bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans
Authors: Troedsson Christofer , Lee Richard F. , Stokes Vivica , Walters Tina L. , Simonelli Paolo , Frischer Marc E. 
Published: May 2008

ABSTRACT

Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (
Callinectes sapidus
) and a parasitic dinoflagellate
Hematodinium
sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.
Detection and Discovery of Crustacean Parasites in Blue Crabs (
Callinectes sapidus
) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography
Authors: Troedsson Christofer , Lee Richard F. , Walters Tina , Stokes Vivica , Brinkley Karrie , Naegele Verena , Frischer Marc E. 
Published: May 2008

ABSTRACT

Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (
Callinectes sapidus
) by the parasitic dinoflagellate
Hematodinium
sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite
Procryptobia sorokini
. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.
Abundance, Depth Distribution, and Composition of Aerobic Bacteriochlorophyll
a
-Producing Bacteria in Four Basins of the Central Baltic Sea
Authors: Salka Ivette , Moulisová Vladimíra , Koblížek Michal , Jost Günter , Jürgens Klaus , Labrenz Matthias 
Published: May 2008

ABSTRACT

The abundance, vertical distribution, and diversity of aerobic anoxygenic phototrophic bacteria (AAP) were studied at four basins of the Baltic Sea. AAP were enumerated by infrared epifluorescence microscopy, and their diversity was analyzed by using
pufM
gene clone libraries. In addition, numbers of CFU containing the
pufM
gene were determined, and representative strains were isolated. Both approaches indicated that AAP reached maximal abundance in the euphotic zone. Maximal AAP abundance was 2.5 × 10
5
cells ml
−1
(11% of total prokaryotes) or 1.0 × 10
3
CFU ml
−1
(9 to 10% of total CFU). Environmental
pufM
clone sequences were grouped into 11 operational taxonomic units phylogenetically related to cultivated members of the
Alpha
-,
Beta
-, and
Gammaproteobacteria
. In spite of varying
pufM
compositions, five clones were present in all libraries. Of these,
Jannaschia
-related clones were always found in relative abundances representing 25 to 30% of the total AAP clones. The abundances of the other clones varied. Clones potentially affiliated with typical freshwater
Betaproteobacteria
sequences were present at three Baltic Sea stations, whereas clones grouping with
Loktanella
represented 40% of the total cell numbers in the Gotland Basin. For three alphaproteobacterial clones, probable
pufM
phylogenetic relationships were supported by 16S rRNA gene analyses of Baltic AAP isolates, which showed nearly identical
pufM
sequences. Our data indicate that the studied AAP assemblages represented a mixture of marine and freshwater taxa, thus characterizing the Baltic Sea as a “melting pot” of abundant, polyphyletic aerobic photoheterotrophic bacteria.
Facilitation of Robust Growth of
Prochlorococcus
Colonies and Dilute Liquid Cultures by “Helper” Heterotrophic Bacteria
Authors: Morris J. Jeffrey , Kirkegaard Robin , Szul Martin J. , Johnson Zackary I. , Zinser Erik R. 
Published: May 2008

ABSTRACT

Axenic (pure) cultures of marine unicellular cyanobacteria of the
Prochlorococcus
genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing
Prochlorococcus
colonies on semisolid agar that improves the level of recovery to approximately 100%.
Prochlorococcus
grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the
Prochlorococcus
cell concentration surpasses a critical threshold, the “helper” heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help
Prochlorococcus
is the reduction of oxidative stress.
Diversity Surveys and Evolutionary Relationships of
aoxB
Genes in Aerobic Arsenite-Oxidizing Bacteria
Authors: Quéméneur Marianne , Heinrich-Salmeron Audrey , Muller Daniel , Lièvremont Didier , Jauzein Michel , Bertin Philippe N. , Garrido Francis , Joulian Catherine 
Published: May 2008

