bluuebunny/crossref_metadata_2025_split huggingface dataset:
C2 Domain Protein MIN1 Promotes Eyespot Organization inChlamydomonas reinhardtii
Authors: Mittelmeier Telsa M. , Berthold Peter , Danon Avihai , Lamb Mary Rose , Levitan Alexander , Rice Michael E. , Dieckmann Carol L. 
Published: October 2008

ABSTRACTAssembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green algaChlamydomonas reinhardtiirequires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast.min1(mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. TheMIN1gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly inChlamydomonas. Inmin1null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth ofmin1cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.
Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes
Authors: Chandler Julian , Vandoros Anthula V. , Mozeleski Brian , Klingbeil Michele M. 
Published: October 2008

ABSTRACT

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from
Trypanosoma brucei
with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic
T. brucei
. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol γ) for replication of mitochondrial DNA,
T. brucei
requires at least three to maintain the complex kDNA network.
In Vitro CpG Methylation Increases the Transformation Efficiency ofBorrelia burgdorferiStrains Harboring the Endogenous Linear Plasmid lp56
Authors: Chen Qiang , Fischer Joshua R. , Benoit Vivian M. , Dufour Nicholas P. , Youderian Philip , Leong John M. 
Published: October 2008

ABSTRACTBorrelia burgdorferiis the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains ofB. burgdorferiexhibit extremely low transformation efficiencies—so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poorB. burgdorferitransformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that forB. burgdorferistrains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation ofB. burgdorferiwas exemplified by the ease of plasmid complementation of aB. burgdorferiB31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kanr) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, theB. burgdorferiB31 A3 BBK32::Kanrmutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.
Flexibility of
Vibrio cholerae
ToxT in Transcription Activation of Genes Having Altered Promoter Spacing
Authors: Bellair Michelle , Withey Jeffrey H. 
Published: October 2008

ABSTRACT

Cholera, a severe diarrheal disease, is caused by ingestion of the gram-negative bacterium
Vibrio cholerae
. Expression of
V. cholerae
virulence factors is highly regulated at the transcriptional and posttranscriptional levels by a complex network of proteins and small noncoding RNAs. The direct activator of transcription of most
V. cholerae
virulence genes is the ToxT protein. ToxT binds to a 13-bp sequence, the toxbox, located upstream of genes in its regulon. However, the organization of toxboxes relative to each other and to the core promoter elements at different genes varies dramatically. At different ToxT-activated genes a single toxbox may be necessary and sufficient for full activation, or pairs of toxboxes organized as either inverted or direct repeats may be required for full activation. Although all toxboxes are located at positions consistent with a class I promoter architecture, the locations of toxboxes relative to the transcription start site also vary from gene to gene. To further assess the ability of ToxT to activate transcription from different configurations relative to the core promoter elements, we constructed promoter-
lacZ
fusions having altered spacing both between toxbox pairs and between the promoter-proximal toxbox and the −35 box at five different ToxT-activated promoters. Our results suggest that that ToxT has remarkable flexibility in its positioning as a transcription activator and that different interactions between ToxT and RNA polymerase occur during transcription activation of promoters having different toxbox configurations.
The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the
l
-Lactate Dehydrogenase LdhA in
Corynebacterium glutamicum
Authors: Engels Verena , Lindner Steffen N. , Wendisch Volker F. 
Published: October 2008

ABSTRACT

The transcriptional regulator SugR from
Corynebacterium glutamicum
represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of
sugR
not only perturbed the growth of
C. glutamicum
on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (
pfkA
), fructose-1,6-bisphosphate aldolase (
fba
), enolase (
eno
), pyruvate kinase (
pyk
), and NAD-dependent
l
-lactate dehydrogenase (
ldhA
). The deletion of
sugR
resulted in increased mRNA levels of
eno
,
pyk
, and
ldhA
in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of
sugR
led to increased LdhA activity under aerobic and under oxygen deprivation conditions,
l
-lactate production by
C. glutamicum
was determined. The overexpression of
sugR
reduced
l
-lactate production by about 25%, and
sugR
deletion increased
l
-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative
l
-lactate dehydrogenase in
C. glutamicum
.
Dimethylsulfoniopropionate-Dependent Demethylase (DmdA) from
Pelagibacter ubique
and
Silicibacter pomeroyi
Authors: Reisch Chris R. , Moran Mary Ann , Whitman William B. 
Published: October 2008

