bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endosymbiotic
Bacteroidales
Bacteria of the Flagellated Protist
Pseudotrichonympha grassii
in the Gut of the Termite
Coptotermes formosanus
Authors: Noda Satoko , Iida Toshiya , Kitade Osamu , Nakajima Hideaki , Kudo Toshiaki , Ohkuma Moriya 
Published: December 2005

ABSTRACT

A unique lineage of bacteria belonging to the order
Bacteroidales
was identified as an intracellular endosymbiont of the protist
Pseudotrichonympha grassii
(
Parabasalia
,
Hypermastigea
) in the gut of the termite
Coptotermes formosanus
. We identified the 16S rRNA,
gyrB
, elongation factor Tu, and
groEL
gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The
Bacteroidales
endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the
Bacteroidales
attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the
Bacteroidales
endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of
Pseudotrichonympha
were very abundant in the gut symbiotic community of the termite.
Development and Characterization of a Subtilin-Regulated Expression System in
Bacillus subtilis
: Strict Control of Gene Expression by Addition of Subtilin
Authors: Bongers Roger S. , Veening Jan-Willem , Van Wieringen Maarten , Kuipers Oscar P. , Kleerebezem Michiel 
Published: December 2005

ABSTRACT

A system for subtilin-regulated gene expression (SURE) in
Bacillus subtilis
that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple
spaRK
into the
amyE
locus of the
B. subtilis
chromosome and a
B. subtilis
168-derived production host in which the
spaRK
genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type
spaS
promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both
spaS
promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both
spaS
promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type
spaS
promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated
spaS
promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the
spaS
promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.
Development and Evaluation of Genome-Probing Microarrays for Monitoring Lactic Acid Bacteria
Authors: Bae Jin-Woo , Rhee Sung-Keun , Park Ja Ryeong , Chung Won-Hyong , Nam Young-Do , Lee Insun , Kim Hongik , Park Yong-Ha 
Published: December 2005

ABSTRACT
The genome-probing microarray (GPM) was developed for quantitative, high-throughput monitoring of community dynamics in lactic acid bacteria (LAB) fermentation through the deposit of 149 microbial genomes as probes on a glass slide. Compared to oligonucleotide microarrays, the specificity of GPM was remarkably increased to a species-specific level. GPM possesses about 10- to 100-fold higher sensitivity (2.5 ng of genomic DNA) than the currently used 50-mer oligonucleotide microarrays. Since signal variation between the different genomes was very low compared to that of cDNA or oligonucleotide-based microarrays, the capacity of global quantification of microbial genomes could also be observed in GPM hybridization. In order to assess the applicability of GPMs, LAB community dynamics were monitored during the fermentation of kimchi, a traditional Korean food. In this work, approximately 100 diverse LAB species could be quantitatively analyzed as actively involved in kimchi fermentation.
Shiga Toxin 2e-Producing
Escherichia coli
Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells
Authors: Sonntag Anne-Katharina , Bielaszewska Martina , Mellmann Alexander , Dierksen Nadine , Schierack Peter , Wieler Lothar H. , Schmidt M. Alexander , Karch Helge 
Published: December 2005

ABSTRACT

Thirteen
Escherichia coli
strains harboring
stx
2e
were isolated from 11,056 human stools. This frequency corresponded to
the presence of the
stx
2e
allele in 1.7% of all
Shiga toxin-producing
E. coli
(STEC) strains. The strains
harboring
stx
2e
were associated with mild diarrhea
(
n
= 9) or asymptomatic infections (
n
=
4). Because STEC isolates possessing
stx
2e
are porcine pathogens, we compared the human
STEC isolates with
stx
2e
-harboring
E.
coli
isolated from piglets with edema disease and postweaning
diarrhea. All pig isolates possessed the gene encoding the F18 adhesin,
and the majority possessed adhesin involved in diffuse adherence; these
adhesins were absent from all the human STEC isolates. In contrast, the
high-pathogenicity island encoding an iron uptake system was found only
in human isolates. Host-specific patterns of interaction with
intestinal epithelial cells were observed. All human isolates adhered
to human intestinal epithelial cell lines T84 and HCT-8 but not to pig
intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates
completely lysed human epithelial cells but not IPEC-J2 cells, to which
most of them adhered. Our data demonstrate that
E. coli
isolates producing Shiga toxin 2e have imported specific virulence and
fitness determinants which allow them to adapt to the specific hosts in
which they cause various forms of
disease.
Directed Evolution of
Streptomyces clavuligerus
Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion
Authors: Wei Chia-Li , Yang Yunn-Bor , Deng Chan-Hui , Liu Wen-Chi , Hsu Jyh-Shing , Lin Yu-Ching , Liaw Shwu-Huey , Tsai Ying-Chieh 
Published: December 2005

ABSTRACT

The deacetoxycephalosporin C synthase from
Streptomyces clavuligerus
was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the
k
cat
/

