bluuebunny/crossref_metadata_2025 huggingface dataset:
Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin
Authors: Borchardt Mark A. , Bertz Phil D. , Spencer Susan K. , Battigelli David A.
Published: February 2003

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms,
Escherichia coli
, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.
Specific Detection of
Arcobacter
and
Campylobacter
Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization
Authors: Moreno Yolanda , Botella Salut , Alonso José Luis , Ferrús María A. , Hernández Manuel , Hernández Javier
Published: February 2003

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting
Arcobacter
and
Campylobacter
strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10
3
cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.
Use of a Promoter Trap To Identify
Bacillus cereus
Genes Regulated by Tomato Seed Exudate and a Rhizosphere Resident,
Pseudomonas aureofaciens
Authors: Dunn Anne K. , Klimowicz Amy K. , Handelsman Jo
Published: February 2003

The goal of this study was to identify genes in
Bacillus cereus
, a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident,
Pseudomonas aureofaciens
. We constructed a
B. cereus
chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene,
gfp
mut3a. The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of
P. aureofaciens
strain 30-84. We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by
P. aureofaciens
culture supernatant. A clone chosen for further study contained an open reading frame, designated
lipA
, that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in
B. subtilis.
Expression of
gusA
under control of the
lipA
promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids. When the wild type and a 10-fold excess of a
lipA
mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the
lipA
mutant was unaffected. This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of
lipA
, a plant-regulated gene in
B. cereus
.
Differences between
Pseudomonas syringae
pv. syringae B728a and
Pantoea agglomerans
BRT98 in Epiphytic and Endophytic Colonization of Leaves
Authors: Sabaratnam Siva , Beattie Gwyn A.
Published: February 2003

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen
Pseudomonas syringae
pv. syringae strain B728a and the nonpathogen
Pantoea agglomerans
strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The
P. agglomerans
strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the
P. agglomerans
strain formed aggregates that often extended over multiple epidermal cells. The
P. syringae
pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the
P. syringae
pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic
P. syringae
pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic
P. syringae
pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the
P. agglomerans
strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the
P. syringae
pv. syringae strain on bean but not with growth of the
P. syringae
pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic
P. syringae
pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic
P. agglomerans
strain were not.
Identification and Characterization of
Lactobacillus helveticus
PepO2, an Endopeptidase with Post-Proline Specificity
Authors: Chen Yo-Shen , Christensen Jeffrey E. , Broadbent Jeffery R. , Steele James L.
Published: February 2003

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of
Lactobacillus helveticus
CNRZ32 by using the synthetic substrate
N
-acetyl-β-casein-(f203-209)-ρ-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated
pepO2
, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that
L. helveticus
PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to
L. helveticus
PepO,
Lactococcus lactis
PepO2,
L. lactis
PepO, and
Lactobacillus rhamnosus
PepO, respectively. Northern hybridization results indicated that the transcript containing
pepO2
was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the
L. helveticus
PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as β-casein(f193-209).
Hydrolysis of Casein-Derived Peptides α
S1
-Casein(f1-9) and β-Casein(f193-209) by
Lactobacillus helveticus
Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase
Authors: Christensen Jeffrey E. , Broadbent Jeffery R. , Steele James L.
Published: February 2003

Peptides derived from hydrolysis of α
S1
-casein(f1-9) [α
S1
-CN(f1-9)] and β-CN(f193-209) with cell extracts of
Lactobacillus helveticus
CNRZ32 and single-peptidase mutants (Δ
pepC
, Δ
pepE
, Δ
pepN
, Δ
pepO
, and Δ
pepX
) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.
Molecular Characterization of Astroviruses by Reverse Transcriptase PCR and Sequence Analysis: Comparison of Clinical and Environmental Isolates from South Africa
Authors: Nadan S. , Walter J. E. , Grabow W. O. K. , Mitchell D. K. , Taylor M. B.
Published: February 2003

