bluuebunny/crossref_metadata_2025 huggingface dataset:
Parenting influences on bullying and victimization
Authors: Baldry Anna C. , Farrington David P.
Published: June 2011

Purpose. The main aim of this research was to investigate the parental styles and personal characteristics of bullies and victims, and to disentangle factors related to bully/victims from factors related to children who were only bullies or only victims.

Method. A self‐report questionnaire on bullying was completed by 113 girls and 125 boys aged 11–14 years in a middle school in Rome.

Results. Over half of all students had bullied others in the previous three months, and nearly half had been victimized. About a quarter of all students were both bullies and victims. Bullies tended to be male and to have low pro‐social behaviour, but these were largely characteristics of children who were only bullies. Victims tended to be female and to have low self‐esteem, but these were largely characteristics of children who were only victims. Children who were both bullies and victims tended to have authoritarian parents, but these were largely characteristics of bully/victims.

Conclusions. It is important to study only bullies, only victims and bully/victims, as well as bullies and victims in general. Personal characteristics were related to only bullies or only victims, whereas parental styles were more related to bully/victims.
Lactobacilli Antagonize the Growth, Motility, and Adherence of Brachyspira pilosicoli: a Potential Intervention against Avian Intestinal Spirochetosis
Authors: Mappley Luke J. , Tchórzewska Monika A. , Cooley William A. , Woodward Martin J. , La Ragione Roberto M.
Published: June 2011

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic
Brachyspira
species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in
Brachyspira
, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of
Brachyspira
they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two
Lactobacillus
strains,
Lactobacillus reuteri
LM1 and
Lactobacillus salivarius
LM2, suppressed the growth of
Brachyspira pilosicoli
B2904 in a pH-dependent manner. In
in vitro
adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal
in vitro
organ culture (IVOC) model, the adherence and invasion of
B. pilosicoli
in epithelial cells were reduced significantly by the presence of lactobacilli (
P
< 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of
B. pilosicoli
, and electron microscopic observations indicated that contact between the lactobacilli and
Brachyspira
was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for
B. pilosicoli
to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.
Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow
Authors: Tomaru Yuji , Takao Yoshitake , Suzuki Hidekazu , Nagumo Tamotsu , Koike Kanae , Nagasaki Keizo
Published: June 2011

Diatoms are one of the most significant primary producers in the ocean, and the importance of viruses as a potential source of mortality for diatoms has recently been recognized. Thus far, eight different diatom viruses infecting the genera
Rhizosolenia
and
Chaetoceros
have been isolated and characterized to different extents. We report the isolation of a novel diatom virus (ClorDNAV), which causes the lysis of the bloom-forming species
Chaetoceros lorenzianus
, and show its physiological, morphological, and genomic characteristics. The free virion was estimated to be ∼34 nm in diameter. The arrangement of virus particles appearing in cross-section was basically a random aggregation in the nucleus. Occasionally, distinctive formations such as a ring-like array composed of 9 or 10 spherical virions or a centipede-like array composed of rod-shaped particles were also observed. The latent period and the burst size were estimated to be <48 h and 2.2 × 10
4
infectious units per host cell, respectively. ClorDNAV harbors a covalently closed circular single-stranded DNA (ssDNA) genome (5,813 nucleotides [nt]) that includes a partially double-stranded DNA region (979 nt). At least three major open reading frames were identified; one showed a high similarity to putative replicase-related proteins of the other ssDNA diatom viruses,
Chaetoceros salsugineum
DNA virus (previously reported as CsNIV) and
Chaetoceros tenuissimus
DNA virus. ClorDNAV is the third member of the closed circular ssDNA diatom virus group, the genus
Bacilladnavirus
.
Molecular Characterization of Noroviruses and Rotaviruses Involved in a Large Outbreak of Gastroenteritis in Northern Italy
Authors: Di Bartolo Ilaria , Monini Marina , Losio Marina Nadia , Pavoni Enrico , Lavazza Antonio , Ruggeri Franco Maria
Published: June 2011

Noroviruses and rotaviruses from a gastroenteritis outbreak affecting >300 people near Garda Lake (Northern Italy) in 2009 were investigated. Characterization of viruses from 40 patient stool samples and 5 environmental samples identified three distinct rotavirus and five norovirus genotypes; two of the latter were detected in both patient and environmental samples.
Common Ancestry and Novel Genetic Traits of Francisella novicida-Like Isolates from North America and Australia as Revealed by Comparative Genomic Analyses
Authors: Siddaramappa Shivakumara , Challacombe Jean F , Petersen Jeannine M , Pillai Segaran , Hogg Geoff , Kuske Cheryl R
Published: June 2011