ABSTRACT

The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria
Pelagibacter ubique
HTCC1062 and
Silicibacter pomeroyi
DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The
P. ubique
enzyme possessed an
M
r
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the
M
r
was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both
S. pomeroyi
and
P. ubique
possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent

K
m

s for DMSP were 13.2 ± 2.0 and 5.4 ± 2.3 mM for the
P. ubique
and
S. pomeroyi
enzymes, respectively. In cell extracts of
S. pomeroyi
DSS-3, the apparent

K
m

for DMSP was 8.6 ± 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown
S. pomeroyi
DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.
Bacterial Flagella Are Firmly Anchored
Authors: Darnton Nicholas C. , Berg Howard C. 
Published: October 2008

ABSTRACT

There are mutants of
Salmonella enterica
(with mutations in
fliF
and
fliL
) that shed flagella when they are swimming in a viscous medium or on the surface of soft agar. Filaments with hooks and the distal rod segment FlgG are recovered. We tried to extract flagellar filaments from such cells by pulling on them with an optical trap but failed, even when we used forces large enough to straighten the filaments. Thus, flagella are firmly anchored.
Site-Directed Mutagenesis Reveals Putative Substrate Binding Residues in the
Escherichia coli
RND Efflux Pump AcrB
Authors: Bohnert Jürgen A. , Schuster Sabine , Seeger Markus A. , Fähnrich Eva , Pos Klaas M. , Kern Winfried V. 
Published: October 2008

ABSTRACT

The
Escherichia coli
multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. We found that F610A was the point mutation that had the most significant impact on substrate MICs, while other targeted mutations, including conversion of phenylalanines 136, 178, 615, 617, and 628 to alanine, had smaller and more variable effects.
Genetic Analysis ofVibrio choleraeMonolayer Formation Reveals a Key Role for ΔΨ in the Transition to Permanent Attachment
Authors: Van Dellen Katrina L. , Houot Laetitia , Watnick Paula I. 
Published: October 2008

ABSTRACTA bacterial monolayer biofilm is a collection of cells attached to a surface but not to each other. Monolayer formation is initiated when a bacterial cell forms a transient attachment to a surface. While some transient attachments are broken, others transition into the permanent attachments that define a monolayer biofilm. In this work, we describe the results of a large-scale, microscopy-based genetic screen forVibrio choleraemutants that are defective in formation of a monolayer biofilm. This screen identified mutations that alter both transient and permanent attachment. Transient attachment was somewhat slower in the absence of flagellar motility. However, flagellar mutants eventually formed a robust monolayer. In contrast, in the absence of the flagellar motor, monolayer formation was severely impaired. A number of proteins that modulate theV. choleraeion motive force were also found to affect the transition from transient to permanent attachment. Using chemicals that dissipate various components of the ion motive force, we discovered that dissipation of the membrane potential (ΔΨ) completely blocks the transition from transient to permanent attachment. We propose that as a bacterium approaches a surface, the interaction of the flagellum with the surface leads to transient hyperpolarization of the bacterial cell membrane. This, in turn, initiates the transition to permanent attachment.
The Biosynthetic Pathway for Synechoxanthin, an Aromatic Carotenoid Synthesized by the Euryhaline, Unicellular Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Graham Joel E. , Bryant Donald A. 
Published: October 2008

