bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of
Helicobacter pylori
Authors: Boneca Ivo G. , Ecobichon Chantal , Chaput Catherine , Mathieu Aurélie , Guadagnini Stéphanie , Prévost Marie-Christine , Colland Frédéric , Labigne Agnès , de Reuse Hilde 
Published: February 2008

ABSTRACT

The
Escherichia coli
-
Helicobacter pylori
shuttle vector pHeL2 was modified to introduce the inducible LacI
q
-p
Tac
system of
E. coli
, in which the promoters were engineered to be under the control of
H. pylori
RNA polymerase. The
amiE
gene promoter of
H. pylori
was taken to constitutively express the LacI
q
repressor. Expression of the reporter gene
lacZ
was driven by either p
Tac
(pILL2150) or a modified version of the
ureI
gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in
H. pylori
. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential
pbp1
and
ftsI
genes encoding penicillin-binding proteins 1 and 3 of
H. pylori
, respectively. Both genes were cloned into pILL2150 and introduced in the parental
H. pylori
strain N6. The chromosomally harbored
pbp1
and
ftsI
genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-
d
-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of
H. pylori
. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of
H. pylori
isolates with different genetic backgrounds.
Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene
Authors: Nozawa-Inoue Mamie , Jien Mercy , Hamilton Nicholas S. , Stewart Valley , Scow Kate M. , Hristova Krassimira R. 
Published: February 2008

ABSTRACTA quantitative real-time PCR assay targeting thepcrAgene, encoding the catalytic subunit of perchlorate reductase, detectedpcrAgenes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. PartialpcrAsequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.
Bacterial Community Structure of Acid-Impacted Lakes: What Controls Diversity?
Authors: Percent Sascha F. , Frischer Marc E. , Vescio Paul A. , Duffy Ellen B. , Milano Vincenzo , McLellan Maggie , Stevens Brett M. , Boylen Charles W. , Nierzwicki-Bauer Sandra A. 
Published: February 2008

ABSTRACT

Although it is recognized that acidification of freshwater systems results in decreased overall species richness of plants and animals, little is known about the response of aquatic microbial communities to acidification. In this study we examined bacterioplankton community diversity and structure in 18 lakes located in the Adirondack Park (in the state of New York in the United States) that were affected to various degrees by acidic deposition and assessed correlations with 31 physical and chemical parameters. The pH of these lakes ranged from 4.9 to 7.8. These studies were conducted as a component of the Adirondack Effects Assessment Program supported by the U.S. Environmental Protection Agency. Thirty-one independent 16S rRNA gene libraries consisting of 2,135 clones were constructed from epilimnion and hypolimnion water samples. Bacterioplankton community composition was determined by sequencing and amplified ribosomal DNA restriction analysis of the clone libraries. Nineteen bacterial classes representing 95 subclasses were observed, but clone libraries were dominated by representatives of the
Actinobacteria
and
Betaproteobacteria
classes. Although the diversity and richness of bacterioplankton communities were positively correlated with pH, the overall community composition assessed by principal component analysis was not. The strongest correlations were observed between bacterioplankton communities and lake depth, hydraulic retention time, dissolved inorganic carbon, and nonlabile monomeric aluminum concentrations. While there was not an overall correlation between bacterioplankton community structure and pH, several bacterial classes, including the
Alphaproteobacteria
, were directly correlated with acidity. These results indicate that unlike more identifiable correlations between acidity and species richness for higher trophic levels, controls on bacterioplankton community structure are likely more complex, involving both direct and indirect processes.
Physiological State, Growth Mode, and Oxidative Stress Play a Role in Cd(II)-Mediated Inhibition of
Nitrosomonas europaea
19718
Authors: Chandran Kartik , Love Nancy G. 
Published: February 2008

ABSTRACT

The goal of this study was to determine the impact of physiological growth states (batch exponential and batch stationary growth) and growth modes (substrate-limited chemostat, substrate-sufficient exponential batch, and substrate-depleted stationary batch growth) on several measures of growth and responses to Cd(II)-mediated inhibition of
Nitrosomonas europaea
strain 19718. The specific oxygen uptake rate (sOUR) was the most sensitive indicator of inhibition among the different responses analyzed, including total cell abundance, membrane integrity, intracellular 16S rRNA/DNA ratio, and
amoA
expression. This observation remained true irrespective of the physiological state, the growth mode, or the mode of Cd(II) exposure. Based on the sOUR, a strong time-dependent exacerbation of inhibition (in terms of an inhibition coefficient [

K
i

]) in exponential batch cultures was observed. Long-term inhibition levels (based on