A comparative analysis was performed with 25 isolates of astroviruses (AstVs) detected in sewage sources and 22 concurrently identified clinical AstV isolates from the Tshwane (Pretoria) Metropolitan Area in South Africa. The samples and specimens were screened for AstVs by using an enzyme immunoassay and/or a reverse transcriptase PCR (RT-PCR) for the highly conserved untranslated region (3′ end) of the genome. The RT-PCR results were confirmed by oligonucleotide probe dot blot hybridization. Viable viruses were propagated in cell cultures for amplification when a minimal specimen was available or indeterminate sequences were obtained. AstV strains were characterized by RT-PCR and partial sequence analysis of the capsid region. The presence of multiple human AstV (HAstV) types in a single sewage sample complicated identification of individual strains, and additional type-specific RT-PCR and sequence analyses of the capsid region were required for characterization. Amplification and characterization of one genotype from a sample, therefore, did not preclude the possibility that a sample harbored additional different genotypes. Genotype and sequence information obtained from AstVs in wastewater samples were compared to information obtained from AstV strains from human stools. HAstV type 1 (HAstV-1), as well as HAstV-3, -5, -6, and -8, were identified among the clinical isolates, and HAstV-1, -2, -3, -4, -5, -7, and -8 were identified among the environmental samples. Phylogenetic analysis demonstrated that HAstV-1, -3, -5, and -8, which were present in human stool and sewage samples, clustered together, indicating that these viruses are closely related. The concurrent presence of identical HAstV strains in wastewater samples and in hospitalized patients suggests that AstVs present in the environment pose a potential risk to communities in which fecally contaminated water is used for recreational and domestic purposes.
Identification of a High-Affinity Phosphate Transporter Gene in a Prasinophyte Alga,Tetraselmis chui, and Its Expression under Nutrient Limitation
Authors: Chung Chih-Ching , Hwang Sheng-Ping L. , Chang Jeng
Published: February 2003

A high-affinity phosphate transporter gene,TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular algaTetraselmis chui. The full-length cDNA ofTcPHOobtained by 5′ and 3′ rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids. The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 fromSaccharomyces cerevisiaeand PHO4 fromNeurospora crassa, respectively. In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4. The expression ofTcPHOmRNA was dependent on phosphate availability. With a low-phosphate treatment, theTcPHOmRNA concentration increased sharply to 2.72 fmol μg of total RNA−1from day 1 to day 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate orp-nitrophenyl phosphate effectively inhibitedTcPHOmRNA expression. In contrast,TcPHOmRNA expression stayed at a low level (range, 0.25 to 0.28 fmol μg of total RNA−1) under low-nitrate conditions. The expression pattern suggests thatTcPHOcan be used as a molecular probe for monitoring phosphorus stress inT. chui.
Monitoring Gene Expression in Mixed Microbial Communities by Using DNA Microarrays
Authors: Dennis Philip , Edwards Elizabeth A. , Liss Steven N. , Fulthorpe Roberta
Published: February 2003

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium
Ralstonia eutropha
JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (
tfdA
and
tfdC
) from populations of JMP134 as low as 10
5
cells/ml was clearly detected against a background of 10
8
cells/ml. Induction of two others (
tfdB
and
tfdE
) was detected from populations of 10
6
cells/ml in the same background; however, the last gene,
tfdF
, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.
Characterization of the Initial Reactions during the Cometabolic Oxidation of Methyl
tert
-Butyl Ether by Propane-Grown
Mycobacterium vaccae
JOB5
Authors: Smith Christy A. , O'Reilly Kirk T. , Hyman Michael R.
Published: February 2003

The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl
tert
-butyl ether (MTBE), by
Mycobacterium vaccae
JOB5 have been characterized. Two products,
tert
-butyl formate (TBF) and
tert
-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1- or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (≥80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other
tert
-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in
M. vaccae
JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (

K
i

= 3.3 to 4.4 μM). Values for

K
s

of 1.36 and 1.18 mM and for
V
max
of 24.4 and 10.4 nmol min
−1
mg of protein
−1
were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by
M. vaccae
JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by
M. vaccae
JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.
Contribution of
pilA
to Competitive Colonization of the Squid
Euprymna scolopes
by
Vibrio fischeri
Authors: Stabb Eric V. , Ruby Edward G.
Published: February 2003

