bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the Variability in the Number of Viable Bacteria after Mild Heat Treatment of Food
Authors: Aguirre J. S. , Pin C. , Rodríguez M. R. , García de Fernando G. D.
Published: October 2009

Variability in the numbers of bacteria remaining in saline solution and whole milk following mild heat treatment has been studied with
Listeria innocua
,
Enterococcus faecalis
,
Salmonella enterica
serovar Enteritidis, and
Pseudomonas fluorescens
. As expected, the most heat-resistant bacterium was
E. faecalis
, while
P. fluorescens
was the least heat resistant, and all bacteria showed greater thermal resistance in whole milk than in saline solution. Despite the differences in the inactivation kinetics of these bacteria in different media, the variability in the final number of bacteria was affected neither by the species nor by the heating substrate, but it did depend on the intensity of the heat treatment. The more severe the heat treatment was, the lower the average number of surviving bacteria but the greater the variability. Our results indicated that the inactivation times for the cells within a population are not identically distributed random variables and that, therefore, the population includes subpopulations of cells with different distributions for the heat resistance parameters. A linear relationship between the variability of the log of the final bacterial concentration and the logarithmic reduction in the size of the bacterial population was found.
Contamination of Soil by Copper Affects the Dynamics, Diversity, and Activity of Soil Bacterial Communities Involved in Wheat Decomposition and Carbon Storage
Authors: Bernard L. , Maron P. A. , Mougel C. , Nowak V. , Lévêque J. , Marol C. , Balesdent J. , Gibiat F. , Ranjard L.
Published: October 2009

A soil microcosm experiment was conducted to evaluate the influence of copper contamination on the dynamics and diversity of bacterial communities actively involved in wheat residue decomposition. In the presence of copper, a higher level of CO
2
release was observed, which did not arise from greater wheat decomposition but from a higher level of stimulation of soil organic matter mineralization (known as the priming effect). Such functional modifications may be related to significant modifications in the diversity of active bacterial populations characterized using the DNA stable-isotope probing approach.
Clonal Relationship among Atypical Enteropathogenic
Escherichia coli
Strains Isolated from Different Animal Species and Humans
Authors: Moura Rodrigo A. , Sircili Marcelo P. , Leomil Luciana , Matté Maria Helena , Trabulsi Luiz R. , Elias Waldir P. , Irino Kinue , Pestana de Castro Antonio F.
Published: October 2009

Forty-nine typical and atypical enteropathogenic
Escherichia coli
(EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.
Occurrence of Putative Pathogenicity Islands in Enterococci from Distinct Species and of Differing Origins
Authors: Semedo-Lemsaddek Teresa , Barreto-Crespo Maria Teresa , Tenreiro Rogério
Published: October 2009

Enterococci isolated from ewe's milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (
Enterococcus faecalis
/non-
E. faecalis
).
Detection and Identification of
tdh
- and
trh
-Positive
Vibrio parahaemolyticus
Strains from Four Species of Cultured Bivalve Molluscs on the Spanish Mediterranean Coast
Authors: Roque Ana , Lopez-Joven Carmen , Lacuesta Beatriz , Elandaloussi Laurence , Wagley Sariqa , Furones M. Dolores , Ruiz-Zarzuela Imanol , de Blas Ignacio , Rangdale Rachel , Gomez-Gil Bruno
Published: October 2009

Presented here is the first report describing the detection of potentially diarrheal
Vibrio parahaemolyticus
strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both
tdh
- and
trh
-positive isolates. Potentially diarrheal
V. parahaemolyticus
strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.
Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays
Authors: van Doorn R. , Klerks M. M. , van Gent-Pelzer M. P. E. , Speksnijder A. G. C. L. , Kowalchuk G. A. , Schoen C. D.
Published: October 2009

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.
Development and Application of Quantitative-PCR Tools for Subgroups of the
Roseobacter
Clade
Authors: Buchan Alison , Hadden Mary , Suzuki Marcelino T.
Published: October 2009