K
m

ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a
k
cat
/

K
m

ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.
Detection ofMycobacterium aviumsubsp.paratuberculosisin Retail Cheeses from Greece and the Czech Republic
Authors: Ikonomopoulos John , Pavlik Ivo , Bartos Milan , Svastova Petra , Ayele Wuhib Yayo , Roubal Petr , Lukas John , Cook Nigel , Gazouli Maria 
Published: December 2005

ABSTRACTWe investigated the presence ofMycobacterium aviumsubsp.paratuberculosisin retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure toM. aviumsubsp.paratuberculosis, albeit at a low level for viable cells.
Novel Primers Reveal Wider Diversity among Marine Aerobic Anoxygenic Phototrophs
Authors: Yutin Natalya , Suzuki Marcelino T. , Béjà Oded 
Published: December 2005

ABSTRACT

Aerobic anoxygenic phototrophic bacteria (AAnPs) were previously proposed to account for up to 11% of marine bacterioplankton and to potentially have great ecological importance in the world's oceans. Our data show that previously used primers based on the M subunit of anoxygenic photosynthetic reaction center genes (
pufM
) do not comprehensively identify the diversity of AAnPs in the ocean. We have designed and tested a new set of
pufM
-specific primers and revealed several new AAnP variants in environmental DNA samples and genomic libraries.
PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample
Authors: Acinas Silvia G. , Sarma-Rupavtarm Ramahi , Klepac-Ceraj Vanja , Polz Martin F. 
Published: December 2005

ABSTRACT

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated.
Taq
DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.
Evaluation of New Broth Media for Microdilution Antibiotic Susceptibility Testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria
Authors: Klare Ingo , Konstabel Carola , Müller-Bertling Sibylle , Reissbrodt Rolf , Huys Geert , Vancanneyt Marc , Swings Jean , Goossens Herman , Witte Wolfgang 
Published: December 2005

ABSTRACT

Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera
Lactobacillus
,
Pediococcus
,
Lactococcus
, and
Bifidobacterium
. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with
l
-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.
Cell-Cell Influences on Bacterial Community Development in Aquatic Biofilms
Authors: McLean Robert J. C. , Barnes Mary B. , Windham M. Katy , Merchant Mubina , Forstner Michael R. J. , Fuqua Clay 
Published: December 2005

ABSTRACT
Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.
Use of PCR Amplification of tRNA Gene-Linked Short Tandem Repeats for Genotyping
Entamoeba histolytica
Authors: Ali Ibne Karim M. , Zaki Mehreen , Clark C. Graham 
Published: December 2005

ABSTRACT

We have developed a reliable method for PCR-based genotyping of
Entamoeba histolytica
based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate
E. histolytica
from
E. dispar
as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with
Entamoeba histolytica
, as well as other aspects of the organism's epidemiology.
Analysis of a Uropathogenic
Escherichia coli
Clonal Group by Multilocus Sequence Typing
Authors: Tartof Sara Y. , Solberg Owen D. , Manges Amee R. , Riley Lee W. 
Published: December 2005

ABSTRACT

Although many strain typing methods exist for pathogenic
Escherichia coli
, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative. However, its applicability to different pathogens in specific epidemiologic contexts is not well understood. Here, we applied a previously established MLST method based on housekeeping genes to a well-characterized collection of uropathogenic
E. coli
isolates to compare the discriminatory ability of this procedure with that of enterobacterial repeat intergenic consensus (ERIC2) PCR, serogrouping, and pulsed-field gel electrophoresis (PFGE). Among 45
E. coli
isolates studied, 17 different multilocus sequence types (ST) were identified. One MLST group (designated ST69 complex) was comprised of 22 isolates, all belonging to uropathogenic and bacteremic
E. coli
strains previously defined as clonal group A (CgA) by ERIC2 PCR. The ST69 strains contained five different serogroups and 14 PFGE types. ERIC2 PCR CgA strains belonging to different MLST groups were also identified. Interestingly, one cow
E. coli
isolate, previously shown by PFGE to be closely related to a human uropathogenic CgA strain, was found to cluster with the ST69 strains. All of the other animal and environmental CgA isolates had different MLST profiles. The discriminatory power of this MLST method based on housekeeping genes appears to be higher than that of ERIC2 PCR but lower than that of PFGE for epidemiologic study of uropathogenic
E. coli
.
Rapid Ped-2E9 Cell-Based Cytotoxicity Analysis and Genotyping of
Bacillus
Species
Authors: Gray Kristen M. , Banada Padmapriya P. , O'Neal Erin , Bhunia Arun K. 
Published: December 2005