Vibrio fischeri
colonizes the squid
Euprymna scolopes
in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and
V. fischeri
strains must compete to occupy this privileged niche. We cloned a
V. fischeri
gene, designated
pilA
, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives,
Vibrio cholerae mshA
and
vcfA
,
pilA
is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame
pilA
deletion mutant, as well as
pilA
mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (
P
< 0.05) but not by an unmarked
pilA
mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of
pilA
mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for
V. fischeri
populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a
pilA
mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain.
V. fischeri
strain ET101, which was isolated from
Euprymna tasmanica
and is outcompeted by ES114, lacks
pilA
; however, 11 other diverse
V. fischeri
isolates apparently possess
pilA
. The competitive defect of
pilA
mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.
In-Field Spatial Variability in the Degradation of the Phenyl-Urea Herbicide Isoproturon Is the Result of Interactions between Degradative
Sphingomonas
spp. and Soil pH
Authors: Bending Gary D. , Lincoln Suzanne D. , Sørensen Sebastian R. , Morgan J. Alun W. , Aamand Jens , Walker Allan
Published: February 2003

Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) [3-(4-isopropylphenyl)-1,1-dimethylurea] has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water. The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom. Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms. Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation. Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the
Sphingomonas
group. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the
Sphingomonas
group. However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading
Sphingomonas
sp. (strain SRS2) isolated from Deep Slade field in an earlier study. Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU. The pH requirements of IPU-degrading strains of
Sphingomonas
spp. could largely account for the spatial variation of IPU degradation rates across the field.
Microbial Community Composition Affects Soil Fungistasis
Authors: de Boer Wietse , Verheggen Patrick , Klein Gunnewiek Paulien J. A. , Kowalchuk George A. , van Veen Johannes A.
Published: February 2003

Most soils inhibit fungal germination and growth to a certain extent, a phenomenon known as soil fungistasis. Previous observations have implicated microorganisms as the causal agents of fungistasis, with their action mediated either by available carbon limitation (nutrient deprivation hypothesis) or production of antifungal compounds (antibiosis hypothesis). To obtain evidence for either of these hypotheses, we measured soil respiration and microbial numbers (as indicators of nutrient stress) and bacterial community composition (as an indicator of potential differences in the composition of antifungal components) during the development of fungistasis. This was done for two fungistatic dune soils in which fungistasis was initially fully or partly relieved by partial sterilization treatment or nutrient addition. Fungistasis development was measured as restriction of the ability of the fungi
Chaetomium globosum
,
Fusarium culmorum
,
Fusarium oxysporum
, and
Trichoderma harzianum
to colonize soils. Fungistasis did not always reappear after soil treatments despite intense competition for carbon, suggesting that microbial community composition is important in the development of fungistasis. Both microbial community analysis and in vitro antagonism tests indicated that the presence of pseudomonads might be essential for the development of fungistasis. Overall, the results lend support to the antibiosis hypothesis.
Computer Simulation of
Clostridium botulinum
Strain 56A Behavior at Low Spore Concentrations
Authors: Zhao L. , Montville T. J. , Schaffner D. W.
Published: February 2003

It is generally assumed that spore behavior is independent of spore concentration, but recently published mathematical models indicate that this is not the case. A Monte Carlo simulation was employed in this study to further examine the independence assumption by evaluating the inherent variance in spore germination data. All simulations were carried out with @Risk software. A total of 500 to 4,000 iterations were needed for each simulation to reach convergence. Lag time and doubling time from a higher inoculum concentration were used to simulate the time to detection (TTD) at a lower inoculum concentration under otherwise identical environmental conditions. The point summaries of the simulated and observed TTDs were recorded for the 26 simulations, with kinetic data at the target inoculum concentration. The ratios of the median (

R
m

= median
obs
/median
sim
) and 90% range (

R
r

= 90% range
obs
/90% range
sim
) were calculated. Most

R
m

and

R
r

values were greater than one, indicating that the simulated TTDs were smaller and more homogeneous than the observed ones.