Specific SYBR green-based quantitative-PCR assays targeting conserved regions in the 16S-23S rRNA internal transcribed spacer regions were developed for five subgroups of the environmentally abundant and biogeochemically active
Roseobacter
clade of marine bacteria. The assays were applied to field samples demonstrating their utility in investigations of abundant
Roseobacter
group phylotypes in the environment.
Low Salinity and High-Level UV-B Radiation Reduce Single-Cell Activity in Antarctic Sea Ice Bacteria
Authors: Martin Andrew , Hall Julie , Ryan Ken
Published: October 2009

Experiments simulating the sea ice cycle were conducted by exposing microbes from Antarctic fast ice to saline and irradiance regimens associated with the freeze-thaw process. In contrast to hypersaline conditions (ice formation), the simulated release of bacteria into hyposaline seawater combined with rapid exposure to increased UV-B radiation significantly reduced metabolic activity.
Phylogenetic Diversity and Metabolic Potential Revealed in a Glacier Ice Metagenome
Authors: Simon Carola , Wiezer Arnim , Strittmatter Axel W. , Daniel Rolf
Published: October 2009

The largest part of the Earth's microbial biomass is stored in cold environments, which represent almost untapped reservoirs of novel species, processes, and genes. In this study, the first metagenomic survey of the metabolic potential and phylogenetic diversity of a microbial assemblage present in glacial ice is presented. DNA was isolated from glacial ice of the Northern Schneeferner, Germany. Pyrosequencing of this DNA yielded 1,076,539 reads (239.7 Mbp). The phylogenetic composition of the prokaryotic community was assessed by evaluation of a pyrosequencing-derived data set and sequencing of 16S rRNA genes. The
Proteobacteria
(mainly
Betaproteobacteria
),
Bacteroidetes
, and
Actinobacteria
were the predominant phylogenetic groups. In addition, isolation of psychrophilic microorganisms was performed, and 13 different bacterial isolates were recovered. Analysis of the 16S rRNA gene sequences of the isolates revealed that all were affiliated to the predominant groups. As expected for microorganisms residing in a low-nutrient environment, a high metabolic versatility with respect to degradation of organic substrates was detected by analysis of the pyrosequencing-derived data set. The presence of autotrophic microorganisms was indicated by identification of genes typical for different ways of carbon fixation. In accordance with the results of the phylogenetic studies, in which mainly aerobic and facultative aerobic bacteria were detected, genes typical for central metabolism of aerobes were found. Nevertheless, the capability of growth under anaerobic conditions was indicated by genes involved in dissimilatory nitrate/nitrite reduction. Numerous characteristics for metabolic adaptations associated with a psychrophilic lifestyle, such as formation of cryoprotectants and maintenance of membrane fluidity by the incorporation of unsaturated fatty acids, were detected. Thus, analysis of the glacial metagenome provided insights into the microbial life in frozen habitats on Earth, thereby possibly shedding light onto microbial life in analogous extraterrestrial environments.
High Ratio of Bacteriochlorophyll Biosynthesis Genes to Chlorophyll Biosynthesis Genes in Bacteria of Humic Lakes
Authors: Eiler Alexander , Beier Sara , Säwström Christin , Karlsson Jan , Bertilsson Stefan
Published: October 2009

Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime.
BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR
Authors: Yutin Natalya , Suzuki Marcelino T. , Rosenberg Mira , Rotem Denisse , Madigan Michael T. , Süling Jörg , Imhoff Johannes F. , Béjà Oded
Published: October 2009

To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the
bchY
gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.
Molecular Characterization of a Novel Peroxidase from the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Ogola Henry Joseph Oduor , Kamiike Takaaki , Hashimoto Naoya , Ashida Hiroyuki , Ishikawa Takahiro , Shibata Hitoshi , Sawa Yoshihiro
Published: October 2009

