bluuebunny/crossref_metadata_2025_split huggingface dataset:
Versatile Dual-Technology System for Markerless Allele Replacement in
Burkholderia pseudomallei
Authors: López Carolina M. , Rholl Drew A. , Trunck Lily A. , Schweizer Herbert P. 
Published: August 2009

ABSTRACT

Burkholderia pseudomallei
is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for
B. pseudomallei
based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a
lacZ
α gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed
gusA
indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or
sacB-
based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an
araC
-

P
BAD
-I

-
sceI
expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δ
asd Escherichia coli
mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and
sacB
-based methods afforded by pEXKm5 in conjunction with
E. coli
RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory
B. pseudomallei
strains. Finally,
sacB
-based counterselection was employed to isolate a defined chromosomal
fabD
(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.
Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several
Agrobacterium tumefaciens
Strains: a Frequent Event?
Authors: Llop Pablo , Murillo Jesús , Lastra Beatriz , López María M. 
Published: August 2009

ABSTRACT

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven
Agrobacterium tumefaciens
strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two
A. tumefaciens
strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS
426
inserted into the
iaaM
gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.
Integrated Capture and Spectroscopic Detection of Viruses
Authors: Vargas Crystal A. , Wilhelm Allison A. , Williams Jeremy , Lucas Pierre , Reynolds Kelly A. , Riley Mark R. 
Published: August 2009

ABSTRACT
The goal of this work is to develop an online monitoring scheme for detection of viruses in flowing drinking water. The approach applies an electrodeposition process that is similar to the use of charged membrane filters previously employed for collection of viruses from aqueous samples. In the present approach, charged materials are driven onto a robust optical sensing element which has high transparency to infrared light. A spectroscopic measurement is performed using the evanescent wave that penetrates no more than 1 μm from the surface of an infrared optical element in an attenuated total reflectance measurement scheme. The infrared measurement provides quantitative information on the amount and identity of material deposited from the water. Initial studies of this sensing scheme used proteins reversibly electrodeposited onto germanium chips. The results of those studies were applied to design a method for collection of viruses onto an attenuated total reflectance crystal. Spectral signatures can be discriminated between three types of protein and two viruses. There is the potential to remove deposited material by reversing the voltage polarity. This work demonstrates a novel and practical scheme for detection of viruses in water systems with potential application to near-continual, automated monitoring of municipal drinking water.
Development of a Single-Plasmid-Based Regulatable Gene Expression System for
Borrelia burgdorferi
Authors: Whetstine Christine R. , Slusser Joyce G. , Zückert Wolfram R. 
Published: August 2009

ABSTRACT

We developed a single-plasmid-based regulatable protein expression system for
Borrelia burgdorferi
. Expression of a target gene is driven by P

ost

, a hybrid
B. burgdorferi ospA
-
tetO
promoter, from a recombinant
B. burgdorferi
plasmid constitutively expressing TetR. The system was tested using the green fluorescent protein (GFP) as a reporter. Under noninducing conditions, recombinant
B. burgdorferi
cells were nonfluorescent, no GFP protein was detected, and residual, small amounts of transcript were detectable only by reverse transcription-PCR but not by Northern blot hybridization. Upon induction with anhydrotetracycline, increasing levels of GFP transcript, protein, and fluorescence were observed. This tight and titratable promoter system will be invaluable for the study of essential borrelial proteins. Since target protein, operator, and repressor are carried by a single plasmid, the system's application is independent of a particular strain background.
A Unique 45-Amino-Acid Region in the Toprim Domain of
Plasmodium falciparum
Gyrase B Is Essential for Its Activity
Authors: Dar Ashraf , Prusty Dhaneswar , Mondal Neelima , Dhar Suman K. 
Published: August 2009