R
r

values departed farther from one than

R
m

values. Ratios obtained when simulating 1 spore with 10,000 spores deviated the farthest from one. Neither ratio was significantly different from the other when simulating 1 spore with 100 spores or simulating 100 spores with 10,000 spores. When kinetic data were not available, the percent positive observed at the 95th percentile of the simulated TTDs was obtained. These simulation results confirmed that the assumption of independence between spores is not valid.
Production by
Clostridium acetobutylicum
ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9
Authors: López-Contreras Ana M. , Martens Aernout A. , Szijarto Nora , Mooibroek Hans , Claassen Pieternel A. M. , van der Oost John , de Vos Willem M.
Published: February 2003

The genome sequence of
Clostridium acetobutylicum
ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as
Clostridium cellulovorans
. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in
Escherichia coli
. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by
C. acetobutylicum
grown on different substrates was studied by Western blotting by using antibodies raised against the purified
E. coli
-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown
C. cellulovorans
cultures, CelG was not detectable in extracellular medium from
C. acetobutylicum
grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.
Rhizobium etli
and
Rhizobium gallicum
Nodulate Common Bean (
Phaseolus vulgaris
) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications
Authors: Silva Claudia , Vinuesa Pablo , Eguiarte Luis E. , Martínez-Romero Esperanza , Souza Valeria
Published: February 2003

The stability of the genetic structure of rhizobial populations nodulating
Phaseolus vulgaris
cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial
rrs
,
glnII
,
nifH
, and
nodB
sequences), along with host range experiments, placed the isolates examined in
Rhizobium etli
bv. phaseoli and
Rhizobium gallicum
bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the
R. etli
and
R. gallicum
populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis,
nifH
copy number determination, and host range experiments. Plasmid profiles and
nifH
hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an
R. gallicum
bv. gallicum population nodulating common bean in America are discussed.
Characterization of Chitinase Genes from an Alkaliphilic Actinomycete,
Nocardiopsis prasina
OPC-131
Authors: Tsujibo Hiroshi , Kubota Takahiro , Yamamoto Mitsugu , Miyamoto Katsushiro , Inamori Yoshihiko
Published: February 2003

An alkaliphilic actinomycete,
Nocardiopsis
prasina
OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of
chiA
encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The
chiB
ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to
Streptomyces
family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in
N.
prasina
OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus
Streptomyces
.
Heat and Osmotic Stress Responses of Probiotic
Lactobacillus rhamnosus
HN001 (DR20) in Relation to Viability after Drying
Authors: Prasad Jaya , McJarrow Paul , Gopal Pramod
Published: February 2003

The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries. In this study we observed that when prestressed with either heat (50°C) or salt (0.6 M NaCl),
Lactobacillus rhamnosus
HN001 (also known as DR20) showed significant (
P
< 0.05) improvement in viability compared with the nonstressed control culture after storage at 30°C in the dried form. To investigate the mechanisms underlying this stress-related viability improvement in
L. rhamnosus
HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing. Several proteins were up- or down-regulated after either heat or osmotic shock treatments. Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway. The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used. The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments. Carbohydrate analysis of cytoplasmic contents showed higher levels (20 ± 3 μg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 ± 3 μg/mg of protein). Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles. Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol.
Desulfonation and Degradation of the Disulfodiphenylethercarboxylates from Linear Alkyldiphenyletherdisulfonate Surfactants
Authors: Schleheck David , Lechner Melanie , Schönenberger René , Suter Marc J.-F. , Cook Alasdair M.
Published: February 2003