Francisella novicida
is a close relative of
Francisella tularensis
, the causative agent of tularemia. The genomes of
F. novicida
-like clinical isolates 3523 (Australian strain) and Fx1 (Texas strain) were sequenced and compared to
F. novicida
strain U112 and
F. tularensis
strain Schu S4. The strain 3523 chromosome is 1,945,310 bp and contains 1,854 protein-coding genes. The strain Fx1 chromosome is 1,913,619 bp and contains 1,819 protein-coding genes. NUCmer analyses revealed that the genomes of strains Fx1 and U112 are mostly colinear, whereas the genome of strain 3523 has gaps, translocations, and/or inversions compared to genomes of strains Fx1 and U112. Using the genome sequence data and comparative analyses with other members of the genus
Francisella
, several strain-specific genes that encode putative proteins involved in RTX toxin production, polysaccharide biosynthesis/modification, thiamine biosynthesis, glucuronate utilization, and polyamine biosynthesis were identified. The RTX toxin synthesis and secretion operon of strain 3523 contains four open reading frames (ORFs) and was named
rtxCABD
. Based on the alignment of conserved sequences upstream of operons involved in thiamine biosynthesis from various bacteria, a putative THI box was identified in strain 3523. The glucuronate catabolism loci of strains 3523 and Fx1 contain a cluster of nine ORFs oriented in the same direction that appear to constitute an operon. Strains U112 and Schu S4 appeared to have lost the loci for RTX toxin production, thiamine biosynthesis, and glucuronate utilization as a consequence of host adaptation and reductive evolution. In conclusion, comparative analyses provided insights into the common ancestry and novel genetic traits of these strains.
Biochemical Analyses of Multiple Endoxylanases from the Rumen Bacterium Ruminococcus albus 8 and Their Synergistic Activities with Accessory Hemicellulose-Degrading Enzymes
Authors: Moon Young Hwan , Iakiviak Michael , Bauer Stefan , Mackie Roderick I. , Cann Isaac K. O.
Published: June 2011

Ruminococcus albus
8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of
R. albus
8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of
R. albus
8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a β-xylosidase, an α-
l
-arabinofuranosidase, and an α-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by
R. albus
8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.
Extent and Variation of Phage-Borne Bacterial 16S rRNA Gene Sequences in Wastewater Environments
Authors: Del Casale Antonio , Flanagan Paul V. , Larkin Michael J. , Allen Christopher C. R. , Kulakov Leonid A.
Published: June 2011

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of
Proteobacteria
,
Bacteroidetes
, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.
Tuning the Specificity of the Recombinant Multicomponent Toluene
o
-Xylene Monooxygenase from Pseudomonas sp. Strain OX1 for the Biosynthesis of Tyrosol from 2-Phenylethanol
Authors: Notomista Eugenio , Scognamiglio Roberta , Troncone Luca , Donadio Giuliana , Pezzella Alessandro , Di Donato Alberto , Izzo Viviana
Published: June 2011

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene
o
-xylene monooxygenase (ToMO) is a BMM isolated from
Pseudomonas
sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of
Escherichia coli
strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.
High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes
Authors: Kütahya Oylum Erkus , Starrenburg Marjo J. C. , Rademaker Jan L. W. , Klaassen Corné H. W. , van Hylckama Vlieg Johan E. T. , Smid Eddy J. , Kleerebezem Michiel
Published: June 2011

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related
Lactococcus lactis
strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82
L. lactis
strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of
L. lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
type-strain-like genotypes and a third novel genomic lineage rooted from the
L. lactis
subsp.
lactis
genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated
L. lactis
subsp.
cremoris
phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of
L. lactis
subsp.
lactis
phenotype from
L. lactis
subsp.
cremoris
phenotype strains within the same
L. lactis
subsp.
cremoris
type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.
Biodiversity and Emerging Biogeography of the Neutrophilic Iron-Oxidizing Zetaproteobacteria
Authors: McAllister Sean M. , Davis Richard E. , McBeth Joyce M. , Tebo Bradley M. , Emerson David , Moyer Craig L.
Published: June 2011