ABSTRACT

DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the
Plasmodium falciparum
gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite
P. falciparum
but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction.
Transcriptional Responses of
Candida albicans
to Epithelial and Endothelial Cells
Authors: Park Hyunsook , Liu Yaoping , Solis Norma , Spotkov Joshua , Hamaker Jessica , Blankenship Jill R. , Yeaman Michael R. , Mitchell Aaron P. , Liu Haoping , Filler Scott G. 
Published: August 2009

ABSTRACT

Candida albicans
interacts with oral epithelial cells during oropharyngeal candidiasis and with vascular endothelial cells when it disseminates hematogenously. We set out to identify
C. albicans
genes that govern interactions with these host cells in vitro. The transcriptional response of
C. albicans
to the FaDu oral epithelial cell line and primary endothelial cells was determined by microarray analysis. Contact with epithelial cells caused a decrease in transcript levels of genes related to protein synthesis and adhesion, whereas contact with endothelial cells did not significantly influence any specific functional category of genes. Many genes whose transcripts were increased in response to either host cell had not been previously characterized. We constructed mutants with homozygous insertions in 22 of these uncharacterized genes to investigate their function during host-pathogen interaction. By this approach, we found that
YCK2
,
VPS51
, and
UEC1
are required for
C. albicans
to cause normal damage to epithelial cells and resist antimicrobial peptides.
YCK2
is also necessary for maintenance of cell polarity.
VPS51
is necessary for normal vacuole formation, resistance to multiple stressors, and induction of maximal endothelial cell damage.
UEC1
encodes a unique protein that is required for resistance to cell membrane stress. Therefore, some
C. albicans
genes whose transcripts are increased upon contact with epithelial or endothelial cells are required for the organism to damage these cells and withstand the stresses that it likely encounters during growth in the oropharynx and bloodstream.
Role of Ferroxidases in Iron Uptake and Virulence of
Cryptococcus neoformans
Authors: Jung Won Hee , Hu Guanggan , Kuo Wayne , Kronstad James W. 
Published: August 2009

ABSTRACT

Iron acquisition is a critical aspect of the virulence of many pathogenic microbes, and iron limitation is an important defense mechanism for mammalian hosts. We are examining mechanisms of iron regulation and acquisition in the fungal pathogen
Cryptococcus neoformans
, and here, we characterize the roles of the ferroxidases Cfo1 and Cfo2. Cfo1 is required for the reductive iron uptake system that mediates the utilization of transferrin, an important iron source for
C. neoformans
during infection. The virulence of a
cfo1
mutant was attenuated in a mouse model of cryptococcosis, and the mutant also displayed increased sensitivities to the antifungal drugs fluconazole and amphotericin B. Wild-type levels of drug sensitivity were restored by the addition of exogenous heme, which suggested that reduced levels of intracellular iron may curtail heme levels and interfere with ergosterol biosynthesis. We constructed green fluorescent protein (GFP) fusion proteins and found elevated expression of Cfo1-GFP upon iron limitation, as well as localization of the fusion to the plasma membrane. Trafficking to this location was disrupted by a defect in the catalytic subunit of cyclic AMP-dependent protein kinase. This result is consistent with findings from studies indicating an influence of the kinase on the expression of protein-trafficking functions in
C. neoformans
.
The Transcription Factor Homolog CTF1 Regulates β-Oxidation in
Candida albicans
Authors: Ramírez Melissa A. , Lorenz Michael C. 
Published: August 2009