K
i

estimates) in metabolically active chemostat and exponential batch cultures were also especially severe and comparable. In contrast, the inhibition level in stationary-phase cultures was 10-fold lower and invariable with exposure time. Different strategies for surviving substrate limitation (a 10-fold increase in
amoA
expression) and starvation (the retention of 16S rRNA levels) in
N. europaea
cultures were observed.
amoA
expression was most negatively impacted by Cd(II) exposure in the chemostat cultures, was less impacted in exponential batch cultures, and was least impacted in stationary batch cultures. Although the
amoA
response was consistent with that of the sOUR, the
amoA
response was not as strong. The intracellular 16S rRNA/DNA ratio, as determined by fluorescence in situ hybridization, also did not uniformly correlate with the sOUR under conditions of inhibition or no inhibition. Finally, Cd(II)-mediated inhibition of
N. europaea
was attributed partially to oxidative stress.
On-Line Monitoring of Microbial Volatile Metabolites by Proton Transfer Reaction-Mass Spectrometry
Authors: Bunge Michael , Araghipour Nooshin , Mikoviny Tomas , Dunkl Jürgen , Schnitzhofer Ralf , Hansel Armin , Schinner Franz , Wisthaler Armin , Margesin Rosa , Märk Tilmann D. 
Published: February 2008

ABSTRACT

A method for analysis of volatile organic compounds (VOCs) from microbial cultures was established using proton transfer reaction-mass spectrometry (PTR-MS). A newly developed sampling system was coupled to a PTR-MS instrument to allow on-line monitoring of VOCs in the dynamic headspaces of microbial cultures. The novel PTR-MS method was evaluated for four reference organisms:
Escherichia coli
,
Shigella flexneri
,
Salmonella enterica
, and
Candida tropicalis
. Headspace VOCs in sampling bottles containing actively growing cultures and uninoculated culture medium controls were sequentially analyzed by PTR-MS. Characteristic marker ions were found for certain microbial cultures:
C. tropicalis
could be identified by several unique markers compared with the other three organisms, and
E. coli
and
S. enterica
were distinguishable from each other and from
S. flexneri
by specific marker ions, demonstrating the potential of this method to differentiate between even closely related microorganisms. Although the temporal profiles of some VOCs were similar to the growth dynamics of the microbial cultures, most VOCs showed a different temporal profile, characterized by constant or decreasing VOC levels or by single or multiple peaks over 24 h of incubation. These findings strongly indicate that the temporal evolution of VOC emissions during growth must be considered if characterization or differentiation based on microbial VOC emissions is attempted. Our study may help to establish the analysis of VOCs by on-line PTR-MS as a routine method in microbiology and as a tool for monitoring environmental and biotechnological processes.
Capacity of Human Nisin- and Pediocin-Producing Lactic Acid Bacteria To Reduce Intestinal Colonization by Vancomycin-Resistant Enterococci
Authors: Millette Mathieu , Cornut Gilbert , Dupont Claude , Shareck François , Archambault Denis , Lacroix Monique 
Published: February 2008

ABSTRACT

This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model.
Lactococcus lactis
MM19 and
Pediococcus acidilactici
MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by
P. acidilactici
is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that
L. lactis
harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of
L. lactis
MM19,
P. acidilactici
MM33,
P. acidilactici
MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that
L. lactis
and
P. acidilactici
MM33A increased the concentrations of total LAB and anaerobes while
P. acidilactici
MM33 decreased the
Enterobacteriaceae
populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In
L. lactis
-fed mice, fecal VRE levels 1.73 and 2.50 log
10
CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the
P. acidilactici
MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log
10
CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human
L. lactis
and
P. acidilactici
nisin- and pediocin-producing strains can reduce VRE intestinal colonization.
Antibiotic Resistance of Enterococci in American Bison (
Bison bison
) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle
Authors: Anderson John F. , Parrish Torrey D. , Akhtar Mastura , Zurek Ludek , Hirt Helmut 
Published: February 2008

ABSTRACT

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by
Enterococcus casseliflavus
in bison (62.4%), while
Enterococcus hirae
was the most common isolate from cattle (39.7%).
Enterococcus faecalis
was the second most common species isolated from bison (16%). In cattle,
E. faecalis
and
Enterococcus faecium
were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by
tetO
and
ermB
, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10
−6
per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.
The
ygaVP
Genes of
Escherichia coli
Form a Tributyltin-Inducible Operon
Authors: Gueuné Hervé , Durand Marie-José , Thouand Gérald , DuBow Michael S. 
Published: February 2008

ABSTRACT

A tributyltin (TBT)
luxAB
transcriptional fusion in
Escherichia coli
revealed that a TBT-activated promoter is located upstream of two cotranscribed orphan genes,
ygaV
and
ygaP
. We demonstrate that transcription from the promoter upstream of
ygaVP
is constitutive in a
ygaVP
mutant, suggesting that YgaV is an autoregulated, TBT-inducible repressor.
Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms
Authors: Briandet R. , Lacroix-Gueu P. , Renault M. , Lecart S. , Meylheuc T. , Bidnenko E. , Steenkeste K. , Bellon-Fontaine M.-N. , Fontaine-Aupart M.-P. 
Published: February 2008

