bluuebunny/crossref_metadata_2025 huggingface dataset:
Using DNA Microarrays To Identify Library-Independent Markers for Bacterial Source Tracking
Authors: Soule Marilyn , Kuhn Edward , Loge Frank , Gay John , Call Douglas R.
Published: March 2006

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome
Enterococcus
microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50
Enterococcus
isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four
Enterococcus
species (
Enterococcus casseliflavus
,
E. faecalis
,
E. hirae
, or
E. mundtii
). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.
Engineering of Cyclodextrin Glucanotransferase on the Cell Surface ofSaccharomyces cerevisiaefor Improved Cyclodextrin Production
Authors: Wang Zhankun , Qi Qingsheng , Wang Peng George
Published: March 2006

The cyclodextrin glucanotransferase (CGTase) gene (cgt) fromBacillus circulans251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface ofSaccharomyces cerevisiaewas confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.
Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon
Thermococcus
sp. Strain NA1
Authors: Lee Hyun Sook , Kim Yun Jae , Bae Seung Seob , Jeon Jeong Ho , Lim Jae Kyu , Jeong Byeong Chul , Kang Sung Gyun , Lee Jung-Hyun
Published: March 2006

Genomic analysis of a hyperthermophilic archaeon,
Thermococcus
sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of
Thermococcus kodakaraensis
KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the
Thermococcus
sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (
Thermococcus
sp. strain NA1 APP), was cloned and expressed in
Escherichia coli
. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(
N
ε
-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (

K
m

, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co
2+
-, Mn
2+
-, or Zn
2+
-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.
Transcriptional and Functional Analysis of Oxalyl-Coenzyme A (CoA) Decarboxylase and Formyl-CoA Transferase Genes from
Lactobacillus acidophilus
Authors: Azcarate-Peril M. Andrea , Bruno-Bárcena Jose M. , Hassan Hosni M. , Klaenhammer Todd R.
Published: March 2006

Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene (
frc
) and an oxalyl coenzyme A decarboxylase gene (
oxc
) was identified in the genome of the probiotic bacterium
Lactobacillus acidophilus
. Physiological analysis of a mutant harboring a deleted version of the
frc
gene confirmed that
frc
expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the
frc
mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for
frc
and
oxc
transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for
frc
and
oxc
genes. With the exception of
Lactobacillus gasseri
and
Bifidobacterium lactis
, none of the other strains harbored genes for oxalate utilization.
Supplementation of the Diet with High-Viscosity Beta-Glucan Results in Enrichment for Lactobacilli in the Rat Cecum
Authors: Snart Jennifer , Bibiloni Rodrigo , Grayson Teresa , Lay Christophe , Zhang Haiyan , Allison Gwen E. , Laverdiere Julie K. , Temelli Feral , Vasanthan Thavaratnam , Bell Rhonda , Tannock Gerald W.
Published: March 2006

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley β-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with
Lactobacillus acidophilus
. Measurements of
L. acidophilus
rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of
Lactobacillus
cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in β-glucan hydrolysates. The results of this study have relevance to the use of purified β-glucan products as dietary supplements for human consumption.
Microbial Communities and Fecal Indicator Bacteria Associated with
Cladophora
Mats on Beach Sites along Lake Michigan Shores
Authors: Olapade Ola A. , Depas Morgan M. , Jensen Erika T. , McLellan Sandra L.
Published: March 2006

