bluuebunny/crossref_metadata_2025_split huggingface dataset:
Copine A Is Required for Cytokinesis, Contractile Vacuole Function, and Development inDictyostelium
Authors: Damer Cynthia K. , Bayeva Marina , Kim Pamela S. , Ho Lilian K. , Eberhardt Eric S. , Socec Catherine I. , Lee Jennifer S. , Bruce Emily A. , Goldman-Yassen Adam E. , Naliboff Lauren C. 
Published: January 2007

ABSTRACTCopines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in theDictyostelium discoideumgenome and focused our studies oncpnA. Our previous localization studies of green fluorescent protein-tagged CpnA inDictyosteliumsuggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created acpnA−knockout strain, and here we report the initial characterization of the mutant phenotype. ThecpnA−cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions,cpnA−cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water,cpnA−cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for thecpnA−cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes exceptcpnFshowed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators ofDictyosteliumdevelopment.
Cellular Processes and Pathways That Protect
Saccharomyces cerevisiae
Cells against the Plasma Membrane-Perturbing Compound Chitosan
Authors: Zakrzewska Anna , Boorsma Andre , Delneri Daniela , Brul Stanley , Oliver Stephen G. , Klis Frans M. 
Published: January 2007

ABSTRACT

Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to chitosan, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with
t
statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG (high-osmolarity glycerol) signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized
Saccharomyces cerevisiae
cells to chitosan. Intriguingly, the GO-defined group of deletion strains belonging to the “cytosolic large ribosomal subunit” was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions.
Spliced Leader RNA Gene Transcription in
Trypanosoma brucei
Requires Transcription Factor TFIIH
Authors: Lee Ju Huck , Nguyen Tu N. , Schimanski Bernd , Günzl Arthur 
Published: January 2007

ABSTRACT

Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL)
trans
splicing and polyadenylation. In
trans
splicing, the SL RNA is consumed through a transfer of its 5′-terminal part to the 5′ end of mRNAs. Since all mRNAs are
trans
spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in
Trypanosoma brucei
, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although
T. brucei
TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a
T. brucei
TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in
T. brucei
affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.
Enhanced Post-Stationary-Phase Survival of a Clinical Thymidine-Dependent Small-Colony Variant of
Staphylococcus aureus
Results from Lack of a Functional Tricarboxylic Acid Cycle
Authors: Chatterjee Indranil , Herrmann Mathias , Proctor Richard A. , Peters Georg , Kahl Barbara C. 
Published: January 2007

ABSTRACT

The mechanisms underlying the persistence of the
Staphylococcus aureus
small-colony variant (SCV) are not fully elucidated. In this study, clinical thymidine-dependent SCVs displayed altered expression of
citB
,
clpC
, and
arcA
genes, reduced acetate catabolization, and enhanced survival. These results implicate the importance of changes in tricarboxylic acid cycle and acetic acid metabolism in SCV survival and persistence.
Products of theEscherichia coliAcid Fitness Island Attenuate Metabolite Stress at Extremely Low pH and Mediate a Cell Density-Dependent Acid Resistance
Authors: Mates Aaron K. , Sayed Atef K. , Foster John W. 
Published: January 2007

ABSTRACTEscherichia colihas an ability, rare among theEnterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes,gadA,gadX,gadW, andgadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protectedE. colifrom organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF- and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes,yhiDandhdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>108CFU per ml). Density-dependent acid resistance does not require any demonstrable secreted factor and may involve cell contact-dependent activation. These findings further define the complex physiology ofE. coliacid resistance.
Mutational Analysis of the
Escherichia coli
DEAD Box Protein CsdA
Authors: Turner Anne-Marie W. , Love Cheraton F. , Alexander Rebecca W. , Jones Pamela G. 
Published: January 2007

