bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the
xplAB
Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by
Gordonia
sp. Strain KTR9
Authors: Indest Karl J. , Jung Carina M. , Chen Hao-Ping , Hancock Dawn , Florizone Christine , Eltis Lindsay D. , Crocker Fiona H. 
Published: August 2010

ABSTRACT

Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain
Gordonia
sp. KTR9. This plasmid carries
xplA
, encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with
xplA
are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator.
Rhodococcus jostii
RHA1 expressing
xplA
from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an
Escherichia coli
strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of
glnA-xplB
,
xplA
, and
xplR
showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.
Comparison of Single- and Multilocus Genetic Diversity in the Protozoan Parasites
Cryptosporidium parvum
and
C. hominis
Authors: Widmer Giovanni , Lee Yongsun 
Published: August 2010

ABSTRACT

The genotyping of numerous isolates of
Cryptosporidium
parasites has led to the definition of new species and a better understanding of the epidemiology of cryptosporidiosis. A single-locus genotyping method based on the partial sequence of a polymorphic sporozoite surface glycoprotein gene (
GP60
) has been favored by many for surveying
Cryptosporidium parvum
and
C. hominis
populations. Since genetically distinct
Cryptosporidium
parasites recombine in nature, it is unclear whether single-locus classifications can adequately represent intraspecies diversity. To address this question, we investigated whether multilocus genotypes of
C. parvum
and
C. hominis
cluster according to the
GP60
genotype.
C. hominis
multilocus genotypes did not segregate according to this marker, indicating that for this species the
GP60
sequence is not a valid surrogate for multilocus typing methods. In contrast, in
C. parvum
the previously described “anthroponotic” genotype was confirmed as a genetically distinct subspecies cluster characterized by a diagnostic GP60 allele. However, as in
C. hominis
, several
C. parvum
GP60 alleles did not correlate with distinct subpopulations. Given the rarity of some
C. parvum
GP60 alleles in our sample, the existence of additional
C. parvum
subgroups with unique GP60 alleles cannot be ruled out. We conclude that with the exception of genotypically distinct
C. parvum
subgroups, multilocus genotyping methods are needed to characterize
C. parvum
and
C. hominis
populations. Unless parasite virulence is controlled at the GP60 locus, attempts to find associations within species or subspecies between GP60 and phenotype are unlikely to be successful.
Transcriptomic Analysis of
Escherichia coli
O157:H7 and K-12 Cultures Exposed to Inorganic and Organic Acids in Stationary Phase Reveals Acidulant- and Strain-Specific Acid Tolerance Responses
Authors: King Thea , Lucchini Sacha , Hinton Jay C. D. , Gobius Kari 
Published: August 2010

ABSTRACT

The food-borne pathogen
Escherichia coli
O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of
E. coli
to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase
E. coli.
Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production.
E. coli
O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic
E. coli
strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While
E. coli
K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.
The Saccharomyces cerevisiae Anaphase-Promoting Complex Interacts with Multiple Histone-Modifying Enzymes To Regulate Cell Cycle Progression
Authors: Turner Emma L. , Malo Mackenzie E. , Pisclevich Marnie G. , Dash Megan D. , Davies Gerald F. , Arnason Terra G. , Harkness Troy A. A. 
Published: August 2010

ABSTRACT

The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G
1
. Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56
Ac
) levels were as reduced as those of total H3, indicating that loading histones with H3K56
Ac
is unaffected in APC mutants. However, under restrictive conditions, H3K9
Ac
and dimethylated H3K79 (H3K79
me2
) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the
apc5

CA

(chromatin assembly) mutant, we found that deletion of
GCN5
or
ELP3
severely hampered
apc5

CA

temperature-sensitive (ts) growth. Further analyses showed that (i) the
elp3
Δ
gcn5
Δ double mutant ts defect was epistatic to that observed in
apc5

CA

cells; (ii)
gcn5
Δ and
elp3
Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in
elp3
Δ
gcn5
Δ cells. Increased expression of
ELP3
and
GCN5
, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed
apc5

CA

defects, while increased
APC5
expression partially suppressed
elp3
Δ
gcn5
Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G
1
and following G
1
arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G
1
. To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.
Heterologous Expression of Candida albicans Cell Wall-Associated Adhesins in Saccharomyces cerevisiae Reveals Differential Specificities in Adherence and Biofilm Formation and in Binding Oral Streptococcus gordonii
Authors: Nobbs Angela H. , Vickerman M. Margaret , Jenkinson Howard F. 
Published: August 2010

ABSTRACT

Colonization and infection of the human host by opportunistic pathogen
Candida albicans
derive from an ability of this fungus to colonize mucosal tissues and prosthetic devices within the polymicrobial communities present. To determine the functions of
C. albicans
cell wall proteins in interactions with host or bacterial molecules,
Saccharomyces cerevisiae
was utilized as a surrogate host to express
C. albicans
cell wall proteins Als3p, Eap1p, Hwp1p, and Rbt1p. Salivary pellicle and fibrinogen were identified as novel substrata for Als3p and Hwp1p, while only Als3p mediated adherence of
S. cerevisiae
to basement membrane collagen type IV. Parental
S. cerevisiae
cells failed to form biofilms on salivary pellicle, polystyrene, or silicone, but cells expressing Als3p or Hwp1p exhibited significant attachment to each surface. Virulence factor Rbt1p also conferred lower-level binding to salivary pellicle and polystyrene.
S. cerevisiae
cells expressing Eap1p formed robust biofilms upon polystyrene surfaces but not salivary pellicle. Proteins Als3p and Eap1p, and to a lesser degree Hwp1p, conferred upon
S. cerevisiae
the ability to bind cells of the oral primary colonizing bacterium
Streptococcus gordonii
. These interactions, which occurred independently of amyloid aggregate formation, provide the first examples of specific
C. albicans
surface proteins serving as receptors for bacterial adhesins.
Streptococcus gordonii
did not bind parental
S. cerevisiae
or cells expressing Rbt1p. Taken collectively, these data suggest that a network of cell wall proteins comprising Als3p, Hwp1p, and Eap1p, with complementary adhesive functions, promotes interactions of
C. albicans
with host and bacterial molecules, thus leading to effective colonization within polymicrobial communities.
The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum
Authors: Phillips Jonathan E. , Gomer Richard H. 
Published: August 2010

ABSTRACT

AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in
Dictyostelium discoideum.
Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that
qkgA
−
cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking
qkgA
, and expression of QkgA-GFP in
qkgA
−
cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.
Phosphatidylinositol 3-Phosphate, an Essential Lipid in Plasmodium, Localizes to the Food Vacuole Membrane and the Apicoplast
Authors: Tawk Lina , Chicanne Gaëtan , Dubremetz Jean-François , Richard Véronique , Payrastre Bernard , Vial Henri J. , Roy Christian , Wengelnik Kai 
Published: August 2010

ABSTRACTPhosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasitePlasmodium falciparumestablishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile ofP. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. ThePlasmodiumgenome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted inPlasmodium berghei, andin vitrogrowth ofP. falciparumwas sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential inPlasmodiumblood stages. For intraparasitic PI3P localization, transgenicP. falciparumthat expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function inPlasmodiumappear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.
Ccq1p and the Condensin Proteins Cut3p and Cut14p Prevent Telomere Entanglements in the Fission Yeast Schizosaccharomyces pombe
Authors: Motwani Tina , Doris Rosemarie , Holmes Scott G. , Flory Mark R. 
Published: August 2010

ABSTRACT

The
Schizosaccharomyces pombe
telomere-associated protein Ccq1p has previously been shown to participate in telomerase recruitment, heterochromatin formation, and suppression of checkpoint activation. Here we characterize a critical role for Ccq1p in mitotic transit. We show that mitotic cells lacking Ccq1p lose minichromosomes at high frequencies but that conditional knockdown of Ccq1p expression results in telomere bridging within one cell cycle. Elevating Ccq1p expression resolves the telomere entanglements caused by decreased Taz1p activity. Ccq1p affects telomere resolution in the absence of changes in telomere size, indicating a role for Ccq1p that is independent of telomere length regulation. Using affinity purification, we identify the condensin proteins Cut3p and Cut14p as candidate Ccq1p interactors in this activity. Condensin loss-of-function disrupts Ccq1p telomeric localization and normal intertelomere clustering, while condensin overexpression relieves the chromosome segregation defects associated with conditional Ccq1p knockdown. These data suggest that Ccq1p and condensins collaborate to mediate resolution of telomeres in mitosis and regulate intertelomeric clustering during interphase.
Integration of Metabolism and Virulence by
Clostridium difficile
CodY
Authors: Dineen Sean S. , McBride Shonna M. , Sonenshein Abraham L. 
Published: August 2010

ABSTRACT

CodY, a global regulatory protein that monitors the nutrient sufficiency of the environment by responding to the intracellular levels of GTP and the branched-chain amino acids, was previously shown to be a potent repressor of toxin gene expression in
Clostridium difficile
during growth in rich medium. In the intestinal tract, such derepression of toxin synthesis would lead to destruction of epithelial cells and the liberation of potential nutrients for the bacterium. CodY is likely to play an important role in regulating overall cellular physiology as well. In this study, DNA microarray analysis and affinity purification of CodY-DNA complexes were used to identify and distinguish the direct and indirect effects of CodY on global gene transcription. A
codY
null mutation resulted in >4-fold overexpression of 146 genes (organized in 82 apparent transcription units) and underexpression of 19 genes. In addition to the toxin genes, genes for amino acid biosynthesis, nutrient transport, fermentation pathways, membrane components, and surface proteins were overexpressed in the
codY
mutant. Genome-wide analysis identified more than 350 CodY binding regions, many of which are likely to correspond to sites of direct CodY-mediated regulation. About 60% of the CodY-repressed transcription units were associated with binding regions. Several of these genes were confirmed to be direct targets of CodY by gel mobility shift and DNase I footprinting assays.
CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in
Caulobacter crescentus
Authors: Braz Vânia S. , da Silva Neto José F. , Italiani Valéria C. S. , Marques Marilis V. 
Published: August 2010

ABSTRACT

Caulobacter crescentus
is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a
C. crescentus
mutant strain with a mini-Tn
5lacZ
transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a
lacZ
reporter gene. β-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent
cztA
gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The
cztR
strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to
tert
-butyl hydroperoxide. The expression of
katG
and
ahpC
, but not that of the superoxide dismutase genes, was increased in the
cztR
mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate
cztA
expression and repress its own transcription.
Domain Analysis of a Modular α-
l
-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium
Fibrobacter succinogenes
S85
Authors: Yoshida Shosuke , Hespen Charles W. , Beverly Robert L. , Mackie Roderick I. , Cann Isaac K. O. 
Published: August 2010

ABSTRACT

Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from
Fibrobacter succinogenes
S85. The recombinant protein showed α-
l
-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The
F. succinogenes
AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and
F. succinogenes
-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher
k
cat
for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the

K
m

for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.
Small Genes under Sporulation Control in the
Bacillus subtilis
genome
Authors: Schmalisch Matthias , Maiques Elisa , Nikolov Lachezar , Camp Amy H. , Chevreux Bastien , Muffler Andrea , Rodriguez Sabrina , Perkins John , Losick Richard 
Published: August 2010

ABSTRACT

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of
Bacillus subtilis
, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor σ
E
or σ
K
. A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor σ
E
, and another was under the control of the motility sigma factor σ
D
.
Brochothrix thermosphactaBacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites
Authors: Kilcher Samuel , Loessner Martin J. , Klumpp Jochen 
Published: August 2010

ABSTRACTBrochothrixbelongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related toListeria,Staphylococcus,Clostridium, andBacillus. Brochothrix thermosphactais a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization ofBrochothrixbacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to theSpounavirinaesubfamily and shares significant homologies withListeriaphage A511,Staphylococcusphage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness toListeriaphages but rather demonstrate relatedness to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed −1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3′ end of a RNA methyltransferase, whereas NF5 integrates into the 5′-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.