A high biomasses of
Cladophora
, a filamentous green alga, is found mainly during the summer along the shores of Lake Michigan. In this study, the abundance and persistence of the fecal indicator bacterium
Escherichia coli
and sulfate-reducing bacteria (SRB) on
Cladophora
mats collected at Lake Michigan beaches were evaluated using both culture-based and molecular analyses. Additionally, 16S rRNA gene cloning and sequencing were used to examine the bacterial community composition. Overall,
E. coli
was detected in all 63 samples obtained from 11 sites, and the average levels at most beaches ranged from 2,700 CFU/100 g (wet weight) of
Cladophora
to 7,500 CFU/100 g of
Cladophora
. However, three beaches were found to have site average
E. coli
densities of 12,800, 21,130, and 27,950 CFU/100 g of
Cladophora
. The
E. coli
levels in the lake water collected at the same time from these three sites were less than the recommended U.S. Environmental Protection Agency limit, 235 CFU/100 ml.
E. coli
also persisted on
Cladophora
mats in microcosms at room temperature for more than 7 days, and in some experiments it persisted for as long as 28 days. The SRB densities on
Cladophora
mats were relatively high, ranging from 4.4 × 10
6
cells/g (6.64 log CFU/g) to 5.73 × 10
6
cells/g (6.76 log CFU/g) and accounting for between 20% and 27% of the total bacterial counts. Partial sequences of the 16S rRNA gene clones revealed a phylogenetically diverse community, in which the
Cytophaga-Flavobacterium-Bacteroides
cluster and the low-G+C-content gram-positive bacteria were the dominant organisms, accounting for 40% and 12.8%, respectively, of the total clone library. These results further reveal the potential public health and ecological significance of
Cladophora
mats that are commonly found along the shoreline of Lake Michigan, especially with regard to the potential to harbor microorganisms associated with fecal pollution and odor-causing bacteria.
The Phytopathogen
Dickeya dadantii
(
Erwinia chrysanthemi
3937) Is a Pathogen of the Pea Aphid
Authors: Grenier Anne-Marie , Duport Gabrielle , Pagès Sylvie , Condemine Guy , Rahbé Yvan
Published: March 2006

Dickeya dadantii
(
Erwinia chrysanthemi
) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from
Bacillus thuringiensis
, which are not present in the close relative
Pectobacterium carotovorum
subsp.
atrosepticum
. The pathogenicity of
D. dadantii
was tested on the pea aphid
Acyrthosiphon pisum
, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A
D. dadantii
mutant with the four cytotoxin genes deleted showed a reduced per os virulence for
A. pisum
, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (
Erwinia aphidicola
), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the
Enterobacteriaceae
. Previously characterized gut symbionts from thrips (
Erwinia
/
Pantoea
group) were also highly pathogenic to the aphid, whereas the potent entomopathogen
Photorhabdus luminescens
was not.
D. dadantii
is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (
Drosophila melanogaster
,
Sitophilus oryzae
, and
Spodoptera littoralis
).
D. dadantii
was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.
Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring
Dehalococcoides
Isolate
Authors: Sung Youlboong , Ritalahti Kirsti M. , Apkarian Robert P. , Löffler Frank E.
Published: March 2006

A novel
Dehalococcoides
isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of
Dehalococcoides
16S rRNA gene and chloroethene reductive dehalogenase gene (i.e.,
vcrA
,
tceA
, and
bvcA
) copy numbers revealed that the culture consisted of multiple, distinct
Dehalococcoides
organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only
vcrA
. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of
Dehalococcoides
cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the
Dehalococcoides
cluster and shares 100% 16S rRNA gene sequence identity with two other
Dehalococcoides
isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE,
cis
-1,2-dichloroethene (
cis
-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE,
cis
-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter
−1
day
−1
, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of
cis
-DCE and VC during TCE-to-ethene dechlorination.
Macroscopic Streamer Growths in Acidic, Metal-Rich Mine Waters in North Wales Consist of Novel and Remarkably Simple Bacterial Communities
Authors: Hallberg Kevin B. , Coupland Kris , Kimura Sakurako , Johnson D. Barrie
Published: March 2006

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were β
-Proteobacteria
. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the β-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The β-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer
Gallionella ferruginea
. FISH analysis using oligonucleotide probes that targeted the two β-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.
Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes
Authors: Ward Linda N. , Bej Asim K.
Published: March 2006

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenicVibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters.V. parahaemolyticushas been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) andtdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype ofV. parahameolyticusO3:K6. TotalV. parahaemolyticuswas targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFUV. parahaemolyticusper g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive fortlhand 4/33 samples that were positive fortdh. This assay specifically and sensitively detected total and pathogenicV. parahaemolyticusand is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.
Chelator-Induced Dispersal and Killing of
Pseudomonas aeruginosa
Cells in a Biofilm
Authors: Banin Ehud , Brady Keith M. , Greenberg E. Peter
Published: March 2006