ABSTRACT

A new primer set was designed to specifically amplify ca. 1,100 bp of
aoxB
genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny.
Alphaproteobacteria
-,
Betaproteobacteria
-, and
Gammaproteobacteria
-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the
aoxB
gene as a functional marker of aerobic As(III) oxidizers.
Use of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy To Identify Microbial Metabolic Products on Carbonate Mineral Surfaces
Authors: Bullen Heather A. , Oehrle Stuart A. , Bennett Ariel F. , Taylor Nicholas M. , Barton Hazel A. 
Published: May 2008

ABSTRACT

This paper demonstrates the use of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to detect microbial metabolic products on carbonate mineral surfaces. By creating an ATR-FTIR spectral database for specific organic acids using ATR-FTIR spectroscopy we were able to distinguish metabolic acids on calcite surfaces following
Escherichia coli
growth. The production of these acids by
E. coli
was verified using high-performance liquid chromatography with refractive index detection. The development of this technique has allowed us to identify microbial metabolic products on carbonate surfaces in nutrient-limited cave environments.
Saccharomyces cerevisiae
Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway
Authors: Izquierdo Alicia , Casas Celia , Mühlenhoff Ulrich , Lillig Christopher Horst , Herrero Enrique 
Published: May 2008

ABSTRACT

Saccharomyces cerevisiae
Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of
GRX6
is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate
GRX7
expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.
Purification and Characterization of Bacteriophage P22 Xis Protein
Authors: Mattis Aras N. , Gumport Richard I. , Gardner Jeffrey F. 
Published: May 2008

ABSTRACT

The temperate bacteriophages λ and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. λ and P22 Xis proteins are both small proteins (λ Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for λ Xis, 11.16). However, the P22 Xis and λ Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the
attP
arms containing the major Xis binding sites from bacteriophages λ, P22, L5, HP1, and P2 and the conjugative transposon Tn
916
. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.
NrdI Essentiality for Class Ib Ribonucleotide Reduction in
Streptococcus pyogenes
Authors: Roca Ignasi , Torrents Eduard , Sahlin Margareta , Gibert Isidre , Sjöberg Britt-Marie 
Published: May 2008

ABSTRACT

The
Streptococcus pyogenes
genome harbors two clusters of class Ib ribonucleotide reductase genes,
nrdHEF
and
nrdF
*
I
*
E
*, and a second stand-alone
nrdI
gene, designated
nrdI2
. We show that both clusters are expressed simultaneously as two independent operons. The NrdEF enzyme is functionally active in vitro, while the NrdE*F* enzyme is not. The NrdF* protein lacks three of the six highly conserved iron-liganding side chains and cannot form a dinuclear iron site or a tyrosyl radical. In vivo, on the other hand, both operons are functional in heterologous complementation in
Escherichia coli
. The
nrdF
*
I
*
E
* operon requires the presence of the
nrdI
* gene, and the
nrdHEF
operon gained activity upon cotranscription of the heterologous
nrdI
gene from
Streptococcus pneumoniae
, while neither
nrdI
* nor
nrdI
2 from
S. pyogenes
rendered it active. Our results highlight the essential role of the flavodoxin NrdI protein in vivo, and we suggest that it is needed to reduce met-NrdF, thereby enabling the spontaneous reformation of the tyrosyl radical. The NrdI* flavodoxin may play a more direct role in ribonucleotide reduction by the NrdF*I*E* system. We discuss the possibility that the
nrdF
*
I
*
E
* operon has been horizontally transferred to
S. pyogenes
from
Mycoplasma
spp.
Formate as the Main Branch Point for Methylotrophic Metabolism in
Methylobacterium extorquens
AM1
Authors: Crowther Gregory J. , Kosály George , Lidstrom Mary E. 
Published: May 2008