Earlier work showed that the biodegradation of a commercial linear monoalkyldiphenyletherdisulfonate surfactant as a carbon source for microbial growth leads to the quantitative formation of corresponding disulfodiphenylether carboxylates (DSDPECs), which were not degraded. α-Proteobacterium strain DS-1 (DSM 13023) catalyzes these reactions. These DSDPECs have now been characterized by high-pressure liquid chromatography coupled via an electrospray interface to a mass spectrometer. DSDPECs were a complex mixture of compounds which indicated catabolism via ω-oxygenation and β-oxidation. DSDPECs were subject to quantitative desulfonation in bacterial cultures in which they served as sole sulfur sources for bacterial growth. On average, one sulfonate group per DSDPEC species was removed, and the organism responsible for this desulfonation was isolated and identified as
Rhodococcus opacus
ISO-5. The products were largely monosulfodiphenylether carboxylate-phenols (MSDPEC-phenols). MSDPEC-phenols were subject to extensive dissimilation by bacteria from activated sludge.
Phylogenetic Diversity of Nitrogenase (
nifH
) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge
Authors: Mehta Mausmi P. , Butterfield David A. , Baross John A.
Published: February 2003

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N
2
gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene
nifH
from hydrothermal vent fluid. A total of 120
nifH
sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor
nifH
genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other
nifH
genes unique to the vent samples include proteobacteria and divergent
Archaea
. All of the
nifH
genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon
Methanococcus thermolithotrophicus
(77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of
nifH
genes detected in hydrothermal vent fluid:
nifH
genes from an anaerobic, hot subseafloor and
nifH
genes from cold, oxygenated deep seawater.
Comparison of Method 1623 and Cell Culture-PCR for Detection of
Cryptosporidium
spp. in Source Waters
Authors: LeChevallier Mark W. , Di Giovanni George D. , Clancy Jennifer L. , Bukhari Zia , Bukhari Shan , Rosen Jeffrey S. , Sobrinho Jose , Frey Michelle M.
Published: February 2003

Analysis of
Cryptosporidium
occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (
P
= 0.06).
Cryptosporidium
oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the
Cryptosporidium
oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the
Cryptosporidium
parvum
isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the
C. parvum
isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious
Cryptosporidium
and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water
Cryptosporidium
levels to meet potable water goals.
Characterization of a Thermostable
d
-Stereospecific Alanine Amidase from
Brevibacillus borstelensis
BCS-1
Authors: Baek Dae Heoun , Kwon Seok-Joon , Hong Seung-Pyo , Kwak Mi-Sun , Lee Mi-Hwa , Song Jae Jun , Lee Seung-Goo , Yoon Ki-Hong , Sung Moon-Hee
Published: February 2003

A gene encoding a new thermostable
d
-stereospecific alanine amidase from the thermophile
Brevibacillus borstelensis
BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards
d
-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards
l
-amino acid amides,
d
-amino acid-containing peptides, and NH
2
-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co
2+
and Mn
2+
. The
k
cat
/

K
m

for
d
-alaninamide was measured as 544.4 ± 5.5 mM
−1
min
−1
at 50°C with 1 mM Co
2+
.
Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a
Dehalococcoides
Species
Authors: He Jianzhong , Ritalahti Kirsti M. , Aiello Michael R. , Löffler Frank E.
Published: February 2003

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54
μ
mol liter
−1
day
−1
, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (

K
S

) for VC was 5.8
μ
M, which was about one-third lower than the concentrations determined for
cis
-DCE and
trans
-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H
2
as electron donor. VC-dechlorinating cultures consumed H
2
to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a
Dehalococcoides
population, and
Dehalococcoides
-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that
Dehalococcoides
populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.
PCR Assay for Identification of
Histoplasma capsulatum
Based on the Nucleotide Sequence of the M Antigen
Authors: Guedes Herbert Leonel de Matos , Guimarães Allan Jefferson , Muniz Mauro de Medeiros , Pizzini Claudia Vera , Hamilton Andrew John , Peralta José Mauro , Deepe, George S. , Zancopé-Oliveira Rosely M.
Published: February 2003