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as
Staphylococcus aureus
. EDTA can also kill planktonic cells of
Proteobacteria
like
Pseudomonas aeruginosa
. In this study we demonstrate that EDTA is a potent
P. aeruginosa
biofilm disrupter. In Tris buffer, EDTA treatment of
P. aeruginosa
biofilms results in 1,000-fold greater killing than treatment with the
P. aeruginosa
antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells.
P. aeruginosa
biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of
P. aeruginosa
cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect
P. aeruginosa
biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.
The
atu
and
liu
Clusters Are Involved in the Catabolic Pathways for Acyclic Monoterpenes and Leucine in
Pseudomonas aeruginosa
Authors: Aguilar J. A. , Zavala A. N. , Díaz-Pérez C. , Cervantes C. , Díaz-Pérez A. L. , Campos-García J.
Published: March 2006

Evidence suggests that the
Pseudomonas aeruginosa
PAO1
gnyRDBHAL
cluster, which is involved in acyclic isoprenoid degradation (A. L. Díaz-Pérez, N. A. Zavala-Hernández, C. Cervantes, and J. Campos-García, Appl. Environ. Microbiol. 70:5102-5110, 2004), corresponds to the
liuRABCDE
cluster (B. Hoschle, V. Gnau, and D. Jendrossek, Microbiology 151:3649-3656, 2005). A
liu
(leucine and isovalerate utilization) homolog cluster was found in the PAO1 genome and is related to the catabolism of acyclic monoterpenes of the citronellol family (AMTC); it was named the
atu
cluster (acyclic terpene utilization), consisting of the
atuCDEF
genes and lacking the hydroxymethyl-glutaryl-coenzyme A (CoA) lyase (HMG-CoA lyase) homolog. Mutagenesis of the
atu
and
liu
clusters showed that both are involved in AMTC and leucine catabolism by encoding the enzymes related to the geranyl-CoA and the 3-methylcrotonyl-CoA pathways, respectively. Intermediary metabolites of the acyclic monoterpene pathway, citronellic and geranic acids, were accumulated, and leucine degradation rates were affected in both
atuF
and
liuD
mutants. The alpha subunit of geranyl-CoA carboxylase and the alpha subunit of 3-methylcrotonyl-CoA carboxylase (α-MCCase), encoded by the
atuF
and
liuD
genes, respectively, were both induced by citronellol, whereas only the α-MCCase subunit was induced by leucine. Both citronellol and leucine also induced a LacZ transcriptional fusion at the
liuB
gene. The
liuE
gene encodes a probable hydroxy-acyl-CoA lyase (probably HMG-CoA lyase), an enzyme with bifunctional activity that is essential for both AMTC and leucine degradation.
P. aeruginosa
PAO1 products encoded by the
liuABCD
cluster showed a higher sequence similarity (77.2 to 79.5%) with the probable products of
liu
clusters from several
Pseudomonas
species than with the
atuCDEF
cluster from PAO1 (41.5%). Phylogenetic studies suggest that the
atu
cluster from
P. aeruginosa
could be the result of horizontal transfer from
Alphaproteobacteria
. Our results suggest that the
atu
and
liu
clusters are bifunctional operons involved in both the AMTC and leucine catabolic pathways.
Use of Single-Point Genome Signature Tags as a Universal Tagging Method for Microbial Genome Surveys
Authors: van der Lelie Daniel , Lesaulnier Celine , McCorkle Sean , Geets Joke , Taghavi Safiyh , Dunn John
Published: March 2006

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes
rpoC
,
uvrB
, and
recA
and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of
Bacillus cereus
and
Bacillus anthracis
. This concept was further used successfully to identify the individual members of a defined microbial community.
Concentration-Dependent Patterns of Leucine Incorporation by Coastal Picoplankton
Authors: Alonso Cecilia , Pernthaler Jakob
Published: March 2006