ABSTRACT

The
Escherichia coli
cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3′ or 5′ extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157→Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the
csdA
null mutant at 15°C. Plasmid-encoded CsdAΔ444, which lacks most of the carboxy-terminal extension, enhanced the growth of a
csdA
null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.
Identification of the Initial Steps in
d
-Lysine Catabolism in
Pseudomonas putida
Authors: Revelles Olga , Wittich Rolf-Michael , Ramos Juan L. 
Published: January 2007

ABSTRACT

Pseudomonas putida
uses
l
-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways δ-aminovalerate is the key metabolite, whereas in the other
l
-lysine is racemized to
d
-lysine, and
l
-pipecolate and α-aminoadipate are the key metabolites. All the genes and enzymes involved in the
d
-lysine pathway, except for those involved in the conversion of
d
-lysine into Δ
1
-piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of
d
-lysine into Δ
1
-piperideine-2-carboxylate can be mediated by a
d
-lysine aminotransferase (PP3590) and a
d
-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of
d
-lysine since its inactivation leads to a marked reduction in the growth rate with
l
- or
d
-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here
amaC
, forms an operon with
dpkA
, the gene encoding the enzyme involved in conversion of Δ
1
-piperideine-2-carboxylate to
l
-pipecolate in the
d
-lysine catabolic pathway. The gene encoding PP3596, called here
amaD
, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The
dpkA amaC
operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent −35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to
l
- or
d
-lysine in the culture medium.
MalE of Group A
Streptococcus
Participates in the Rapid Transport of Maltotriose and Longer Maltodextrins
Authors: Shelburne Samuel A. , Fang Han , Okorafor Nnaja , Sumby Paul , Sitkiewicz Izabela , Keith David , Patel Payal , Austin Celest , Graviss Edward A. , Musser James M. , Chow Dar-Chone 
Published: January 2007

ABSTRACT

Study of the maltose/maltodextrin binding protein MalE in
Escherichia coli
has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A
Streptococcus
(GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for
E. coli
MalE. Moreover, unlike
E. coli
MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied
E. coli
counterpart.
Phage 3396 from a
Streptococcus dysgalactiae
subsp.
equisimilis
Pathovar May Have Its Origins in
Streptococcus pyogenes
Authors: Davies Mark R. , McMillan David J. , Van Domselaar Gary H. , Jones Malcolm K. , Sriprakash Kadaba S. 
Published: January 2007

ABSTRACT

Streptococcus dysgalactiae
subsp.
equisimilis
strains (group G streptococcus [GGS]) are largely defined as commensal organisms, which are closely related to the well-defined human pathogen, the group A streptococcus (GAS). While lateral gene transfers are emerging as a common theme in these species, little is known about the mechanisms and role of these transfers and their effect on the population structure of streptococci in nature. It is now becoming evident that bacteriophages are major contributors to the genotypic diversity of GAS and, consequently, are pivotal to the GAS strain structure. Furthermore, bacteriophages are strongly associated with altering the pathogenic potential of GAS. In contrast, little is know about phages from GGS and their role in the population dynamics of GGS. In this study we report the first complete genome sequence of a GGS phage, Φ3396. Exhibiting high homology to the GAS phage Φ315.1, the chimeric nature of Φ3396 is unraveled to reveal evidence of extensive ongoing genetic diversity and dissemination of streptococcal phages in nature. Furthermore, we expand on our recent findings to identify inducible Φ3396 homologues in GAS from a region of endemicity for GAS and GGS infection. Together, these findings provide new insights into not only the population structure of GGS but also the overall population structure of the streptococcal genus and the emergence of pathogenic variants.
Properties of
R
-Citramalyl-Coenzyme A Lyase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of
Chloroflexus aurantiacus
Authors: Friedmann Silke , Alber Birgit E. , Fuchs Georg 
Published: January 2007