Members of the neutrophilic iron-oxidizing candidate class
Zetaproteobacteria
have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length
Zetaproteobacteria
small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel
Zetaproteobacteria
isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the
Proteobacteria
and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of
Zetaproteobacteria
biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the
Zetaproteobacteria
have a strong biogeographic signal.
Advanced Multiplex PCR Assay for Differentiation of Brucella Species
Authors: Kang Sung-Il , Her Moon , Kim Jong Wan , Kim Ji-Yeon , Ko Kyung Yuk , Ha Yun-Mi , Jung Suk Chan
Published: June 2011

Two new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates
Brucella canis
and
Brucella microti
from
Brucella suis
strains and also may differentiate all of the 10
Brucella
species.
Calicivirus Removal in a Membrane Bioreactor Wastewater Treatment Plant
Authors: Sima Laura C. , Schaeffer Julien , Le Saux Jean-Claude , Parnaudeau Sylvain , Elimelech Menachem , Le Guyader Françoise S.
Published: June 2011

To evaluate membrane bioreactor wastewater treatment virus removal, a study was conducted in southwest France. Samples collected from plant influent, an aeration basin, membrane effluent, solid sludge, and effluent biweekly from October 2009 to June 2010 were analyzed for calicivirus (norovirus and sapovirus) by real-time reverse transcription-PCR (RT-PCR) using extraction controls to perform quantification. Adenovirus and
Escherichia coli
also were analyzed to compare removal efficiencies. In the influent, sapovirus was always present, while the norovirus concentration varied temporally, with the highest concentration being detected from February to May. All three human norovirus genogroups (GI, GII, and GIV) were detected in effluent, but GIV was never detected in effluent; GI and GII were detected in 50% of the samples but at low concentrations. In the effluent, sapovirus was identified only once. An adenovirus titer showing temporal variation in influent samples was identified only twice in effluent.
E. coli
was always below the limit of detection in the effluent. Overall, the removal of calicivirus varied from 3.3 to greater than 6.8 log units, with no difference between the two main genogroups. Our results also demonstrated that the viruses are blocked by the membrane in the treatment plant and are removed from the plant as solid sludge.
Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture
Authors: Ghafoor Aamir , Hay Iain D. , Rehm Bernd H. A.
Published: June 2011

Pseudomonas aeruginosa
is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 Δ
pslA
Δ
alg8
overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of
P. aeruginosa
.
An Alkaline Phosphatase/Phosphodiesterase, PhoD, Induced by Salt Stress and Secreted Out of the Cells of Aphanothece halophytica, a Halotolerant Cyanobacterium
Authors: Kageyama Hakuto , Tripathi Keshawanand , Rai Ashwani K. , Cha-um Suriyan , Waditee-Sirisattha Rungaroon , Takabe Teruhiro
Published: June 2011

Alkaline phosphatases (APases) are important enzymes in organophosphate utilization. Three prokaryotic APase gene families, PhoA, PhoX, and PhoD, are known; however, their functional characterization in cyanobacteria largely remains to be clarified. In this study, we cloned the
phoD
gene from a halotolerant cyanobacterium,
Aphanothece halophytica
(

phoD
Ap

). The deduced protein, PhoD

Ap

, contains Tat consensus motifs and a peptidase cleavage site at the N terminus. The PhoD

Ap

enzyme was activated by Ca
2+
and exhibited APase and phosphodiesterase (APDase) activities. Subcellular localization experiments revealed the secretion and processing of PhoD

Ap

in a transformed cyanobacterium. Expression of the

phoD
Ap

gene in
A. halophytica
cells was upregulated not only by phosphorus (P) starvation but also under salt stress conditions. Our results suggest that
A. halophytica
cells possess a PhoD that participates in the assimilation of P under salinity stress.
In VitroFermentation of Linear and α-1,2-Branched Dextrans by the Human Fecal Microbiota
Authors: Sarbini Shahrul R. , Kolida Sofia , Naeye Thierry , Einerhand Alexandra , Brison Yoann , Remaud-Simeon Magali , Monsan Pierre , Gibson Glenn R. , Rastall Robert A.
Published: June 2011