Coastal pelagic environments are believed to feature concentration gradients of dissolved organic carbon at a microscale, and they are characterized by pronounced seasonal differences in substrate availability for the heterotrophic picoplankton. Microbial taxa that coexist in such habitats might thus differ in their ability to incorporate substrates at various concentrations. We investigated the incorporation patterns of leucine in four microbial lineages from the coastal North Sea at concentrations between 0.1 and 100 nM before and during a spring phytoplankton bloom. Community bulk incorporation rates and the fraction of leucine-incorporating cells in the different populations were analyzed. Significantly fewer bacterial cells incorporated the amino acid before (13 to 35%) than during (23 to 47%) the bloom at all but the highest concentration. The incorporation rate per active cell in the prebloom situation was constant above 0.1 nM added leucine, whereas it increased steeply with substrate concentration during the bloom. At both time points, a high proportion of members of the
Roseobacter
clade incorporated leucine at all concentrations (55 to 80% and 86 to 94%, respectively). In contrast, the fractions of leucine-incorporating cells increased substantially with substrate availability in bacteria from the SAR86 clade (8 to 31%) and from DE cluster 2 of the
Flavobacteria-Sphingobacteria
(14 to 33%). The incorporation patterns of marine
Euryarchaeota
were between these extremes (30 to 56% and 48 to 70%, respectively). Our results suggest that the contribution of microbial taxa to the turnover of particular substrates may be concentration dependent. This may help us to understand the specific niches of coexisting populations that appear to compete for the same resources.
Evidence for Distinct
l
-Methionine Catabolic Pathways in the Yeast
Geotrichum candidum
and the Bacterium
Brevibacterium linens
Authors: Arfi Kenza , Landaud Sophie , Bonnarme Pascal
Published: March 2006

Tracing experiments were carried out to identify volatile and nonvolatile
l
-methionine degradation intermediates and end products in the yeast
Geotrichum candidum
and in the bacterium
Brevibacterium linens
, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium
B. linens
is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast
G. candidum
produces, we examined whether the
l
-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and α-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of
l
-methionine catabolism. However, the
l
-methionine degradation pathways differ in the first steps of
l
-methionine degradation.
l
-Methionine degradation is initiated by a one-step degradation process in the bacterium
B. linens
, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast
G. candidum
. Since
G. candidum
develops earlier than
B. linens
during the ripening process, MOBA and MHBA generated by
G.candidum
could also be used as precursors for VSC production by
B. linens
.
Heterologous Expression of BetL, a Betaine Uptake System, Enhances the Stress Tolerance ofLactobacillus salivariusUCC118
Authors: Sheehan Vivien M. , Sleator Roy D. , Fitzgerald Gerald F. , Hill Colin
Published: March 2006

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strainLactobacillus salivariusUCC118. Following nisin induction, strains expressingbetLexhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.
Inactivation of Single-Celled
Ascaris suum
Eggs by Low-Pressure UV Radiation
Authors: Brownell Sarah A. , Nelson Kara L.
Published: March 2006

Intact and decorticated single-celled
Ascaris suum
eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m
2
for intact eggs and from 0 to 500 J/m
2
for decorticated eggs. With a UV fluence of 500 J/m
2
, 0.44- ± 0.20-log inactivation (mean ± 95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m
2
resulted in 2.23- ± 0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here,
Ascaris
eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m
2
), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m
2
), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80- ± 0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m
2
, suggesting that the outer eggshell layers protected
A. suum
eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-μm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the
Ascaris
eggs (∼20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.
Effect of Transport at Ambient Temperature on Detection and Isolation of
Vibrio cholerae
from Environmental Samples
Authors: Alam Munirul , Sadique Abdus , Nur-A-Hasan  , Bhuiyan Nurul A. , Nair G. Balakrish , Siddique A. K. , Sack David A. , Ahsan Sunjukta , Huq Anwar , Sack R. Bradley , Colwell Rita R.
Published: March 2006