ABSTRACT

Bacillus
species causing food-borne disease produce multiple toxins eliciting gastroenteritis. Toxin assays with mammalian cell cultures are reliable but may take 24 to 72 h to complete and also lack sensitivity. Here, a sensitive and rapid assay was developed using a murine hybridoma Ped-2E9 cell model.
Bacillus
culture supernatants containing toxins were added to a Ped-2E9 cell line and analyzed for cytotoxicity with an alkaline phosphatase release assay. Most
Bacillus cereus
strains produced positive cytotoxicity results within 1 h, and data were comparable to those obtained with the standard Chinese hamster ovary (CHO)-based cytotoxicity assay, which took about 72 h to complete. Moreover, the Ped-2E9 cell assay had 25- to 58-fold-higher sensitivity than the CHO assay. Enterotoxin-producing
Bacillus thuringiensis
also gave positive results with Ped-2E9 cells, while several other
Bacillus
species were negative. Eight isolates from food suspected of
Bacillus
contamination were also tested, and only one strain, which was later confirmed as
B. cereus
, gave a positive result. In comparison with two commercial diarrheal toxin assay kits (BDE-VIA and BCET-RPLA), the Ped-2E9 assay performed more reliably. Toxin fractions of >30 kDa showed the highest degree of cytotoxicity effects, and heat treatment significantly reduced the toxin activity, indicating the involvement of a heat-labile high-molecular-weight component in Ped-2E9 cytotoxicity. PCR results, in most cases, were in agreement with the cytotoxic potential of each strain. Ribotyping was used to identify cultures and indicated differences for several previously reported isolates. This Ped-2E9 cell assay could be used as a rapid (∼1-h) alternative to current methods for sensitive detection of enterotoxins from
Bacillus
species.
Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis
Authors: DiStefano Daniel J. , Kraiouchkine Nikolai , Mallette Laura , Maliga Marianne , Kulnis Gregory , Keller Paul M. , Clark H. Fred , Shaw Alan R. 
Published: December 2005

ABSTRACT
Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ∼92% of samples (3, 17, 19).
Use of Recombinant Antigens for Early Postnatal Diagnosis of Congenital Toxoplasmosis
Authors: Buffolano Wilma , Beghetto Elisa , Del Pezzo Mariassunta , Spadoni Andrea , Di Cristina Manlio , Petersen Eskild , Gargano Nicola 
Published: December 2005

ABSTRACT

The main objective of this work was to improve the early serologic diagnosis of toxoplasmosis in children at risk of congenital infection by using recombinant antigens. Serum samples from 104 infants born to mothers with primary
Toxoplasma gondii
infection acquired during pregnancy, of which 35 were congenitally infected and 22 had clinical silent toxoplasmosis at birth, were included. Immunoglobulin M (IgM), IgG, and IgG subtype antibodies against epitopes carried by fragments of
T. gondii MIC2
,
MIC3
,
MIC4
,
M2AP
,
AMA1
, and
SAG1
gene products were measured by performing parallel enzyme immunoassays (Rec-ELISAs). Recombinant antigens preferentially reacted with IgG antibodies from infected infants compared to uninfected subjects (
P
< 0.0001), indicating that sera from infected children recognized a more diverse repertoire of antigens than sera transferred over the placenta from the mothers. Using two serial samples collected within 3 months of life, it was possible to demonstrate a neosynthesis of specific anti-MIC2 and anti-SAG1 immunoglobulin G, mainly of the IgG2 subtype, in 13 out of 20 infants with congenital toxoplasmosis. IgM antibodies in 97% of infected infants reacted with at least one of the recombinant antigens, confirming the diagnosis of congenital infection as soon as 2 months after birth (
P
< 0.0001). The use of recombinant antigens is effective in distinguishing
T. gondii
-infected from uninfected infants and shows that assays based on recombinant antigens improve the diagnosis of newborns with congenital toxoplasmosis.
Genetic Diversity and Recombination of Porcine Sapoviruses
Authors: Wang Qiu-Hong , Han Myung Guk , Funk Julie A. , Bowman Gary , Janies Daniel A. , Saif Linda J. 
Published: December 2005

ABSTRACT
Sapoviruses (SaVs) are emerging enteric pathogens that cause diarrhea in humans and animals. Human SaVs are genetically variable and have been classified into four genogroups (GI, -II, -IV, and -V). To date, only two genetically similar porcine SaV strains have been reported that belong to GIII. To investigate the genetic diversity of porcine SaVs and their genetic relatedness to human strains, we sequenced 286 nucleotides (nt) of the RNA-dependent RNA polymerase (RdRp) region of nine porcine SaVs detected from field pig fecal samples collected in U.S. swine farms during the period from 1999 to 2003. One strain (Po/SaV/MI-QW19/2002/US) was most closely related to human GII SaVs. We also sequenced 3 kb of the viral genome, including the partial RdRp (766 to 790 nt), the complete capsid, the ORF2 and the 3′-untranslated region of four strains representative for the positive farms or for the distinct genetic clusters. From the sequence analysis of the complete capsid, we identified a potential new genogroup of porcine SaVs, with Po/SaV/OH-JJ681/00/US as the representative strain. Furthermore, two potential porcine SaV recombinants were identified. To our knowledge this is the first report of a porcine SaV strain more closely related genetically to human SaVs and the occurrence of porcine SaV recombinants. The presence of porcine SaVs more similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants if intragenogroup human strains exist.