bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Level Molecular Diversity of Copper-Zinc Superoxide Dismutase Genes among and within Species of Arbuscular Mycorrhizal Fungi
Authors: Corradi Nicolas , Ruffner Beat , Croll Daniel , Colard Alexandre , Horák Ales , Sanders Ian R. 
Published: February 2009

ABSTRACTIn the ecologically important arbuscular mycorrhizal fungi (AMF),Sod1encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at theSod1locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of theSod1gene in six AMF species, as well as 14Glomus intraradicesisolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. TheSod1gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy inG. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates ofG. intraradicesfrom a natural population harbor very divergent orthologousSod1sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.
Quantitative Measurement of Varicella-Zoster Virus Infection by Semiautomated Flow Cytometry
Authors: Gates Irina V. , Zhang Yuhua , Shambaugh Cindy , Bauman Meredith A. , Tan Charles , Bodmer Jean-Luc 
Published: February 2009

ABSTRACT
Varicella-zoster virus (VZV; human herpesvirus 3) is the etiological cause of chickenpox and, upon reactivation from latency, zoster. Currently, vaccines are available to prevent both diseases effectively. A critical requirement for the manufacturing of safe and potent vaccines is the measurement of the biological activity to ensure proper dosing and efficacy, while minimizing potentially harmful secondary effects induced by immunization. In the case of live virus-containing vaccines, such as VZV-containing vaccines, biological activity is determined using an infectivity assay in a susceptible cellular host in vitro. Infectivity measurements generally rely on the enumeration of plaques by visual inspection of an infected cell monolayer. These plaque assays are generally very tedious and labor intensive and have modest throughput and high associated variability. In this study, we have developed a flow cytometry assay to measure the infectivity of the attenuated vaccine strain (vOka/Merck) of VZV in MRC-5 cells with improved throughput. The assay is performed in 96-well tissue culture microtiter plates and is based on the detection and quantification of infected cells expressing VZV glycoproteins on their surfaces. Multiple assay parameters have been investigated, including specificity, limit of detection, limit of quantification, range of linear response, signal-to-noise ratio, and precision. This novel assay appears to be in good concordance with the classical plaque assay results and therefore provides a viable, higher-throughput alternative to the plaque assay.
NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin
Authors: Kontkanen Hanna , Westerholm-Parvinen Ann , Saloheimo Markku , Bailey Michael , Rättö Marjaana , Mattila Ismo , Mohsina Marzia , Kalkkinen Nisse , Nakari-Setälä Tiina , Buchert Johanna 
Published: February 2009

ABSTRACTThree cutinase gene-like genes from the basidiomyceteCoprinopsis cinerea(Coprinus cinereus) found with a similarity search were cloned and expressed inTrichoderma reeseiunder the control of an induciblecbh1promoter. The selected transformants of all three polyesterase constructs showed activity withp-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−lpurified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2to C10) fatty acid esters ofp-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.
Characterization ofrrdA, a TetR Family Protein Gene Involved in the Regulation of Secondary Metabolism inStreptomyces coelicolor
Authors: Ou Xijun , Zhang Bo , Zhang Lin , Zhao Guoping , Ding Xiaoming 
Published: February 2009

ABSTRACTStreptomycesnot only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production inStreptomyces coelicolorM145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized generrdA(regulator ofredD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, therrdAnull mutant exhibited increased Red production and decreased Act production. A high level ofrrdAexpression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controllingredDmRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene,actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.
High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes
Authors: Karsi Attila , Gülsoy Nagihan , Corb Erin , Dumpala Pradeep R. , Lawrence Mark L. 
Published: February 2009

ABSTRACTA high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogenEdwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to theE. ictalurigenome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.
A Vaccine against Rumen Methanogens Can Alter the Composition of Archaeal Populations
Authors: Williams Yvette J. , Popovski Sam , Rea Suzanne M. , Skillman Lucy C. , Toovey Andrew F. , Northwood Korinne S. , Wright André-Denis G. 
Published: February 2009

ABSTRACT

The objectives of this study were to formulate a vaccine based upon the different species/strains of methanogens present in sheep intended to be immunized and to determine if a targeted vaccine could be used to decrease the methane output of the sheep. Two 16S rRNA gene libraries were used to survey the methanogenic archaea in sheep prior to vaccination, and methanogens representing five phylotypes were found to account for >52% of the different species/strains of methanogens detected. A vaccine based on a mixture of these five methanogens was then formulated, and 32 sheep were vaccinated on days 0, 28, and 103 with either a control or the anti-methanogen vaccine. Enzyme-linked immunosorbent assay analysis revealed that each vaccination with the anti-methanogen formulation resulted in higher specific immunoglobulin G titers in plasma, saliva, and rumen fluid. Methane output levels corrected for dry-matter intake for the control and treatment groups were not significantly different, and real-time PCR data also indicated that methanogen numbers were not significantly different for the two groups after the second vaccination. However, clone library data indicated that methanogen diversity was significantly greater in sheep receiving the anti-methanogen vaccine and that the vaccine may have altered the composition of the methanogen population. A correlation between 16S rRNA gene sequence relatedness and cross-reactivity for the methanogens (
R
2
= 0.90) also exists, which suggests that a highly specific vaccine can be made to target specific strains of methanogens and that a more broad-spectrum approach is needed for success in the rumen. Our data also suggest that methanogens take longer than 4 weeks to adapt to dietary changes and call into question the validity of experimental results based upon a 2- to 4-week acclimatization period normally observed for bacteria.
Ser67Asp and His68Asp Substitutions in
Candida parapsilosis
Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction
Authors: Zhang Rongzhen , Xu Yan , Sun Ying , Zhang Wenchi , Xiao Rong 
Published: February 2009

ABSTRACT

A short-chain carbonyl reductase (SCR) from
Candida parapsilosis
catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (
S
)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (
R
)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (
R
)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the
k
cat
/

K
m

value when NADH and NADPH were used as the cofactors, respectively, but maintaining a
k
cat
value essentially the same with respect to wild-type SCR. The ratio of

K
d

(dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.
In Situ Hydrogen and Nitrous Oxide as Indicators of Concomitant Fermentation and Denitrification in the Alimentary Canal of the EarthwormLumbricus terrestris
Authors: Wüst Pia K. , Horn Marcus A. , Drake Harold L. 
Published: February 2009

ABSTRACTThe earthworm gut is a unique microzone in aerated soils that has been proposed to selectively stimulate ingested soil microorganisms by its in situ conditions, which include anoxia, high water content, a near-neutral pH, and high concentrations of organic compounds. The central objective of this study was to resolve potential links between in situ conditions and anaerobic microbial activities during the gut passage ofLumbricus terrestris. Both H2and N2O were emitted by living earthworms, and in situ microsensor analyses revealed both H2and N2O in the O2-free gut center. The highest H2concentrations occurred in foregut and midgut regions, whereas the highest N2O concentrations occurred in crop/gizzard and hindgut regions. Thus, H2-producing fermentations were more localized in the foregut and midgut, whereas denitrification was more localized in the crop/gizzard and hindgut. Moisture content, total carbon, and total nitrogen were highest in the foregut and decreased from the anterior to posterior end of the gut. Nitrite, ammonium, and iron(II) concentrations were highest in the crop/gizzard and decreased from the anterior to posterior end of the alimentary canal. Concentrations of soluble organic compounds were indicative of distinct fermentation processes along the alimentary canal, with maximal concentrations of organic acids (e.g., acetate and butyrate) occurring in the midgut. These findings suggest that earthworms (i) contribute to the terrestrial cycling of carbon and nitrogen via anaerobic microbial activities in the alimentary canal and (ii) constitute a mobile source of reductant (i.e., emitted H2) for microbiota in aerated soils.
Pea Aphid as both Host and Vector for the Phytopathogenic Bacterium
Pseudomonas syringae
Authors: Stavrinides John , McCloskey Jodi K. , Ochman Howard 
Published: February 2009

ABSTRACT

Aphids are widespread agricultural pests that are capable of disseminating plant viral diseases; however, despite coming into frequent contact with epiphytic bacteria, aphids are considered to have no role in bacterial transmission. Here, we demonstrate the ability of pea aphids to vector the phytopathogen
Pseudomonas syringae
pv. syringae B728a (PsyB728a). While feeding on plants colonized by epiphytic bacteria, aphids acquire the bacteria, which colonize the digestive tract, multiply, and are excreted in the aphid honeydew, resulting in inoculation of the phyllosphere with up to 10
7
phytopathogenic bacteria per cm
2
. Within days of ingesting bacteria, aphids succumb to bacterial sepsis, indicating that aphids serve as an alternative, nonplant host for PsyB728a. The related strain
Pseudomonas syringae
pv. tomato DC3000 is >1,000-fold less virulent than PsyB728a in the pea aphid, suggesting that PsyB728a possesses strain-specific pathogenicity factors that allow it to exploit aphids as hosts. To identify these factors, we performed a mutagenesis screen and recovered PsyB728a mutants that were hypovirulent, including one defective in a gene required for flagellum formation and motility. These interactions illustrate that aphids can also vector bacterial pathogens and that even seemingly host-restricted pathogens can have alternative host specificities and lifestyles.
Differences in Hydrogenase Gene Expression between
Methanosarcina acetivorans
and
Methanosarcina barkeri
Authors: Guss Adam M. , Kulkarni Gargi , Metcalf William W. 
Published: February 2009