ABSTRACT

In serine cycle methylotrophs, methylene tetrahydrofolate (H
4
F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H
4
F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H
4
F to generate methylene H
4
F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H
4
F via H
4
F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H
4
F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H
4
F and formate are unable to grow on methanol, suggesting that this route for methylene H
4
F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in
Methylobacterium extorquens
AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H
4
F synthesis pathway through formate. These results suggested that the methylene H
4
F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in
M. extorquens
AM1.
Rad50 Is Not Essential for the Mre11-Dependent Repair of DNA Double-Strand Breaks inHalobacteriumsp. Strain NRC-1
Authors: Kish A. , DiRuggiero J. 
Published: May 2008

ABSTRACTThe genome of the halophilic archaeonHalobacteriumsp. strain NRC-1 encodes homologs of the eukaryotic Mre11 and Rad50 proteins, which are involved in the recognition and end processing of DNA double-strand breaks in the homologous recombination repair pathway. We have analyzed the phenotype ofHalobacteriumdeletion mutants lackingmre11and/orrad50after exposure to UV-C radiation, an alkylating agent (N-methyl-N′-nitro-N-nitrosoguanidine), and γ radiation, none of which resulted in a decrease in survival of the mutant strains compared to that of the background strain. However, a decreased rate of repair of DNA double-strand breaks in strains lacking themre11gene was observed using pulsed-field gel electrophoresis. These observations led to the hypothesis that Mre11 is essential for the repair of DNA double-strand breaks inHalobacterium, whereas Rad50 is dispensable. This is the first identification of a Rad50-independent function for the Mre11 protein, and it represents a shift in theArchaeaaway from the eukaryotic model of homologous recombination repair of DNA double-strand breaks.
Genetic and Functional Characterization of the Type IV Secretion System inWolbachia
Authors: Rancès Edwige , Voronin Denis , Tran-Van Van , Mavingui Patrick 
Published: May 2008

ABSTRACTA type IV secretion system (T4SS) is used by many symbiotic and pathogenic intracellular bacteria for the successful infection of and survival, proliferation, and persistence within hosts. In this study, the presence and function of the T4SS inWolbachiastrains were investigated by a combination of genetic screening and immunofluorescence microscopy. Two operons ofvirB-virD4loci were found in the genome ofWolbachia pipientisstrainwAtab3, from the HymenopteraAsobara tabida, and strainwRi, infectingDrosophila simulans. One operon consisted of fivevirgenes (virB8,virB9,virB10,virB11, andvirD4) and the downstreamwspBlocus. The other operon was composed of three genes (virB3,virB4, andvirB6) and included four additional open reading frames (orf1toorf4) orientated in the same direction. In cell culture and insect hosts infected with differentWolbachiastrains, the bona fidevirgenes were polycistronically transcribed, together with the downstream adjacent loci, notably, asvirB8tovirD4andwspBand asvirB3,virB4,virB6, andorf1toorf4. Two peptides encompassing conserved C and N termini of theWolbachiaVirB6 protein were used for the production of polyclonal antibodies. Anti-VirB6 antibodies could detect the corresponding recombinant protein by chemifluorescence on Western blots of total proteins fromEscherichia colitransformants andWolbachiastrains cultured in cell lines. Using immunofluorescence microscopy, we further demonstrated that the VirB6 protein was produced byWolbachiastrains in ovaries of insects harboringwAtab3 orwRi and cell lines infected withwAlbB orwMelPop. As VirB6 is known to associate with other VirB proteins to form a membrane-spanning structure, this finding suggests that a T4SS may function inWolbachia.
Complex Regulation of the DnaJ Homolog CbpA by the Global Regulators σ
S
and Lrp, by the Specific Inhibitor CbpM, and by the Proteolytic Degradation of CbpM
Authors: Chenoweth Matthew R. , Wickner Sue 
Published: May 2008