The open reading frame alr1585 of
Anabaena
sp. strain PCC 7120 encodes a heme-dependent peroxidase (
Anabaena
peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in
Escherichia coli
. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min
−1
, respectively. The apparent

K
m

and
k
cat
/

K
m

values for Reactive Blue 5 were 3.6 μM and 1.2 × 10
7
M
−1
s
−1
, respectively, while the apparent

K
m

and
k
cat
/

K
m

values for H
2
O
2
were 5.8 μM and 6.6 × 10
6
M
−1
s
−1
, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.
Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment
Authors: Gittel Antje , Sørensen Ketil Bernt , Skovhus Torben Lund , Ingvorsen Kjeld , Schramm Andreas
Published: October 2009

Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80°C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with
Firmicutes
(
Bacteria
) and
Thermococcales
(
Archaea
). Potential heterotrophic nitrate reducers (
Deferribacterales
) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of
dsrAB
genes revealed that archaeal SRP (
Archaeoglobus
) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (
Desulfacinum
and
Desulfotomaculum
). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [
35
S]sulfate incubations at 80°C. Sulfate reduction was found at 60°C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i)
Archaeoglobus
can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.
Generation of Amylosucrase Variants That Terminate Catalysis of Acceptor Elongation at the Di- or Trisaccharide Stage
Authors: Schneider Jens , Fricke Christin , Overwin Heike , Hofmann Birgit , Hofer Bernd
Published: October 2009

An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites −1 to +3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.
Metabolic Engineering of
Saccharomyces cerevisiae
for Astaxanthin Production and Oxidative Stress Tolerance
Authors: Ukibe Ken , Hashida Keisuke , Yoshida Nobuyuki , Takagi Hiroshi
Published: October 2009

The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast
Saccharomyces cerevisiae
by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast
Xanthophyllomyces dendrorhous
encoding phytoene desaturase (
crtI
product) and bifunctional phytoene synthase/lycopene cyclase (
crtYB
product) resulted in the accumulation of a small amount of β-carotene in
S. cerevisiae
. Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from
S. cerevisiae
(the
BTS1
gene product) increased the intracellular β-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the
X. dendrorhous crtS
gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with
X. dendrorhous
CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the β-carotene-producing yeast cells transformed by the bacterial genes
crtW
and
crtZ
, encoding β-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on
S. cerevisiae
cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.
CO Dehydrogenase Genes Found in Metagenomic Fosmid Clones from the Deep Mediterranean Sea
Authors: Martin-Cuadrado Ana-Belen , Ghai Rohit , Gonzaga Aitor , Rodriguez-Valera Francisco
Published: October 2009

The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years, the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement for heterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metagenomes from the ocean's photic zone, where CO is continuously generated by organic matter photolysis. We have also found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomic library generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databases indicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on the surfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with which this gene was found indicates that this energy-generating metabolism would be at least as important in the bathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or origins deep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organic matter. The identities of the microbes that carry out these processes were not established, but they seem to be representatives of either
Bacteroidetes
or
Chloroflexi
.
Mechanically Ventilated Broiler Sheds: a Possible Source of AerosolizedSalmonella,Campylobacter, andEscherichia coli
Authors: Chinivasagam H. N. , Tran T. , Maddock L. , Gale A. , Blackall P. J.
Published: October 2009

This study assessed the levels of two key pathogens,SalmonellaandCampylobacter, along with the indicator organismEscherichia coliin aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated byE. coli. The typical levels ofE. coliin litter were ∼108CFU g−1and, as a consequence, were in the range of 102to 104CFU m−3in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels.Salmonellawas only present intermittently in litter and at lower levels (103to 105most probable number [MPN] g−1) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m−3) and once outside (2.3 MPN m−3). TheSalmonellaserovars isolated in litter were generally also isolated from aerosols and dust, with theSalmonellaserovars Chester and Sofia being the dominant serovars across these interfaces.Campylobacterwas detected late in the production cycle, in litter at levels of around 107MPN g−1.Campylobacterwas detected only once inside the shed and then at low levels of 2.2 MPN m−3. Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.
Strain-Specific Genotyping ofBifidobacterium animalissubsp.lactisby Using Single-Nucleotide Polymorphisms, Insertions, and Deletions
Authors: Briczinski Elizabeth P. , Loquasto Joseph R. , Barrangou Rodolphe , Dudley Edward G. , Roberts Anastasia M. , Roberts Robert F.
Published: October 2009

Several probiotic strains ofBifidobacterium animalissubsp.lactisare widely supplemented into food products and dietary supplements due to their documented health benefits and ability to survive within the mammalian gastrointestinal tract and acidified dairy products. The strain specificity of these characteristics demands techniques with high discriminatory power to differentiate among strains. However, to date, molecular approaches, such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR, have been ineffective at achieving strain separation due to the monomorphic nature of this subspecies. Previously, sequencing and comparison of twoB. animalissubsp.lactisgenomes (DSMZ 10140 and Bl-04) confirmed this high level of sequence similarity, identifying only 47 single-nucleotide polymorphisms (SNPs) and four insertions and/or deletions (INDELs) between them. In this study, we hypothesized that a sequence-based typing method targeting these loci would permit greater discrimination between strains than previously attempted methods. Sequencing 50 of these loci in 24 strains ofB. animalissubsp.lactisrevealed that a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. In addition, the presence of a nonsynonymous SNP within the gene encoding a putative glucose uptake protein was found to correlate with the ability of certain strains to transport glucose and to grow rapidly in a medium containing glucose as the sole carbon source. The method reported here can be used in clinical, regulatory, and commercial applications requiring identification ofB. animalissubsp.lactisat the strain level.
Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities
Authors: Schloss Patrick D. , Westcott Sarah L. , Ryabin Thomas , Hall Justine R. , Hartmann Martin , Hollister Emily B. , Lesniewski Ryan A. , Oakley Brian B. , Parks Donovan H. , Robinson Courtney J. , Sahl Jason W. , Stres Blaz , Thallinger Gerhard G. , Van Horn David J. , Weber Carolyn F.
Published: October 2009

mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.
Cadherin Fragments from
Anopheles gambiae
Synergize
Bacillus thuringiensis
Cry4Ba's Toxicity against
Aedes aegypti
Larvae
Authors: Park Youngjin , Hua Gang , Abdullah Mohd Amir F. , Rahman Khalidur , Adang Michael J.
Published: October 2009

A peptide from cadherin AgCad1 of
Anopheles gambiae
larvae was reported as a synergist of
Bacillus thuringiensis
subsp.
israelensis
Cry4Ba's toxicity to the
Anopheles
mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to
Aedes aegypti
larvae, but a
Diabrotica virgifera virgifera
cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval
A. aegypti
brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against
A. aegypti
.
Influence of Environmental Gradients on the Abundance and Distribution of
Mycobacterium
spp. in a Coastal Lagoon Estuary
Authors: Jacobs John , Rhodes Matt , Sturgis Brian , Wood Bob
Published: October 2009

Environmental mycobacteria are of increasing concern in terms of the diseases they cause in both humans and animals. Although they are considered to be ubiquitous in aquatic environments, few studies have examined their ecology, and no ecological studies of coastal marine systems have been conducted. This study uses indirect gradient analysis to illustrate the strong relationships that exists between coastal water quality and the abundance of
Mycobacterium
spp. within a U.S. mid-Atlantic embayment.
Mycobacterium
species abundance and water quality conditions (based on 16 physical and chemical variables) were examined simultaneously in monthly samples obtained at 18 Maryland and Virginia coastal bay stations from August 2005 to November 2006 (
n
= 212). A quantitative molecular assay for
Mycobacterium
spp. was evaluated and applied, allowing for rapid, direct enumeration. By using indirect gradient analysis (environmental principal-components analysis), a strong linkage between eutrophic conditions, characterized by low dissolved-oxygen levels and elevated nutrient concentrations, and mycobacteria was determined. More specifically, a strong nutrient response was noted, with all nitrogen components and turbidity measurements correlating positively with abundance (
r
values of >0.30;
P
values of <0.001), while dissolved oxygen showed a strong negative relationship (
r
= −0.38;
P
= 0.01). Logistic regression models developed using salinity, dissolved oxygen, and total nitrogen showed a high degree of concordance (83%). These results suggest that coastal restoration and management strategies designed to reduce eutrophication may also reduce total mycobacteria in coastal waters.
Identification of Two Aflatrem Biosynthesis Gene Loci inAspergillus flavusand Metabolic Engineering ofPenicillium paxilliTo Elucidate Their Function
Authors: Nicholson Matthew J. , Koulman Albert , Monahan Brendon J. , Pritchard Beth L. , Payne Gary A. , Scott Barry
Published: October 2009

Aflatrem is a potent tremorgenic toxin produced by the soil fungusAspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search ofAspergilluscomplete genome sequence data identified putative aflatrem gene clusters in the genomes ofA. flavusandAspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first,ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes,atmG,atmC, andatmM, and the second,ATM2, is telomere distal on chromosome 7 and contains five genes,atmD,atmQ,atmB,atmA, andatmP. Reverse transcriptase PCR inA. flavusdemonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer ofatmPandatmQintoPenicillium paxilli paxPandpaxQdeletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis inA. flavusbased on these reconstitution experiments inP. paxilliand identification of putative intermediates in wild-type cultures ofA. flavus.
Leishmania majorMPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage
Authors: Morales Miguel A. , Pescher Pascale , Späth Gerald F.
Published: October 2009

During the infectious cycle, protozoan parasites of the genusLeishmaniaundergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlyingLeishmaniastage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activatedLeishmania majorprotein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We establishedL. majorandLeishmania donovanilines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenicL. donovanicells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle.
Role of Intracellular Polysaccharide in Persistence of
Streptococcus mutans
Authors: Busuioc Monica , Mackiewicz Katarzyna , Buttaro Bettina A. , Piggot Patrick J.
Published: October 2009

Intracellular polysaccharide (IPS) is accumulated by
Streptococcus mutans
when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of
S. mutans
. Here we show that inactivation of the
glgA
gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of
S. mutans
grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a
glgA
mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.
Structure-Function Analysis of
Escherichia coli
MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme
Authors: Shi Rong , Villarroya Magda , Ruiz-Partida Rafael , Li Yunge , Proteau Ariane , Prado Silvia , Moukadiri Ismaïl , Benítez-Páez Alfonso , Lomas Rodrigo , Wagner John , Matte Allan , Velázquez-Campoy Adrián , Armengod M.-Eugenia , Cygler Miroslaw
Published: October 2009

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of
Escherichia coli
MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.
Differential Modulation ofBurkholderia cenocepaciaVirulence and Energy Metabolism by the Quorum-Sensing Signal BDSF and Its Synthase
Authors: Deng Yinyue , Boon Calvin , Eberl Leo , Zhang Lian-Hui
Published: October 2009

Burkholderia cenocepaciaproduces the moleculecis-2-dodecenoic acid (BDSF), which was previously shown to play a role in antagonism against the fungal pathogenCandida albicansby interfering with its morphological transition. In this study, we show that production of BDSF is under stringent transcriptional control and the molecule accumulates in a cell density-dependent manner, typically found with quorum-sensing (QS) signals.B. cenocepaciamutant strain J2315 with a deleted Bcam0581 gene, which encodes an enzyme essential for BDSF production, exhibited a growth defect in minimal medium but not in rich medium, decreased virulence gene expression, and attenuated virulence in a zebrafish infection model. Exogenous addition of BDSF to the mutant rescues virulence gene expression but fails to restore its growth defect in minimal medium. We show that Bcam0581, but not BDSF, is associated withB. cenocepaciaATP biogenesis. We also provide evidence that some of the BDSF-regulated genes are also controlled by the acyl-homoserine-lactone-dependent QS system and are thus coregulated by two cell-to-cell signaling systems. These data demonstrate that in addition to the role in cross-kingdom signal interference, BDSF and its synthase are also important for the virulence and physiology ofB. cenocepacia.
CbpA: a Polarly Localized Novel Cyclic AMP-Binding Protein in
Pseudomonas aeruginosa
Authors: Endoh Takayuki , Engel Joanne N.
Published: October 2009

In
Pseudomonas aeruginosa
, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the
Escherichia coli
catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in
P. aeruginosa
PAO1, which we herein refer to as CbpA, for
c
AMP-
b
inding
p
rotein
A
. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the
cbpA
promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of
cbpA
did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.
Identification of a Stress-Induced Factor ofCorynebacterineaeThat Is Involved in the Regulation of the Outer Membrane Lipid Composition
Authors: Meniche Xavier , Labarre Cécile , de Sousa-d'Auria Célia , Huc Emilie , Laval Françoise , Tropis Marielle , Bayan Nicolas , Portevin Damien , Guilhot Christophe , Daffé Mamadou , Houssin Christine
Published: October 2009

Corynebacterineaeare gram-positive bacteria that possess a true outer membrane composed of mycolic acids and other lipids. Little is known concerning the modulation of mycolic acid composition and content in response to changes in the bacterial environment, especially temperature variations. To address this question, we investigated the function of the Rv3802c gene, a gene conserved inCorynebacterineaeand located within a gene cluster involved in mycolic acid biosynthesis. We showed that the Rv3802 ortholog is essential inMycobacterium smegmatis, while itsCorynebacterium glutamicumortholog, NCgl2775, is not. We provided evidence that the NCgl2775 gene is transcriptionally induced under heat stress conditions, and while the corresponding protein has no detectable activity under normal growth conditions, the increase in its expression triggers an increase in mycolic acid biosynthesis concomitant with a decrease in phospholipid content. We demonstrated that these lipid modifications are part of a larger outer membrane remodeling that occurs in response to exposure to a moderately elevated temperature (42°C). In addition to showing an increase in the ratio of saturated corynomycolates to unsaturated corynomycolates, our results strongly suggested that the balance between mycolic acids and phospholipids is modified inside the outer membrane following a heat challenge. Furthermore, we showed that these lipid modifications help the bacteria to protect against heat damage. The NCgl2775 protein and its orthologs thus appear to be a protein family that plays a role in the regulation of the outer membrane lipid composition ofCorynebacterineaeunder stress conditions. We therefore propose to name this protein family the envelope lipids regulation factor (ElrF) family.
Phylogenetic relationships in the tribes Schizopetaleae and Thelypodieae (Brassicaceae) based on nuclear ribosomal ITS region and plastidndhF DNA sequences
Authors: Warwick Suzanne I. , Sauder Connie A. , Mayer Michael S. , Al-Shehbaz Ihsan A.
Published: October 2009

Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region and plastid gene ndhF of 95 species, represented by 147 accessions, were used to determine the tribal limits, monophyly status, and phylogenetic intra-tribal relationships of genera within the New World tribe Schizopetaleae (formerly Thelypodieae; Brassicaceae). Maximum parsimony, Bayesian, and maximum-likelihood analyses all support the separate recognition of three distinct and generally well-supported clades. The largest corresponds to the North–South American Thelypodieae (27 genera: Catadysia , Caulanthus , Chaunanthus , Chilocardamum , Chlorocrambe , Coelophragmus , Dictyophragmus , Dryopetalon , Englerocharis , Hesperidanthus , Mostacillastrum , Neuontobotrys , Polypsecadium , Pringlea , Pterygiosperma , Romanschulzia , Sibara , Sibaropsis , Stanleya , Streptanthella , Streptanthus , Thelypodiopsis , Thelypodium , Thysanocarpus , Warea , Weberbauera , and Werdermannia ). The remaining six South American Schizopetaleae genera were divided into two clades: SCHIZ I of four genera ( Aschersoniodoxa , Brayopsis , Eudema , and Onuris ) and SCHIZ II Schizopetaleae s. str.: Mathewsia , and Schizopetalon ). Resolution within the Thelypodieae clade was limited in both the ITS- and ndhF-based phylogenies. Based on ITS sequence data, elements of the Old World tribe Brassiceae were sister to the Thelypodieae, whereas the ndhF data strongly supported the Sisymbrieae as sister to the Thelypodieae, and the Brassiceae as sister to both tribes. Sister groups to the Schizopetaleae clades I and II were not clearly resolved. Morphological and cytological data support the separation of these three clades. In SCHIZ II, the sepals are always erect to form a closed tube, and the trichomes are mostly dendritic, although other types also occur. In both the Thelypodieae and SCHIZ I clades, the sepals are variously oriented but never form a closed tube, whereas the trichomes are either absent, or simple, forked, or rarely dendritic. The latter clade differs from both SCHIZ II and Thelypodieae by lacking trichomes or rarely having forked ones and by having exclusively basal, entire leaves. Thelypodieae circumscription should be restricted in future to the Thelypodieae clade, Schizopetaleae circumscription to the SCHIZ II clade, while a new tribe is needed for the SCHIZ I clade.
The effects of temperature, photoperiod, and vernalization on regrowth and flowering competence inEuphorbia esula(Euphorbiaceae) crown buds
Authors: Foley M. E. , Anderson J. V. , Horvath D. P.
Published: October 2009

The herbaceous perennial weed Euphorbia esula  L. (Euphorbiaceae) reproduces by vegetative and sexual means, characteristics that are key to its persistence and survival. In this study, we examined environmental effects on regrowth and flowering under controlled conditions to further validate field observations and set the stage for the future identification of molecular mechanisms involved in the regulation of these processes. Shoot cuttings were exposed to different combinations of decreasing temperatures, decreasing photoperiods, and vernalization, in growth chambers. Subsequently, shoots were removed and regrowth and flowering from new shoots were monitored in a warm temperature greenhouse under long-day conditions. Vernalization alone has no effect on regrowth and flowering. Plants required decreasing temperature followed by vernalization for rapid regrowth and flowering. Decreasing photoperiod at a constant temperature with or without vernalization had no significant effect on regrowth and flowering. In conjunction with previous field research, the results suggest that a gradually decreasing temperature is required as one of the components for flowering competence and vernalization is determinate for reproductive development under long-day conditions.
Patterns of defence gene expression in the tomato–Verticilliuminteraction
Authors: Robb Jane , Castroverde Christian D.M. , Shittu Hakeem O. , Nazar Ross N.
Published: October 2009

In a tomato plant infected by Verticillium dahliae , race 1, compatibility or incompatibility appears to be determined in the stem, but little is known about the genes that either regulate or effect critical cellular events. In the present study, microarray and RT-PCR analyses were used to assess changes in tomato mRNA populations during both interactions. Initially, a commercially available DNA chip was used to screen gene expression at a single critical time point after inoculation of resistant and susceptible plants. From the results, the most-affected genes were selected to develop a tomato Verticillium response (TVR) DNA chip for detailed analyses of gene expression for 15 d after inoculation. Taken together, over half of the genes on the TVR array exhibited one of three distinct patterns of change, one reflecting a resistant phenotype and two being consistent with a susceptible phenotype. Of particular interest was a cluster of strongly expressed genes belonging to groups 2 and 3 that appeared to be co-ordinately down regulated in infected resistant plants relative to susceptible. Many of these genes encode pathogenesis related (PR) proteins. The data demonstrate that even though complex, the biological system can be standardized sufficiently to allow the reproducible analysis of gene expression in a whole plant system and provide patterns of transcriptional variation that can be used to assess the significance of specific genes in pathogenesis and resistance.