The major diagnostic antigens of
Histoplasma capsulatum
var.
capsulatum
are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. The gene encoding the M antigen has previously been sequenced, and its sequence has significant overall homology to those of the genes for fungal catalases. Regions of the M-antigen gene with little or no homology were used to design four oligonucleotide sequences for application in the PCR detection and identification of
H. capsulatum
var.
capsulatum
. The PCR correctly identified the 31
H. capsulatum
var.
capsulatum
strains isolated from human, animal, and soil specimens and 1
H. capsulatum
var.
duboisii
isolate. PCR products of 111 and 279 bp were amplified with primers Msp1F-Msp1R and Msp2F-Msp2R, respectively. No amplification product was obtained from DNA extracted from an
H. capsulatum
var.
farciminosum
isolate. The specificity of the PCR with the M-antigen-derived primers was confirmed by the total absence of amplification products when genomic DNA from
Paracoccidioides brasiliensis
,
Candida
spp.,
Sporothrix schenckii
,
Cryptococcus neoformans
,
Blastomyces dermatitidis
,
Coccidioides immitis
,
Aspergillus niger
, and
Aspergillus fumigatus
were applied in the reaction. This rapid, sensitive, and specific assay provides a way to identify typical and atypical isolates of
H. capsulatum
var.
capsulatum
.
Diversity of Bacterial Populations on the Tongue Dorsa of Patients with Halitosis and Healthy Patients
Authors: Kazor C. E. , Mitchell P. M. , Lee A. M. , Stokes L. N. , Loesche W. J. , Dewhirst F. E. , Paster B. J.
Published: February 2003

The primary purpose of the present study was to compare the microbial profiles of the tongue dorsa of healthy subjects and subjects with halitosis by using culture-independent molecular methods. Our overall goal was to determine the bacterial diversity on the surface of the tongue dorsum as part of our ongoing efforts to identify all cultivable and not-yet-cultivated species of the oral cavity. Tongue dorsum scrapings were analyzed from healthy subjects with no complaints of halitosis and subjects with halitosis, defined as an organoleptic score of 2 or more and volatile sulfur compound levels greater than 200 ppb. 16S rRNA genes from DNA isolated from tongue dorsum scrapings were amplified by PCR with universally conserved bacterial primers and cloned into
Escherichia coli
. Typically, 50 to 100 clones were analyzed from each subject. Fifty-one strains isolated from the tongue dorsa of healthy subjects were also analyzed. Partial sequences of approximately 500 bases of cloned inserts from the 16S rRNA genes of isolates were compared with sequences of known species or phylotypes to determine species identity or closest relatives. Nearly complete sequences of about 1,500 bases were obtained for potentially novel species or phylotypes. In an analysis of approximately 750 clones, 92 different bacterial species were identified. About half of the clones were identified as phylotypes, of which 29 were novel to the tongue microbiota. Fifty-one of the 92 species or phylotypes were detected in more than one subject. Those species most associated with healthy subjects were
Streptococcus salivarius
,
Rothia mucilaginosa
, and an uncharacterized species of
Eubacterium
(strain FTB41).
Streptococcus salivarius
was the predominant species in healthy subjects, as it represented 12 to 40% of the total clones analyzed from each healthy subject. Overall, the predominant microbiota on the tongue dorsa of healthy subjects was different from that on the tongue dorsa of subjects with halitosis. Those species most associated with halitosis were
Atopobium parvulum
, a phylotype (clone BS095) of
Dialister
,
Eubacterium sulci
, a phylotype (clone DR034) of the uncultivated phylum TM7,
Solobacterium moorei
, and a phylotype (clone BW009) of
Streptococcus
. On the basis of our ongoing efforts to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species that colonize the oral cavity, there are now over 600 species.
Serotyping of
Listeria monocytogenes
by Enzyme-Linked Immunosorbent Assay and Identification of Mixed-Serotype Cultures by Colony Immunoblotting
Authors: Palumbo Jeffrey D. , Borucki Monica K. , Mandrell Robert E. , Gorski Lisa
Published: February 2003

Routine analysis of
Listeria monocytogenes
by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of
L. monocytogenes
antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101
L. monocytogenes
isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of
L. monocytogenes
were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.
Quantitative Assay of Hepatitis C Virus RNA Using an Automated Extraction System for Specific Capture with Probes and Paramagnetic Particle Separation
Authors: Miyachi Hayato , Masukawa Atsuko , Asai Satomi , Miura Toshiaki , Tamatsukuri Shigeru , Hirose Toru , Ando Yasuhiko
Published: February 2003

A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850,000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log10(IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R2= 0.972, n= 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.