitative PCR analyses. We conclude that metabolites, in this case diphenols, which might be produced by the sponge
S. domuncula
are utilized by the sponge surface-associated bacterium for energy generation. This rationale will help to further uncover the symbiotic pathways between sponges and their associated “nonculturable” microorganisms; our approach is flanked by the establishment of an EST (expressed sequence tags) database in our laboratory.
Urease-Encoding Genes in Ammonia-Oxidizing Bacteria
Authors: Koper Teresa E. , El-Sheikh Amal F. , Norton Jeanette M. , Klotz Martin G.
Published: April 2004

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the β-proteobacterium
Nitrosospira
sp. strain NpAV and the γ-proteobacterium
Nitrosococcus oceani
. In both organisms, all seven urease genes were contiguous: the three structural urease genes
ureABC
were preceded and succeeded by the accessory genes
ureD
and
ureEFG
, respectively. Green fluorescent protein reporter gene fusions revealed that the
ure
genes were under control of a single operon promoter upstream of the
ureD
gene in
Nitrosococcus oceani
. Southern analyses revealed two copies of
ureC
in the
Nitrosospira
sp. strain NpAV genome, while a single copy of the
ure
operon was detected in the genome of
Nitrosococcus oceani.
The
ureC
gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further
ureC
sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB,
ureC
genes were sequenced from several β-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from β-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other β-proteobacteria. Instead, it appears that urease in β-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of γ- and β-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for
ureC
from β-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for
ure
genes from autotrophic AOB.
Purification and Characterization of a Feruloyl Esterase from the Intestinal BacteriumLactobacillus acidophilus
Authors: Wang Xiaokun , Geng Xin , Egashira Yukari , Sanada Hiroo
Published: April 2004

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium,Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). The metal ions Cu2+and Fe3+(at a concentration of 5 mmol liter−1) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-l-arabinofuranose (FAA) as a substrate, the enzyme exhibited aKmof 0.0953 mmol liter−1and aVmaxof 86.27 mmol liter−1min−1mg−1of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA fromO-(5-O-feruloyl-α-l-arabinofuranosyl)-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.
Real-Time Quantitative PCR for Assessment of Abundance of
Pseudoalteromonas
Species in Marine Samples
Authors: Skovhus Torben L. , Ramsing Niels B. , Holmström Carola , Kjelleberg Staffan , Dahllöf Ingela
Published: April 2004

A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of
Pseudoalteromonas
species in marine samples is presented. PCR primers targeting a
Pseudoalteromonas
-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus
Pseudoalteromonas
were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of
Pseudoalteromonas
ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of
Pseudoalteromonas
compared to all
Eubacteria
were 1.55% on the green alga
Ulva lactuca
, 0.10% on the tunicate
Ciona intestinalis
, and 0.06% on the green alga
Ulvaria fusca
.
Genetic Markers Unique to
Listeria monocytogenes
Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages
Authors: Evans Matthew R. , Swaminathan Bala , Graves Lewis M. , Altermann Eric , Klaenhammer Todd R. , Fink Ryan C. , Kernodle Sheri , Kathariou Sophia
Published: April 2004

A small number of closely related strains of
Listeria monocytogenes
serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to
inlA
, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.
Genome Shuffling Improves Degradation of the Anthropogenic Pesticide Pentachlorophenol by
Sphingobium chlorophenolicum
ATCC 39723
Authors: Dai MingHua , Copley Shelley D.
Published: April 2004

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by
Sphingobium chlorophenolicum
, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and
S. chlorophenolicum
cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by
S. chlorophenolicum
. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.
Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria
Authors: Rahman Mokhlasur , Kühn Inger , Rahman Motiur , Olsson-Liljequist Barbro , Möllby Roland
Published: April 2004

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log
2
dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.
Spatial Distribution of Marine Crenarchaeota Group I in the Vicinity of Deep-Sea Hydrothermal Systems
Authors: Takai Ken , Oida Hanako , Suzuki Yohey , Hirayama Hisako , Nakagawa Satoshi , Nunoura Takuro , Inagaki Fumio , Nealson Kenneth H. , Horikoshi Koki
Published: April 2004

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.
Identification, Detection, and Spatial Resolution of
Clostridium
Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor
Authors: Burrell P. C. , O'Sullivan C. , Song H. , Clarke W. P. , Blackall L. L.
Published: April 2004

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by
Firmicutes
phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to
Clostridium stercorarium
), clone group 2 (most closely related to
Clostridium thermocellum
), and clone group 5 (most closely related to
Bacteroides cellulosolvens
) comprised sequences in
Clostridium
group III. Clone group 3 sequences were in
Clostridium
group XIVa (most closely related to
Clostridium
sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with
Clostridium
group VI. This monophyletic group comprises a new
Clostridium
cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.