ABSTRACT

CbpA is a DnaJ homolog that functions as a DnaK cochaperone. Several cellular processes, including growth at low and high temperatures and septum formation during cell division, require either CbpA or DnaJ. CbpA is encoded in an operon with the gene for CbpM, which is a specific in vivo and in vitro inhibitor of CbpA. Here, we have cooverexpressed CbpA with CbpM in a Δ
cbpAM
Δ
dnaJ
strain and examined the resulting phenotypes. Under these conditions, sufficient free CbpA activity was present to support growth at low temperatures, but not at high temperatures. Defects in cell division and in λ replication were also partially complemented by CbpA when cooverexpressed with CbpM. Utilizing reporter fusions, we demonstrated that the
cbpAM
operon was maximally transcribed at the transition from exponential growth to stationary phase. Transcription was controlled by the σ
S
and Lrp global regulators, and both leucine availability and growth temperature influenced transcription. CbpA and CbpM accumulated to similar levels in stationary phase, ∼2,300 monomers per cell. When not bound to CbpA, CbpM was unstable and was degraded by the Lon and ClpAP proteases. These data demonstrate that CbpA activity is controlled at multiple levels.
The Major Facilitator Superfamily-Type Transporter YmfE and the Multidrug-Efflux Activator Mta Mediate Bacillibactin Secretion inBacillus subtilis
Authors: Miethke Marcus , Schmidt Sarah , Marahiel Mohamed A. 
Published: May 2008

ABSTRACTHigh-affinity iron acquisition inBacillus subtilisis mediated via the bacillibactin catechole siderophore pathway. Three of the four essential pathway steps, bacillibactin synthesis, Fe-bacillibactin uptake, and Fe-bacillibactin hydrolysis have been characterized previously. The functional and regulatory components for bacillibactin secretion, the second step of the siderophore pathway, remained unknown. In this study, the screening of aB. subtilisexporter mutant library led to the identification of the YmfE major facilitator superfamily (MFS)-type transporter as a target for bacillibactin export. Analysis of iron-limitedymfEmutant cultures displayed an eightfold reduced bacillibactin secretion and, on the other hand, a 25-fold increased secretion of the bacillibactin precursor 2,3-dihydroxybenzoate. Investigation of the regulatory aspect revealed that bacillibactin secretion is, in contrast to all other components of the pathway, independent of the ferric uptake repressor Fur. Indeed, the MerR-type transcriptional regulator Mta was found to activate both bacillibactin secretion andymfEgene expression, exposing Mta as an additional regulatory member of the bacillibactin pathway.
Interaction of DevR with Multiple Binding Sites Synergistically Activates Divergent Transcription of
narK2
-Rv1738 Genes in
Mycobacterium tuberculosis
Authors: Chauhan Santosh , Tyagi Jaya Sivaswami 
Published: May 2008

ABSTRACT

Under hypoxic conditions or upon exposure to low concentrations of nitric oxide, DevR transcriptional regulator mediates the activation of ∼50 genes that are believed to assist in dormancy development in
Mycobacterium tuberculosis
. Most of the strongly induced genes are characterized by the presence of one to four copies of a Dev box-like sequence at an upstream location. Among these are several gene pairs that are transcribed in opposite directions. This arrangement could provide for coordinated control of the adjacent genes under inducing conditions. In this work, we report the first detailed analysis of DevR-mediated hypoxic regulation of
narK2
-Rv1738 genes that are oppositely oriented in
M. tuberculosis
. Phosphorylated DevR interacts with intergenic sequences and protects ∼80 bp of DNA that contains three binding sites, designated Dev boxes D1, D2, and D3. The hypoxia-specific transcription start points of
narK2
and Rv1738 were mapped, and it was noted that the −35 elements of both promoters overlapped with the proximally placed Dev box. DevR bound cooperatively to adjacently placed D2 and D3 boxes while binding to D1 was independent of DevR interaction with the D2 and D3 boxes. Mutational analysis and green fluorescent protein reporter assays established that the three Dev boxes function synergistically to mediate maximal transcriptional induction of both
narK2
and Rv1738 in hypoxic cultures of
M. tuberculosis
. Analysis of
narK2
promoter activity indicates that it is under negative regulation in addition to DevR-mediated positive regulation and also reveals differences between
M. tuberculosis
and
Mycobacterium bovis
BCG.