bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTWe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.