ABSTRACT

Carbon starvation is one of the many stresses to which microbial pathogens are subjected while in the host. Pathways necessary for the utilization of alternative carbon sources, such as gluconeogenesis, the glyoxylate cycle, and β-oxidation of fatty acids, have been shown to be required for full virulence in several systems, including the fungal pathogen
Candida albicans
. We have investigated the regulatory network governing alternative carbon metabolism in this organism through characterization of transcriptional regulators identified based on the model fungi,
Saccharomyces cerevisiae
and
Aspergillus nidulans
.
C. albicans
has homologs of the ScCAT8/AnFacB and ScADR1/AnAmdX transcription factors that regulate induction of genes encoding the proteins of gluconeogenesis, the glyoxylate cycle, and ethanol utilization. Surprisingly,
C. albicans
mutants lacking
CAT8
or
ADR1
have no apparent phenotypes and do not regulate genes for key enzymes of these pathways. Fatty acid degradation and peroxisomal biogenesis are controlled by nonhomologous regulators, OAF1/PIP2 in
S. cerevisiae
and FarA/FarB in
A. nidulans
;
C. albicans
is missing OAF1 and PIP2 and, instead, has a single homolog of the Far proteins, CTF1. We have shown that
CTF1
is required for growth on lipids and for expression of genes necessary for β-oxidation, such as
FOX2
.
ctf1
Δ/
ctf1
Δ (
ctf1
Δ/Δ) strains do not, however, show the pleiotropic phenotypes observed for
fox2
Δ/Δ mutants. The
ctf1
Δ/Δ mutant confers a mild attenuation in virulence, like the
fox2
Δ/Δ mutant. Thus, phenotypic and genotypic observations highlight important differences in the regulatory network for alternative carbon metabolism in
C. albicans
compared to the paradigms developed in other model fungi.
Aneuploid Chromosomes Are Highly Unstable during DNA Transformation of
Candida albicans
Authors: Bouchonville Kelly , Forche Anja , Tang Karen E. S. , Selmecki Anna , Berman Judith 
Published: August 2009

ABSTRACT

Candida albicans
strains tolerate aneuploidy, historically detected as karyotype alterations by pulsed-field gel electrophoresis and more recently revealed by array comparative genome hybridization, which provides a comprehensive and detailed description of gene copy number. Here, we first retrospectively analyzed 411 expression array experiments to predict the frequency of aneuploidy in different strains. As expected, significant levels of aneuploidy were seen in strains exposed to stress conditions, including UV light and/or sorbose treatment, as well as in strains that are resistant to antifungal drugs. More surprisingly, strains that underwent transformation with DNA displayed the highest frequency of chromosome copy number changes, with strains that were initially aneuploid exhibiting ∼3-fold more copy number changes than strains that were initially diploid. We then prospectively analyzed the effect of lithium acetate (LiOAc) transformation protocols on the stability of trisomic chromosomes. Consistent with the retrospective analysis, the proportion of karyotype changes was highly elevated in strains carrying aneuploid chromosomes. We then tested the hypothesis that stresses conferred by heat and/or LiOAc exposure promote chromosome number changes during DNA transformation procedures. Indeed, a short pulse of very high temperature caused frequent gains and losses of multiple chromosomes or chromosome segments. Furthermore, milder heat exposure over longer periods caused increased levels of loss of heterozygosity. Nonetheless, aneuploid chromosomes were also unstable when strains were transformed by electroporation, which does not include a heat shock step. Thus, aneuploid strains are particularly prone to undergo changes in chromosome number during the stresses of DNA transformation protocols.
Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium
Photobacterium profundum
SS9
Authors: El-Hajj Ziad W. , Tryfona Theodora , Allcock David J. , Hasan Fariha , Lauro Federico M. , Sawyer Lindsay , Bartlett Douglas H. , Ferguson Gail P. 
Published: August 2009

ABSTRACT

The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium
Photobacterium profundum
SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn
10
) and FL28 (PBPRA1039::mini-Tn
10
), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to
Escherichia coli
DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in
E. coli
mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an
E. coli dnaA
(Cs)
diaA
double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an
E. coli dnaA
(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in
P. profundum
SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.
Tetracycline-Associated Transcriptional Regulation of Transfer Genes of the
Bacteroides
Conjugative Transposon CTnDOT
Authors: Jeters Robert T. , Wang Gui-Rong , Moon Kyung , Shoemaker Nadja B. , Salyers Abigail A. 
Published: August 2009

ABSTRACT

Many human colonic
Bacteroides
spp. harbor a conjugative transposon, CTnDOT, which carries two antibiotic resistance genes,
tetQ
and
ermF
. A distinctive feature of CTnDOT is that its excision and transfer are stimulated by tetracycline. Regulation of the genes responsible for excision has been described previously. We provide here the first characterization of the regulation of CTnDOT transfer (
tra
) genes. Reverse transcription-PCR analysis of the region containing the
tra
genes showed that these genes are regulated at the transcriptional level. Surprisingly, increased production of
tra
gene mRNA in tetracycline-stimulated cells was mediated by the proteins encoded by the excision genes. Previous studies have shown that expression of the excision gene operon is controlled by the regulatory protein RteC. Accordingly, it was possible that RteC was also regulating
tra
gene expression and that the excision proteins were only accessory proteins. However, placing the excision gene operon under the control of a heterologous promoter showed that the excision proteins alone could activate
tra
gene expression and that RteC was not directly involved. We also found a second level of
tra
gene control. The transfer of CTnDOT was inhibited by a DNA segment that included only a portion of the 3′ end of one of the excision genes (
exc
). This segment contained a small open reading frame,
rteR
. By replacing the codons encoding the first two amino acids of the putative protein product of this open reading frame with stop codons, we showed that the
rteR
gene might encode a small regulatory RNA. RteR acted in
trans
to reduce the number of
tra
transcripts in a way that was independent of the excision proteins. The repressive effect of RteR was not the result of decreased stability of the
tra
mRNA. Instead, RteR appears to be modulating the level of
tra
gene expression in some more direct fashion. The complex regulatory system that controls and links the expression of CTnDOT excision and transfer genes may be designed to ensure stable maintenance of CTnDOT in nature by reducing the fitness toll it takes on the cell that harbors it.
Organophosphate Hydrolase in
Brevundimonas diminuta
Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway
Authors: Gorla Purushotham , Pandey Jay Prakash , Parthasarathy Sunil , Merrick Mike , Siddavattam Dayananda 
Published: August 2009

ABSTRACT

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of
Brevundimonas diminuta
. Expression of the precursor form of OPH carrying a C-terminal His tag in an
opd
-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type
opd
gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in
B. diminuta
. The localization of OPH to the periplasmic face of the inner membrane in
B. diminuta
was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.
Physical fitness levels among European adolescents: the HELENA study
Authors: Ortega F B , Artero E G , Ruiz J R , España-Romero V , Jiménez-Pavón D , Vicente-Rodriguez G , Moreno L A , Manios Y , Béghin L , Ottevaere C , Ciarapica D , Sarri K , Dietrich S , Blair S N , Kersting M , Molnar D , González-Gross M , Gutiérrez Á , Sjöström M , Castillo M J ,   on behalf of the HELENA study
Published: August 2009

ObjectiveTo report sex- and age-specific physical fitness levels in European adolescents.MethodsA sample of 3428 adolescents (1845 girls) aged 12.5–17.49 years from 10 European cities in Austria, Belgium, France, Germany, Greece (an inland city and an island city), Hungary, Italy, Spain and Sweden was assessed in the Healthy Lifestyle in Europe by Nutrition in Adolescence study between 2006 and 2008. The authors assessed muscular fitness, speed/agility, flexibility and cardiorespiratory fitness using nine different fitness tests: handgrip, bent arm hang, standing long jump, Bosco jumps (squat jump, counter movement jump and Abalakov jump), 4×10-m shuttle run, back-saver sit and reach and 20-m shuttle run tests.ResultsThe authors derived sex- and age-specific normative values for physical fitness in the European adolescents using the LMS statistical method and expressed as tabulated percentiles from 10 to 100 and as smoothed centile curves (P5, P25, P50, P75and P95). The figures showed greater physical fitness in the boys, except for the flexibility test, and a trend towards increased physical fitness in the boys as their age increased, whereas the fitness levels in the girls were more stable across ages.ConclusionsThe normative values hereby provided will enable evaluation and correct interpretation of European adolescents' fitness status.