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescentin situhybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases inBifidobacteriumpopulations. The degree of α-1,2 branching did not influence theBifidobacteriumpopulations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for theBacteroides-Prevotellagroup, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and theFaecalibacterium prausnitziigroup, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.
The Transcriptional Response of Listeria monocytogenes during Adaptation to Growth on Lactate and Diacetate Includes Synergistic Changes That Increase Fermentative Acetoin Production
Authors: Stasiewicz Matthew J. , Wiedmann Martin , Bergholz Teresa M.
Published: June 2011

The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of
Listeria monocytogenes
. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two
L. monocytogenes
strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of
L. monocytogenes
.
Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp.
lactis
Strains of the L. lactis subsp.
lactis
and L. lactis subsp.
cremoris
Genotypes, Isolated from Starter-Free Cheeses Made of Raw Milk
Authors: Fernández Elena , Alegría Ángel , Delgado Susana , Martín M. Cruz , Mayo Baltasar
Published: June 2011

Twenty
Lactococcus lactis
strains with an
L. lactis
subsp.
lactis
phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the
L. lactis
subsp.
lactis
and
L. lactis
subsp.
cremoris
genotypes (
lactis
and
cremoris
genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the
cremoris
genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely,
atpA
,
rpoA
,
pheS
,
pepN
,
bcaT
,
pepX
, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the
cremoris
or
lactis
genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the
cremoris
group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical
cremoris
genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the
cremoris
genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the
lactis
and
cremoris
genotypes of phenotypic
Lactococcus lactis
subsp.
lactis
actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.
Development of a Method for Markerless Gene Deletion in Pseudomonas putida
Authors: Graf Nadja , Altenbuchner Josef
Published: June 2011

We developed a negative counterselection system for
Pseudomonas putida
based on uracil phosphoribosyltransferase (UPRTase) and sensitivity against the antimetabolite 5-fluorouracil (5-FU). We constructed a
P. putida
strain that is resistant to 5-FU and constructed vectors for the deletion of the surface adhesion protein gene, the flagellum biosynthesis operon, and two endonuclease genes. The genes were efficiently disrupted and left a markerless chromosomal in-frame deletion.
Analysis of Insertion Sequences in Thermophilic Cyanobacteria: Exploring the Mechanisms of Establishing, Maintaining, and Withstanding High Insertion Sequence Abundance
Authors: Nelson William C. , Wollerman Lori , Bhaya Devaki , Heidelberg John F.
Published: June 2011

Insertion sequences (ISs) are simple mobile genetic elements capable of relocating within a genome. Through this transposition activity, they are known to create mutations which are mostly deleterious to the cell, although occasionally they are beneficial. Two closely related isolates of thermophilic
Synechococcus
species from hot spring microbial mats are known to harbor a large number of diverse ISs. To explore the mechanism of IS acquisition within natural populations and survival in the face of high IS abundance, we examined IS content and location in natural populations of
Synechococcus
by comparing metagenomic data to the genomes of fully sequenced cultured isolates. The observed IS distribution in the metagenome was equivalent to the distribution in the isolates, indicating that the cultured isolates are appropriate models for the environmental population. High sequence conservation between IS families shared between the two isolates suggests that ISs are able to move between individuals within populations and between species via lateral gene transfer, consistent with models for IS family accumulation. Most IS families show evidence of recent activity, and interruption of critical genes in some individuals was observed, demonstrating that transposition is an ongoing mutational force in the populations.
Addition of Genes for Cellobiase and Pectinolytic Activity in Escherichia coli for Fuel Ethanol Production from Pectin-Rich Lignocellulosic Biomass
Authors: Edwards Meredith C. , Henriksen Emily DeCrescenzo , Yomano Lorraine P. , Gardner Brian C. , Sharma Lekh N. , Ingram Lonnie O. , Doran Peterson Joy
Published: June 2011