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of
Vibrio cholerae
, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of
V. cholerae
. For water and plankton samples held at ambient temperatures ranging from 31°C to 35°C for 20 h, the total counts did not increase significantly but the number of culturable
V. cholerae
increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for
V. cholerae
increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of
V. cholerae
O1 and improved detection of
V. cholerae
O1
rfb
and
ctxA
also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic
V. cholerae
from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.
Generation of Novel-Substrate-Accepting Biphenyl Dioxygenases through Segmental Random Mutagenesis and Identification of Residues Involved in Enzyme Specificity
Authors: Zielinski Marco , Kahl Silke , Standfuß-Gabisch Christine , Cámara Beatriz , Seeger Michael , Hofer Bernd
Published: March 2006

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of
Burkholderia xenovorans
LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.
Evaluation of 23S rRNA PCR Primers for Use in Phylogenetic Studies of Bacterial Diversity
Authors: Hunt Dana E. , Klepac-Ceraj Vanja , Acinas Silvia G. , Gautier Clement , Bertilsson Stefan , Polz Martin F.
Published: March 2006

The availability of a diverse set of 23S rRNA gene sequences enabled evaluation of the specificity of 39 previously published and 4 newly designed primers specific for bacteria. An extensive clone library constructed using an optimized primer pair resulted in similar gene richness but slightly differing coverage of some phylogenetic groups, compared to a 16S rRNA gene library from the same environmental sample.
In Situ Gene Expression by
Vibrio vulnificus
Authors: Smith Ben , Oliver James D.
Published: March 2006

Strains of
Vibrio vulnificus
incubated in situ in natural estuarine waters during warm months continued to express
katG
(periplasmic catalase),
rpoS
(stress sigma factor),
tufA
(elongation factor),
wza
, and
wzb
(capsule synthesis).
vvhA
(hemolysin) was differentially expressed between environmental and clinical isolates. These results paralleled our in vitro findings.
Resistance Determinants of a Highly Arsenic-Resistant Strain of
Leptospirillum ferriphilum
Isolated from a Commercial Biooxidation Tank
Authors: Tuffin I. Marla , Hector Stanton B. , Deane Shelly M. , Rawlings Douglas E.
Published: March 2006

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant
Leptospirillum ferriphilum
Fairview strain. One set is located on a transposon, Tn
LfArs
, and is related to the previously identified Tn
AtcArs
from
Acidithiobacillus caldus
isolated from the same arsenopyrite biooxidation tank as
L. ferriphilum
. Tn
LfArs
conferred resistance to arsenite and arsenate and was transpositionally active in
Escherichia coli
. Tn
LfArs
and Tn
AtcArs
were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in
E. coli
and appeared to be poorly expressed in both
L. ferriphilum
and
E. coli
.
Thermophilic Lifestyle for an Uncultured Archaeon from Hydrothermal Vents: Evidence from Environmental Genomics
Authors: Moussard Hélène , Henneke Ghislaine , Moreira David , Jouffe Vincent , López-García Purificacion , Jeanthon Christian
Published: March 2006

We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon
Thermoplasma acidophilum
, providing evidence for a thermophilic way of life of this group of uncultured archaea.
Recovery of
Escherichia coli
from Soil after Addition of Sterile Organic Wastes
Authors: Unc Adrian , Gardner Julie , Springthorpe Susan
Published: March 2006

Laboratory batch tests indicate that addition of sterile municipal sewage biosolids to clay soil from four depths increases the numbers of
Escherichia coli
isolates recoverable in EC-MUG broth (EC broth with 4-methylumbelliferyl-β-glucuronide). This effect was most marked for the deeper soil layers, with increases of about 2.6 orders of magnitude in
E. coli
most probable number.
Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method
Authors: Vincze Eva , Bowra Steve
Published: March 2006

Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in
Mesorhizobium loti
without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.
Novel Technique of Quantitative Nested Real-Time PCR Assay for
Mycobacterium tuberculosis
DNA
Authors: Takahashi Teruyuki , Nakayama Tomohiro
Published: March 2006