ABSTRACT

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting
Stenotrophomonas maltophilia
biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of
Lactococcus lactis
but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as “active” phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.
Microcyclamide Biosynthesis in Two Strains ofMicrocystis aeruginosa: from Structure to Genes and Vice Versa
Authors: Ziemert Nadine , Ishida Keishi , Quillardet Philippe , Bouchier Christiane , Hertweck Christian , de Marsac Nicole Tandeau , Dittmann Elke 
Published: February 2008

ABSTRACTComparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacteriumMicrocystis aeruginosaNIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain,M. aeruginosaPCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria.
Molecular Variation of Satellite DNAβ Molecules Associated with
Malvastrum yellow vein virus
and Their Role in Pathogenicity
Authors: Guo Wei , Jiang Tong , Zhang Xian , Li Guixin , Zhou Xueping 
Published: February 2008

ABSTRACT

Previous studies have found that the diversity of begomovirus-associated DNAβ satellites is related to host and geographical origin. In this study, we have cloned and sequenced 20 different isolates of DNAβ molecules associated with
Malvastrum yellow vein virus
(MYVV) isolated from
Malvastrum coromandelianum
plants in different geographical locations of Yunnan Province, China. Analyses of their molecular variation indicate that the satellites are clustered together according to their geographical location but that they have only limited sequence diversity. Infectivity tests using infectious clones of MYVV and its associated DNAβ molecule indicate that MYVV DNAβ is indispensable for symptom induction in
Nicotiana benthamiana
,
N. glutinosa
,
Petunia hybrida
, and
M. coromandelianum
plants. Furthermore, we showed that MYVV interacts functionally with heterologous DNAβ molecules in
N. benthamiana
plants.
Use of
Drosophila
S2 Cells as a Model for Studying
Ehrlichia chaffeensis
Infections
Authors: Luce-Fedrow Alison , Von Ohlen Tonia , Boyle Daniel , Ganta Roman R. , Chapes Stephen K. 
Published: February 2008

ABSTRACT

Ehrlichia chaffeensis
is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that
Drosophila
S2 cells are permissive for the growth of
E. chaffeensis
. We saw morulae (aggregates of bacteria) by microscopy, detected the
E. chaffeensis
16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect
E. chaffeensis
in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for
E. chaffeensis
through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of
E. chaffeensis
. The use of a
Drosophila
system has the potential to serve as a model system for studying
Ehrlichia
due to its completed genome, ease of genetic manipulation, and the availability of mutants.
Molecular Analysis as an Aid To Assess the Public Health Risk of Non-O157 Shiga Toxin-Producing
Escherichia coli
Strains
Authors: Coombes Brian K. , Wickham Mark E. , Mascarenhas Mariola , Gruenheid Samantha , Finlay B. Brett , Karmali Mohamed A. 
Published: February 2008

ABSTRACT

Shiga toxin-producing
Escherichia coli
(STEC) strains are commensal bacteria in cattle with high potential for environmental and zoonotic transmission to humans. Although O157:H7 is the most common STEC serotype, there is growing concern over the emergence of more than 200 highly virulent non-O157 STEC serotypes that are globally distributed, several of which are associated with outbreaks and/or severe human illness such as hemolytic-uremic syndrome (HUS) and hemorrhagic colitis. At present, the underlying genetic basis of virulence potential in non-O157 STEC is unknown, although horizontal gene transfer and the acquisition of new pathogenicity islands are an expected origin. We used seropathotype classification as a framework to identify genetic elements that distinguish non-O157 STEC strains posing a serious risk to humans from STEC strains that are not associated with severe and epidemic disease. We report the identification of three genomic islands encoding non-LEE effector (
nle
) genes and 14 individual
nle
genes in non-O157 STEC strains that correlate independently with outbreak and HUS potential in humans. The implications for transmissible zoonotic spread and public health are discussed. These results and methods offer a molecular risk assessment strategy to rapidly recognize and respond to non-O157 STEC strains from environmental and animal sources that might pose serious public health risks to humans.
Biochemical and Physiological Characterization of the Pyruvate Formate-Lyase Pfl1 of
Chlamydomonas reinhardtii
, a Typically Bacterial Enzyme in a Eukaryotic Alga
Authors: Hemschemeier Anja , Jacobs Jessica , Happe Thomas 
Published: February 2008

ABSTRACT

The unicellular green alga
Chlamydomonas reinhardtii
has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in
C. reinhardtii
was predicted from formate secretion and the homology of the deduced protein of the
PFL1
gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the
C. reinhardtii PFL1
cDNA in
Escherichia coli
and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient
E. coli
strain secretes formate when expressing the
PFL1
cDNA of
C. reinhardtii
. We also examined the Pfl1 fermentation pathway of
C. reinhardtii
under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.