bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Performance Liquid Chromatography Analyses of Pyoverdin Siderophores Differentiate among Phytopathogenic Fluorescent
Pseudomonas
Species
Authors: Bultreys Alain , Gheysen Isabelle , Wathelet Bernard , Maraite Henri , de Hoffmann Edmond 
Published: February 2003

ABSTRACT

The relationship of pyoverdins produced by 41 pathovars of
Pseudomonas syringae
and by phytopathogenic
Pseudomonas
species was investigated. A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins. Nonfluorescent strains were found in
Pseudomonas amygdali
,
Pseudomonas meliae
,
Pseudomonas fuscovaginae
, and
P. syringae. Pseudomonas agarici
and
Pseudomonas marginalis
produced typical pyoverdins. Among the arginine dihydrolase-negative fluorescent
Pseudomonas
species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species. Within this group, the oxidase-negative species
Pseudomonas viridiflava
and
Pseudomonas ficuserectae
and the pathovars of
P. syringae
produced the same atypical pyoverdin, whereas the oxidase-positive species
Pseudomonas cichorii
produced a similar atypical pyoverdin that contained a glycine instead of a serine. The more distantly related species
Pseudomonas asplenii
and
Pseudomonas fuscovaginae
both produced a less similar atypical pyoverdin. The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two β-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based. The peptide chain influenced the chelation of iron more in atypical pyoverdins. Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification.
Oligonucleotide Microarray for the Study of Functional Gene Diversity in the Nitrogen Cycle in the Environment
Authors: Taroncher-Oldenburg Gaspar , Griner Erin M. , Francis Chris A. , Ward Bess B. 
Published: February 2003

ABSTRACT

The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene
nirS
in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (
amoA
,
nifH
,
nirK
, and
nirS
) and detecting different variants of the same gene,
nirK
, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64
nirS
sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The
nirS
oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 10
7
copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACTA 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.