ABSTRACT

Methanosarcina acetivorans
C2A encodes three putative hydrogenases, including one cofactor F
420
-linked (
frh
) and two methanophenazine-linked (
vht
) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of
Methanosarcina barkeri
and
Methanosarcina mazei
shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in
M. acetivorans
were aligned with those in other
Methanosarcina
species to identify conserved transcription and translation signals. The
M. acetivorans vht
promoter region is well conserved among the sequenced
Methanosarcina
species, while the second
vht
-type homolog (here called
vhx
) and
frh
promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both
M. acetivorans
and
M. barkeri
strains carrying reporter gene fusions to each of the
M. acetivorans
and
M. barkeri
hydrogenase promoters. Generally, the
M. acetivorans
gene fusions are not expressed in either organism, suggesting that
cis
-acting mutations inactivated the
M. acetivorans
promoters. The
M. barkeri
hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that
M. acetivorans
possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the
M. acetivorans
hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data.
A Widely Conserved Gene Cluster Required for Lactate Utilization inBacillus subtilisand Its Involvement in Biofilm Formation
Authors: Chai Yunrong , Kolter Roberto , Losick Richard 
Published: February 2009

ABSTRACTWe report that catabolism ofl-lactate inBacillus subtilisdepends on the previously uncharacterizedyvfV-yvfW-yvbY(herein renamedlutABC) operon, which is inferred to encode three iron-sulfur-containing proteins. The operon is under the dual control of a GntR-type repressor (LutR, formerly YvfI) and the master regulator for biofilm formation SinR and is induced during growth in response tol-lactate. Operons with high similarity tolutABCare present in the genomes of a variety of gram-positive and gram-negative bacteria, raising the possibility that LutABC is a widely conserved and previously unrecognized pathway for the utilization ofl-lactate or related metabolites.
Rrp2, a σ
54
-Dependent Transcriptional Activator of
Borrelia burgdorferi
, Activates
rpoS
in an Enhancer-Independent Manner
Authors: Blevins Jon S. , Xu Haijun , He Ming , Norgard Michael V. , Reitzer Larry , Yang X. Frank 
Published: February 2009

ABSTRACT

Rrp2 is the sole σ
54
-dependent transcriptional activator present in the
Borrelia burgdorferi
genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates σ
54
-dependent
rpoS
expression without an apparent upstream enhancer element commonly associated with other σ
54
-dependent transcriptional activators.
A Conserved α-Helix Essential for a Type VI Secretion-Like System of
Francisella tularensis
Authors: Bröms Jeanette E. , Lavander Moa , Sjöstedt Anders 
Published: February 2009

ABSTRACT

Francisella tularensis
harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include
iglA
and
iglB
encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of
F. tularensis
LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved α-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast
Saccharomyces cerevisiae
but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for
Francisella
pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of
Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica
serovar Typhimurium, and
Escherichia coli
were also shown to interact in yeast, and the interaction was dependent on preservation of the same α-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for
Francisella
pathogenicity, and the same interaction is conserved in other human pathogens.
HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in
Corynebacterium diphtheriae
Authors: Allen Courtni E. , Schmitt Michael P. 
Published: February 2009

ABSTRACT

Many human pathogens, including
Corynebacterium diphtheriae
, the causative agent of diphtheria, use host compounds such as heme and hemoglobin as essential iron sources. In this study, we examined the
Corynebacterium hmu
hemin transport region, a genetic cluster that contains the
hmuTUV
genes encoding a previously described ABC-type hemin transporter and three additional genes, which we have designated
htaA
,
htaB
, and
htaC
. The
hmu
gene cluster is composed of three distinct transcriptional units. The
htaA
gene appears to be part of an iron- and DtxR-regulated operon that includes
hmuTUV
, while
htaB
and
htaC
are transcribed from unique DtxR-regulated promoters. Nonpolar deletion of either
htaA
or the
hmuTUV
genes resulted in a reduced ability to use hemin as an iron source, while deletion of
htaB
had no effect on hemin iron utilization in
C. diphtheriae
. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they share some sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembrane regions. Protein localization studies with
C. diphtheriae
showed that HtaA is associated predominantly with the cell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-rich broth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growth medium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting that these proteins may function as cell surface hemin receptors in
C. diphtheriae
.