Ethanologenic
Escherichia coli
strain KO11 was sequentially engineered to contain the
Klebsiella oxytoca
cellobiose phosphotransferase genes (
casAB
) as well as a pectate lyase (
pelE
) from
Erwinia chrysanthemi
, yielding strains LY40A (
casAB
) and JP07 (
casAB
pelE
), respectively. To obtain an effective secretion of PelE, the Sec-dependent pathway
out
genes from
E. chrysanthemi
were provided on a cosmid to strain JP07 to construct strain JP07C. Finally, oligogalacturonide lyase (
ogl
) from
E. chrysanthemi
was added to produce strain JP08C.
E. coli
strains LY40A, JP07, JP07C, and JP08C possessed significant cellobiase activity in cell lysates, while only strains JP07C and JP08C demonstrated extracellular pectate lyase activity. Fermentations conducted by using a mixture of pure sugars representative of the composition of sugar beet pulp (SBP) showed that strains LY40A, JP07, JP07C, and JP08C were able to ferment cellobiose, resulting in increased ethanol production from 15 to 45% in comparison to that of KO11. Fermentations with SBP at very low fungal enzyme loads during saccharification revealed significantly higher levels of ethanol production for LY40A, JP07C, and JP08C than for KO11. JP07C ethanol yields were not considerably higher than those of LY40A; however, oligogalacturonide polymerization studies showed an increased breakdown of biomass to small-chain (degree of polymerization, ≤6) oligogalacturonides. JP08C achieved a further breakdown of polygalacturonate to monomeric sugars, resulting in a 164% increase in ethanol yields compared to those of KO11. The addition of commercial pectin methylesterase (PME) further increased JP08C ethanol production compared to that of LY40A by demethylating the pectin for enzymatic attack by pectin-degrading enzymes.
Identification and Characterization of Genes Required for Cell-to-Cell Fusion in Neurospora crassa
Authors: Fu Ci , Iyer Priyadarshini , Herkal Amrita , Abdullah Julia , Stout Angela , Free Stephen J.
Published: June 2011

A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the
Neurospora crassa
single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (
mik-1
,
mek-1
,
mak-1
,
nrc-1
,
mek-2
,
mak-2
,
rac-1
,
pp2A
,
so/ham-1
,
ham-2
,
ham-3
,
ham-5
,
ham-9
, and
mob3
). The screening experiments also identified four transcription factors that are required for cell fusion (
adv-1
,
ada-3
,
rco-1
, and
snf5
). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (
amph-1
,
ham-10
,
pkr1
). Three of the genes identified by the screening procedure,
ham-6
,
ham-7
, and
ham-8
, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion.
Genomic Characterization of Neoparamoeba pemaquidensis (Amoebozoa) and Its Kinetoplastid Endosymbiont
Authors: Tanifuji Goro , Kim Eunsoo , Onodera Naoko T. , Gibeault Rebecca , Dlutek Marlena , Cawthorn Richard J. , Fiala Ivan , Lukeš Julius , Greenwood Spencer J. , Archibald John M.
Published: June 2011

We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan
Neoparamoeba pemaquidensis
. The genome of this “
Ichthyobodo
-related organism” was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp.
The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans
Authors: Hall Rebecca A. , Turner Kara J. , Chaloupka James , Cottier Fabien , De Sordi Luisa , Sanglard Dominique , Levin Lonny R. , Buck Jochen , Mühlschlegel Fritz A.
Published: June 2011

Living as a commensal,
Candida albicans
must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on
C. albicans
, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C
12
-homoserine lactone, a quorum-sensing molecule secreted by
Pseudomonas aeruginosa
, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the
Candida
adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the
C. albicans
hyphal repressor, Sfl1p. Deletion of
SFL1
did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in
C. albicans
is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the
Burkholderia cenocepacia
diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C
12
-homoserine lactone, may be used by other quorum-sensing molecules.
Functional Analyses of Two Acetyl Coenzyme A Synthetases in the Ascomycete Gibberella zeae
Authors: Lee Seunghoon , Son Hokyoung , Lee Jungkwan , Min Kyunghun , Choi Gyung Ja , Kim Jin-Cheol , Lee Yin-Won
Published: June 2011

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) inGibberella zeaerevealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACSgenesACS1andACS2), to identify alternative acetyl-CoA production mechanisms for ACL. TheACS1deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene,ACS2, has accessorial functions forACS1and has compensatory functions forACLas a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle ofG. zeaeand has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.
Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity
Authors: Basenko Evelina , Topcu Zeki , McEachern Michael J.
Published: June 2011

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for
RAD52
for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.
The Essential β-Barrel Assembly Machinery Complex Components BamD and BamA Are Required for Autotransporter Biogenesis
Authors: Rossiter Amanda E. , Leyton Denisse L. , Tveen-Jensen Karina , Browning Douglas F. , Sevastsyanovich Yanina , Knowles Timothy J. , Nichols Katie B. , Cunningham Adam F. , Overduin Michael , Schembri Mark A. , Henderson Ian R.
Published: June 2011

Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.
Genome-Wide Identification of
In Vivo
Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum
Authors: Toyoda Koichi , Teramoto Haruhiko , Inui Masayuki , Yukawa Hideaki
Published: June 2011

Corynebacterium glutamicum
GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the
C. glutamicum
genome are predicted
in silico
, studies on the physiological function of GlxR have been hindered by the severe growth defects of a
glxR
mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as
in vivo
GlxR-binding sites.
In vitro
binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of
cyaB
, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the
cyaB
mutant, allowing the
cyaB
mutant to grow much better than the
glxR
mutant.
Importance of a Hydrophobic Pocket for Peptide Binding in Lactococcal OppA
Authors: Berntsson Ronnie P.-A. , Thunnissen Andy-Mark W. H. , Poolman Bert , Slotboom Dirk-Jan
Published: June 2011

Lactococcal oligopeptide-binding protein A (OppA) binds peptides with widely varied lengths and sequences. We previously hypothesized that a hydrophobic pocket in OppA preferentially binds a hydrophobic peptide side chain and thus determines its binding register. Two crystal structures of OppA with different nonapeptides now indeed show binding in different registers.
Crystal Structure of the Pseudomonas aeruginosa Virulence Factor Regulator
Authors: Cordes Timothy J. , Worzalla Gregory A. , Ginster Aaron M. , Forest Katrina T.
Published: June 2011

Virulence factor regulator (Vfr) enhances
Pseudomonas aeruginosa
pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prevents the growth of
P. aeruginosa
.
Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403
Authors: Pudlik Agata M. , Lolkema Juke S.
Published: June 2011

Citrate metabolism in resting cells of
Lactococcus lactis
IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706–714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of
L. lactis
when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of
l
-lactate, indicating exchange between oxaloacetate and
l
-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of
L. lactis
is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.
RcnB Is a Periplasmic Protein Essential for Maintaining Intracellular Ni and Co Concentrations in Escherichia coli
Authors: Blériot Camille , Effantin Géraldine , Lagarde Florence , Mandrand-Berthelot Marie-Andrée , Rodrigue Agnès
Published: June 2011

Nickel and cobalt are both essential trace elements that are toxic when present in excess. The main resistance mechanism that bacteria use to overcome this toxicity is the efflux of these cations out of the cytoplasm. RND (resistance-nodulation-cell division)- and MFS (major facilitator superfamily)-type efflux systems are known to export either nickel or cobalt. The RcnA efflux pump, which belongs to a unique family, is responsible for the detoxification of Ni and Co inEscherichia coli. In this work, the role of the geneyohN, which is located downstream ofrcnA, is investigated.yohNis cotranscribed withrcnA, and its expression is induced by Ni and Co. Surprisingly, in contrast to the effect of deletingrcnA, deletion ofyohNconferred enhanced resistance to Ni and Co inE. coli, accompanied by decreased metal accumulation. We show that YohN is localized to the periplasm and does not bind Ni or Co ions directly. Physiological and genetic experiments demonstrate that YohN is not involved in Ni import. YohN is conserved among proteobacteria and belongs to a new family of proteins; consequently,yohNhas been renamedrcnB.We show that the enhanced resistance ofrcnBmutants to Ni and Co and their decreased Ni and Co intracellular accumulation are linked to the greater efflux of these ions in the absence ofrcnB. Taken together, these results suggest that RcnB is required to maintain metal ion homeostasis, in conjunction with the efflux pump RcnA, presumably by modulating RcnA-mediated export of Ni and Co to avoid excess efflux of Ni and Co ions via an unknown novel mechanism.
Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus
Authors: Gómez Cristina , Horna Dina H. , Olano Carlos , Palomino-Schätzlein Martina , Pineda-Lucena Antonio , Carbajo Rodrigo J. , Braña Alfredo F. , Méndez Carmen , Salas José A.
Published: June 2011

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the
Streptomyces lydicus
streptolydigin gene cluster
slgE1-slgE2-slgE3
are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to
slgE3
, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in
S. lydicus
. The expression of
slgE3
is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of
slgE1-slgE2
led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either
slgE3
or
slgE1-slgE2
in
S. lydicus
led to an increase in the yield of streptolydigin.