The diagnosis of tuberculous meningitis (TBM) remains a complex issue because the most widely used conventional diagnostic tools, such as culture and PCR assay for cerebrospinal fluid (CSF) samples, are unable to rapidly detect
Mycoba
ct
erium tuberculosis
with sufficient sensitivity in the acute phase of TBM. Based on TaqMan PCR, we designed a novel technique consisting of an internally controlled quantitative nested real-time (QNRT) PCR assay that provided a marked improvement in detection sensitivity and quantification. We applied this novel technique to quantitatively detect
M. tuberculosis
DNA in CSF samples from patients with suspected TBM. For use as the internal control in the measurement of the
M. tuberculosis
DNA copy numbers in the QNRT-PCR assay, the original mutation (M) plasmid, which included an artificial random 22-nucleotide sequence within an inserted DNA fragment of the MPB64 gene of
M. tuberculosis
, was prepared. The QNRT-PCR assay showed high sensitivity and specificity that were approximately equivalent to those of the conventional nested PCR assay. Moreover, the QNRT-PCR assay made it possible to precisely and quantitatively detect the initial copy number of
M. tuberculosis
DNA in CSF samples. Therefore, compared to the conventional PCR assay, the QNRT-PCR assay can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. To establish the superiority of this novel technique in TBM diagnosis, it will be necessary to accumulate data from a larger number of patients with suspected TBM.
Use of Oligoarrays for Characterization of Community-Onset Methicillin-Resistant
Staphylococcus aureus
Authors: Koessler Thibaud , Francois Patrice , Charbonnier Yvan , Huyghe Antoine , Bento Manuela , Dharan Sasi , Renzi Gesuele , Lew Daniel , Harbarth Stephan , Pittet Didier , Schrenzel Jacques
Published: March 2006

Until recently, methicillin-resistant
Staphylococcus aureus
(MRSA) was considered the prototype of a hospital-acquired bacterial pathogen. However, recent reports have shown that MRSA has now emerged in the community. Characterization of specific markers for distinguishing the origin of isolates could contribute to improved knowledge of MRSA epidemiology. The release of whole-genome sequences of hospital- and community-acquired
S. aureus
strains allowed the development of whole-genome content analysis techniques, including microarrays. We developed a microarray composed of 8,191 open reading frame-specific oligonucleotides covering >99% of the four sequenced
S. aureus
genomes (N315, Mu50, MW2, and COL) to evaluate gene contents of hospital- and community-onset
S. aureus
strains. In parallel, pulsed-field gel electrophoresis, variable number of tandem repeats, antibiogram, staphylococcal cassette chromosome-
mec
element typing, and presence of the Panton-Valentine leukocidin gene were evaluated in a collection of 15 clinical isolates. Clusters obtained with microarrays showed a high degree of similarity with those obtained by pulsed-field gel electrophoresis or variable number of tandem repeats. Clusters clearly segregated hospital-onset strains from community-onset strains. Moreover, the microarray approach allowed definition of novel marker genes and chromosomal regions specific for given groups of isolates, thus providing better discrimination and additional information compared to pulsed-field gel electrophoresis and variable number of tandem repeats. Finally, the comparative genome hybridization approach unraveled the occurrence of multiple horizontal transfer events leading to community-onset MRSA as well as the need for a specific genetic background in recipient strains for both the acquisition and the stability of the
mec
element.
High-Level Ciprofloxacin Resistance from Point Mutations in
gyrA
and
parC
Confined to Global Hospital-Adapted Clonal Lineage CC17 of
Enterococcus faecium
Authors: Leavis Helen L. , Willems Rob J. L. , Top Janetta , Bonten Marc J. M.
Published: March 2006