ABSTRACT

The autotrophic CO
2
fixation pathway (3-hydroxypropionate cycle) in
Chloroflexus aurantiacus
results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO
2
acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO
2
) to β-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both
S-
and
R-
citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the
S-
and
R-
citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth.
S-
Citramalyl-CoA lyase activity was found to be due to
l
-malyl-CoA lyase/β-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO
2
fixation pathway, forms acetyl-CoA and glyoxylate from
l
-malyl-CoA, and condenses glyoxylate with propionyl-CoA to β-methylmalyl-CoA.
R-
Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg
2
or Mn
2+
ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in
C. aurantiacus
. Genes similar to those involved in CO
2
fixation in
C. aurantiacus
, including an
R-
citramalyl-CoA lyase gene, were found in
Roseiflexus
sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the γ-proteobacterium
Congregibacter litoralis
. This may indicate that part of the reactions may be involved in a different metabolic process.
Functional Interactions between Coexisting Toxin-Antitoxin Systems of the
ccd
Family in
Escherichia coli
O157:H7
Authors: Wilbaux Myriam , Mine Natacha , Guérout Anne-Marie , Mazel Didier , Van Melderen Laurence 
Published: January 2007

ABSTRACT

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the
ccd
TA system of the F plasmid (
ccd
F
) located in the chromosomal backbone of the pathogenic O157:H7
Escherichia coli
strain (
ccd
O157
). The
ccd
F
and the
ccd
O157
systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the
ccd
F
system. We have shown that the chromosomal
ccd
O157
system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB
O157
toxin targets the DNA gyrase, and the CcdA
O157
antitoxin is degraded by the Lon protease. The
ccd
O157
chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of
ccd
F
.
ccd
O157
is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the
ccd
F
system is able to mediate postsegregational killing in an
E. coli
strain harboring the
ccd
O157
system in its chromosome. This shows that the plasmidic
ccd
F
system is functional in the presence of its chromosomal counterpart.
Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance
Authors: Matsumi Rie , Manabe Kenji , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki 
Published: January 2007

ABSTRACTWe have developed a gene disruption system in the hyperthermophilic archaeonThermococcus kodakaraensisusing the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted theT. kodakaraensisamylopullulanase gene (apuTk) or a gene cluster which includesapuTkand genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-typeT. kodakaraensisKOD1 cells, and transformants exhibiting resistance to 4 μM simvastatin were isolated. The transformants exhibited growth in the presence of 20 μM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at thehmgTklocus when the endogenoushmgTkgene was used. This could be avoided by using the corresponding gene fromPyrococcus furiosus(hmgPf) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ΔapuTkstrain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that ApuTkis a major polysaccharide-degrading enzyme inT. kodakaraensis.
The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in
Bacillus cereus
F4430/73
Authors: Zigha Assia , Rosenfeld Eric , Schmitt Philippe , Duport Catherine 
Published: January 2007

ABSTRACT

Glucose-grown cells of
Bacillus cereus
respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the
B. cereus
genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the
Bacillus subtilis
Fnr redox regulator. The
fnr
gene of the food-borne pathogen
B. cereus
F4430/73 has been cloned and partially characterized. We showed that
fnr
was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of
fnr
was down-regulated in the presence of O
2
and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of
B. cereus fnr
abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on
B. cereus
fermentative pathways. More importantly, the
fnr
mutation strongly decreased the expression of PlcR-dependent
hbl
and
nhe
genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that
fnr
is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression.
The Quorum-Sensing Hybrid Histidine Kinase LuxN of
Vibrio harveyi
Contains a Periplasmically Located N Terminus
Authors: Jung Kirsten , Odenbach Tina , Timmen Melanie 
Published: January 2007

ABSTRACT

Hydropathy profile analyses of the amino acid sequence of the quorum-sensing hybrid histidine kinase LuxN of
Vibrio harveyi
predict a periplasmic location of the N terminus. To test this, two-hybrid proteins consisting of LuxN and an N-terminally fused maltose-binding protein with or without a leader sequence were analyzed with regard to the enzymatic activities of LuxN, protease accessibility, and complementation of an
Escherichia coli malE
mutant. The results strongly support a periplasmic location of the N terminus, implying that LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane.