bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Tools for Select-Agent-Compliant Manipulation of
Burkholderia pseudomallei
Authors: Choi Kyoung-Hee , Mima Takehiko , Casart Yveth , Rholl Drew , Kumar Ayush , Beacham Ifor R. , Schweizer Herbert P. 
Published: December 2007

ABSTRACT

Because of
Burkholderia pseudomallei
's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated

Escherichia coli P
BAD

promoter and kanamycin (
nptI
) and zeocin (
ble
) selection markers from the constitutive
Burkholderia thailandensis
ribosomal
P
S12
or synthetic bacterial
P
EM7
promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of
nptII
and
ble
selection markers contained on
FRT
or
loxP
cassettes. Finally, expression of Tn
7
site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for
B. pseudomallei
. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked Δ(
amrRAB
-
oprA
) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different
B. pseudomallei
strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn
7
element carrying the
amrAB
-
oprA
operon. The new tools allow routine select-agent-compliant genetic manipulations of
B. pseudomallei
and other
Burkholderia
species.
Melanin-Based High-Throughput Screen for
l
-Tyrosine Production in
Escherichia coli
Authors: Santos Christine Nicole S. , Stephanopoulos Gregory 
Published: December 2007

ABSTRACT

We present the development of a simple, high-throughput screen for identifying bacterial strains capable of
l
-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link
l
-tyrosine production in
Escherichia coli
with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular
l
-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for
l
-tyrosine-overproducing strains.
Genomic Differences between
Fibrobacter succinogenes
S85 and
Fibrobacter intestinalis
DR7, Identified by Suppression Subtractive Hybridization
Authors: Qi M. , Nelson K. E. , Daugherty S. C. , Nelson W. C. , Hance I. R. , Morrison M. , Forsberg C. W. 
Published: December 2007

ABSTRACT

Fibrobacter
is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in
Fibrobacter succinogenes
S85 but absent from
F. intestinalis
DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to
F. intestinalis
were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the
F. succinogenes
genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of
F. succinogenes
genomic DNA, were identified that lack similarity to those of
F. intestinalis
chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to
F. succinogenes
. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in
F. succinogenes
S85 was either missing from or not functional in
F. intestinalis
DR7, which explains the requirement of vitamin B
12
for the growth of the
F. intestinalis
species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by
Fibrobacter
genera appear to have an important role in energy metabolism.
Determination of Glycosyltransferase Specificities for the
Escherichia coli
O111 O Antigen by a Generic Approach
Authors: Stevenson Gordon , Dieckelmann Manuela , Reeves Peter R. 
Published: December 2007

ABSTRACT

We describe a bacterial strain developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. For proof of principle for the approach, the strain was used to determine the specificity of the
Escherichia coli
O111 O-antigen GT genes.
ABotrytis cinereaEmopamil Binding Domain Protein, Required for Full Virulence, Belongs to a Eukaryotic Superfamily Which Has Expanded in Euascomycetes
Authors: Gioti A. , Pradier J. M. , Fournier E. , Le Pêcheur P. , Giraud C. , Debieu D. , Bach J. , Leroux P. , Levis C. 
Published: December 2007

ABSTRACTA previous transcriptomic analysis of 3,032 fungal genes identified theBotrytis cinerea PIE3(BcPIE3) gene to be up-regulated early in planta (A. Gioti, A. Simon, P. Le Pêcheur, C. Giraud, J. M. Pradier, M. Viaud, and C. Levis, J. Mol. Biol. 358:372-386, 2006). In the present study, BcPIE3was disrupted in order to determine its implication in pathogenicity. BcPIE3 was shown to be a virulence factor, since the ΔBcPIE3 mutant was blocked during the colonization of tomato and bean leaves, giving lesions reduced in size by at least 74%. Within the emopamil binding domain (EBD), BcPIE3 shows significant structural similarities to mammalian emopamil binding proteins (EBPs). Mammalian EBPs function as sterol isomerases, but an analysis of the sterol content and the results of growth inhibition experiments with the ΔBcPIE3 strain indicated that BcPIE3 is dispensable for ergosterol biosynthesis. The systematic identification of EBD-containing proteins included in public databases showed that these proteins constitute a protein superfamily present only in eukaryotes. Phylogenetic analysis showed that the ancestral EBD-encoding gene was duplicated in the common ancestor of animals and fungi after the split from plants. Finally, we present evidence that the EBP phylogenetic clade of this superfamily has further expanded exclusively in euascomycetes, especially inB. cinerea, which contains three copies of theEBPgene.
Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine
Authors: Agarwal Ameeta K. , Xu Tao , Jacob Melissa R. , Feng Qin , Lorenz Michael C. , Walker Larry A. , Clark Alice M. 
Published: December 2007

ABSTRACT

Sampangine, a plant-derived alkaloid found in the
Annonaceae
family, exhibits strong inhibitory activity against the opportunistic fungal pathogens
Candida albicans
,
Cryptococcus neoformans
, and
Aspergillus fumigatus
. In the present study, transcriptional profiling experiments coupled with analyses of mutants were performed in an effort to elucidate its mechanism of action. Using
Saccharomyces cerevisiae
as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the
hem1
Δ mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed with
C. albicans
to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of
C. albicans
and
S. cerevisiae
to low oxygen, significant correlations were observed between the two transcription profiles, suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.
Protein Kinase A, TOR, and Glucose Transport Control the Response to Nutrient Repletion in
Saccharomyces cerevisiae
Authors: Slattery Matthew G. , Liko Dritan , Heideman Warren 
Published: December 2007

ABSTRACT

Nutrient repletion leads to substantial restructuring of the transcriptome in
Saccharomyces cerevisiae
. The expression levels of approximately one-third of all
S. cerevisiae
genes are altered at least twofold when a nutrient-depleted culture is transferred to fresh medium. Several nutrient-sensing pathways are known to play a role in this process, but the relative contribution that each pathway makes to the total response has not been determined. To better understand this, we used a chemical-genetic approach to block the protein kinase A (PKA), TOR (target of rapamycin), and glucose transport pathways, alone and in combination. Of the three pathways, we found that loss of PKA produced the largest effect on the transcriptional response; however, many genes required both PKA and TOR for proper nutrient regulation. Those genes that did not require PKA or TOR for nutrient regulation were dependent on glucose transport for either nutrient induction or repression. Therefore, loss of these three pathways is sufficient to prevent virtually the entire transcriptional response to fresh medium. In the absence of fresh medium, activation of the cyclic AMP/PKA pathway does not induce cellular growth; nevertheless, PKA activation induced a substantial fraction of the PKA-dependent genes. In contrast, the absence of fresh medium strongly limited gene repression by PKA. These results account for the signals needed to generate the transcriptional responses to glucose, including induction of growth genes required for protein synthesis and repression of stress genes, as well as the classical glucose repression and hexose transporter responses.
Blastocystis rattiContains Cysteine Proteases That Mediate Interleukin-8 Response from Human Intestinal Epithelial Cells in an NF-κB-Dependent Manner
Authors: Puthia Manoj K. , Lu Jia , Tan Kevin S. W. 
Published: December 2007

ABSTRACTBlastocystisis a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association ofBlastocystiswith gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associatedBlastocystiswith intestinal inflammation, and it has been shown thatBlastocystishas potential to modulate the host immune response.Blastocystisis also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by whichBlastocystisactivates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases ofBlastocystis rattiWR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-κB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced byB. rattiWR1. We also show for the first time that the central vacuole ofBlastocystismay function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.
Cryptococcus neoformans
Capsular Polysaccharide and Exopolysaccharide Fractions Manifest Physical, Chemical, and Antigenic Differences
Authors: Frases Susana , Nimrichter Leonardo , Viana Nathan B. , Nakouzi Antonio , Casadevall Arturo 
Published: December 2007

ABSTRACT

The human pathogenic fungus
Cryptococcus neoformans
has a large polysaccharide (PS) capsule and releases copious amounts of PS into cultures and infected tissues. The capsular PS is a major virulence factor that can elicit protective antibody responses. PS recovered from culture supernatants has historically provided an ample and convenient source of material for structural and immunological studies. Two major assumptions in such studies are that the structural features of the exopolysaccharide material faithfully mirror those of capsular PS and that the isolation methods do not change PS properties. However, a comparison of exopolysaccharide made by two isolation techniques with capsular PS stripped from cells with gamma radiation or dimethyl sulfoxide revealed significant differences in glycosyl composition, mass, size, charge, viscosity, circular-dichroism spectra, and reactivity with monoclonal antibodies. Our results strongly suggest that exopolysaccharides and capsular PS are structurally different. A noteworthy finding was that PS made by cetyltrimethylammonium bromide precipitation had a larger mass and a different conformation than PS isolated by concentration and filtration, suggesting that the method most commonly used to purify glucuronoxylomannan alters the PS. Hence, the method used to isolate PS can significantly influence the structural and antigenic properties of the product. Our findings have important implications for current views of the relationship between capsular PS and exopolysaccharides, for the generation of PS preparations suitable for immunological studies, and for the formulation of PS-based vaccines for the prevention of cryptococcosis.
Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in
Saccharomyces cerevisiae
Authors: Fei Weihua , Alfaro Gabriel , Muthusamy Baby-Periyanayaki , Klaassen Zachary , Graham Todd R. , Yang Hongyuan , Beh Christopher T. 
Published: December 2007

ABSTRACT

The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (
Saccharomyces cerevisiae
) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.
Regulation of
Cryptococcus neoformans
Capsule Size Is Mediated at the Polymer Level
Authors: Yoneda Aki , Doering Tamara L. 
Published: December 2007

ABSTRACT

The fungal pathogen
Cryptococcus neoformans
regulates its polysaccharide capsule depending on environmental stimuli. To investigate whether capsule polymers change under different growth conditions, we analyzed shed capsules at physiological concentrations without physical perturbation. Our results indicate that regulation of capsule size is mediated at the level of individual polysaccharide molecules.
Function of a Mycobacterial Major Facilitator Superfamily Pump Requires a Membrane-Associated Lipoprotein
Authors: Farrow Mary F. , Rubin Eric J. 
Published: December 2007

ABSTRACT

The
lprG
-Rv1410c operon is critical for the survival of
Mycobacterium tuberculosis
during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in
Mycobacterium smegmatis
is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from
M. tuberculosis
. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type
M. smegmatis
and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the
lprG
-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.
RhaU of
Rhizobium leguminosarum
Is a Rhamnose Mutarotase
Authors: Richardson Jason S. , Carpena Xavi , Switala Jack , Perez-Luque Rosa , Donald Lynda J. , Loewen Peter C. , Oresnik Ivan J. 
Published: December 2007

ABSTRACT

Of the nine genes comprising the
l
-rhamnose operon of
Rhizobium leguminosarum
,
rhaU
has not been assigned a function. The construction of a Δ
rhaU
strain revealed a growth phenotype that was slower than that of the wild-type strain, although the ultimate cell yields were equivalent. The transport of
l
-rhamnose into the cell and the rate of its phosphorylation were unaffected by the mutation. RhaU exhibits weak sequence similarity to the formerly hypothetical protein YiiL of
Escherichia coli
that has recently been characterized as an
l
-rhamnose mutarotase. To characterize RhaU further, a His-tagged variant of the protein was prepared and subjected to mass spectrometry analysis, confirming the subunit size and demonstrating its dimeric structure. After crystallization, the structure was refined to a 1.6-Å resolution to reveal a dimer in the asymmetric unit with a very similar structure to that of YiiL. Soaking a RhaU crystal with
l
-rhamnose resulted in the appearance of β-
l
-rhamnose in the active site.
Transcriptional Regulation of the
Clostridium cellulolyticum cip-cel
Operon: a Complex Mechanism Involving a Catabolite-Responsive Element
Authors: Abdou Laetitia , Boileau Céline , de Philip Pascale , Pagès Sandrine , Fiérobe Henri-Pierre , Tardif Chantal 
Published: December 2007

ABSTRACT

The
cip-cel
cluster of genes plays an important role in the catabolism of the substrate cellulose by
Clostridium cellulolyticum
. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene,
cipC
. We analyzed the regulation of the
cipC
promoter using a transcriptional fusion approach. A single promoter is located between nucleotides −671 and −643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position −194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.
Large-Scale Transposon Mutagenesis ofPhotobacterium profundumSS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure
Authors: Lauro Federico M. , Tran Khiem , Vezzi Alessandro , Vitulo Nicola , Valle Giorgio , Bartlett Douglas H. 
Published: December 2007

ABSTRACTMicroorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacteriumPhotobacterium profundumstrain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.
Anaerobic Metabolism of Catechol by the Denitrifying Bacterium
Thauera aromatica
—a Result of Promiscuous Enzymes and Regulators?
Authors: Ding Bin , Schmeling Sirko , Fuchs Georg 
Published: December 2007

ABSTRACT

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium
Thauera aromatica
that was grown with CO
2
as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the
para
position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.