To substantiate a common genetic background of ciprofloxacin-resistant
Enterococcus faecium
, 32 ciprofloxacin-resistant (Cip
r
) and 31 ciprofloxacin-susceptible (Cip
s
) isolates from outbreaks, clinical infections, surveillances, and animals from 10 different countries were genotyped by multilocus sequence typing. Additionally, susceptibilities to ampicillin and vancomycin and the presence of
esp
were determined and the quinolone resistance-determining regions of
parC
,
gyrA
,
parB
, and
gyrE
were sequenced. High-level Cip
r
(MIC ≥ 64 μg/ml) due to point mutations in the quinolone resistance-determining region was unique to a distinct hospital-adapted genetic complex in
E. faecium
, previously designated CC17. Low-level Cip
r
(MIC = 4 μg/ml) in non-CC17 strains is not attributable to point mutations in any subunit of the topoisomerase genes, and the mechanism of resistance remains unclear. Acquisition of mutations in
parC
and
gyrA
, leading to high-level Cip
r
, is, in addition to ampicillin resistance and the presence of a putative pathogenicity island, another cumulative step in hospital adaptation of CC17.
Molecular Subtyping of Poultry-Associated Type AClostridium perfringensIsolates by Repetitive-Element PCR
Authors: Siragusa G. R. , Danyluk M. D. , Hiett K. L. , Wise M. G. , Craven S. E.
Published: March 2006

Clostridium perfringensstrains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates ofC. perfringenswere obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates wereC. perfringensalpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtypingC. perfringenslibraries from specific epidemiological or production environment settings.
Molecular Diagnosis of
Bordetella pertussis
Infection by Evaluation of Formalin-Fixed Tissue Specimens
Authors: Tatti Kathleen M. , Wu Kai-Hui , Sanden Gary N. , Greer Patricia , Sumner John , Guarner Jeannette , Paddock Christopher D. , Zaki Sherif R.
Published: March 2006

Formalin-fixed lung or trachea tissue specimens from four infants and one adolescent who died of respiratory illness were tested for
Bordetella pertussis
by conventional and real-time PCR assays.
B. pertussis
was confirmed in all cases. PCR can be an invaluable retrospective diagnostic tool for evaluating archival tissues from patients with suspected fatal pertussis.
Insertion Sequence IS
900
Revisited
Authors: Semret Makeda , Turenne Christine Y. , Behr Marcel A.
Published: March 2006

Many studies investigating
Mycobacterium avium
subsp.
paratuberculosis
in Crohn's disease have used molecular detection of IS
900
in clinical samples, but some have described polymorphisms in IS
900
as variants of this organism. Analysis of 23
M. avium
subsp.
paratuberculosis
isolates revealed that IS
900
is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS
900
-like sequences is not sufficient as a proxy for
M. avium
subsp.
paratuberculosis
.
PCR as a Confirmatory Technique for Laboratory Diagnosis of Malaria
Authors: Johnston Stephanie P. , Pieniazek Norman J. , Xayavong Maniphet V. , Slemenda Susan B. , Wilkins Patricia P. , da Silva Alexandre J.
Published: March 2006

We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of
Plasmodium
spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.
Establishment of ST30 as the Predominant Clonal Type among Community-Associated Methicillin-Resistant
Staphylococcus aureus
Isolates in Singapore
Authors: Hsu Li-Yang , Koh Yin-Ling , Chlebicka Nidhi Loomba , Tan Thean-Yen , Krishnan Prabha , Lin Raymond Tzer-Pin , Tee Nancy , Barkham Timothy , Koh Tse-Hsien
Published: March 2006

The number of infections attributable to community-associated methicillin-resistant
Staphylococcus aureus
(CA-MRSA) in Singapore is progressively increasing. Most cases in the past 2 years were caused by Panton-Valentine leukocidin-positive isolates belonging to sequence type 30, according to multilocus sequence typing. This has clearly become the predominant sequence type among CA-MRSA isolates in Singapore.
Sensitive Line Probe Assay That Simultaneously Detects Mutations Conveying Resistance to Lamivudine and Adefovir
Authors: Hussain Munira , Fung Scott , Libbrecht Evelien , Sablon Erwin , Cursaro Carmela , Andreone Pietro , Lok Anna S. F.
Published: March 2006

The INNO-LiPA HBV DR v2 assay is designed to detect hepatitis B virus mutations conveying resistance to lamivudine and adefovir. Our study confirms that this assay can simultaneously detect the presence of lamivudine and adefovir resistance mutations in clinical samples, has a high degree of concordance with sequencing, and can detect mutants earlier.