ABSTRACT

The euryhaline, unicellular cyanobacterium
Synechococcus
sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in
Synechococcus
sp. strain PCC 7002 to verify their function. The
cruE
gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The
cruH
gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.
Loss of RNase R Induces Competence Development in
Legionella pneumophila
Authors: Charpentier Xavier , Faucher Sébastien P. , Kalachikov Sergey , Shuman Howard A. 
Published: October 2008

ABSTRACT

RNase R is a processive 3′-5′ exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3′-5′ exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen
Legionella pneumophila
. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of
Legionella pneumophila
under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, the loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at a lower temperature, the loss of RNase R had a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the
rnr
mutant was found to be independent of previously identified competence regulators in
Legionella pneumophila
. We suggest that a physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability.
Structure of
l
-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic
l
-Ascorbate Utilization Pathway of
Escherichia coli
: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold
Authors: Shi Rong , Pineda Marco , Ajamian Eunice , Cui Qizhi , Matte Allan , Cygler Miroslaw 
Published: October 2008

ABSTRACT

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of
l
-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce
l
-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (
l
-xylulose-5-phosphate 3-epimerase) and UlaF (
l
-ribulose-5-phosphate 4-epimerase), yielding
d
-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from
Escherichia coli
O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn
2+
, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the
l
-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.
Discovering Functional Novelty in Metagenomes: Examples from Light-Mediated Processes
Authors: Singh Amoolya H. , Doerks Tobias , Letunic Ivica , Raes Jeroen , Bork Peer 
Published: October 2008

ABSTRACT
The emerging coverage of diverse habitats by metagenomic shotgun data opens new avenues of discovering functional novelty using computational tools. Here, we apply three different concepts for predicting novel functions within light-mediated microbial pathways in five diverse environments. Using phylogenetic approaches, we discovered two novel deep-branching subfamilies of photolyases (involved in light-mediated repair) distributed abundantly in high-UV environments. Using neighborhood approaches, we were able to assign seven novel functional partners in luciferase synthesis, nitrogen metabolism, and quorum sensing to BLUF domain-containing proteins (involved in light sensing). Finally, by domain analysis, for RcaE proteins (involved in chromatic adaptation), we predict 16 novel domain architectures that indicate novel functionalities in habitats with little or no light. Quantification of protein abundance in the various environments supports our findings that bacteria utilize light for sensing, repair, and adaptation far more widely than previously thought. While the discoveries illustrate the opportunities in function discovery, we also discuss the immense conceptual and practical challenges that come along with this new type of data.
Oxidant-Responsive Induction of the
suf
Operon, Encoding a Fe-S Assembly System, through Fur and IscR in
Escherichia coli
Authors: Lee Kyung-Chang , Yeo Won-Sik , Roe Jung-Hye 
Published: October 2008

ABSTRACT

The
suf
operon encoding a Fe-S assembly system is induced by peroxides through activators OxyR and IscR in
Escherichia coli
. For apo-IscR to bind, oxidation-mediated dissociation of Fur is required. Therefore, a peroxide-responsive signal is transduced through OxyR, IscR, and Fur to achieve oxidation-sensitive and maximal induction of this operon.
Avoidance of smoking: the impact of warning labels in Brazil
Authors: Nascimento B E M , Oliveira L , Vieira A S , Joffily M , Gleiser S , Pereira M G , Cavalcante T , Volchan E 
Published: October 2008

Background:Research on human emotion shows that pictures drive the activity of specialised brain networks affecting attitude and behaviour. Pictorial warnings on cigarette packages are considered one of the most effective ways to convey information on the health consequences of smoking. However, few studies have evaluated the effectiveness of warning labels to elicit avoidance of smoking.Objectives:To investigate the impact of pictorial health warnings conveyed by the Brazilian tobacco control programme through a well-established psychometric tool designed for studies on emotion and behaviour.Methods:Graphic Brazilian cigarette warnings labels were evaluated. They consisted of the two sets of warning pictures displayed in 2002–4 (n = 9) and 2004–8 (n = 10). Pleasant, unpleasant and neutral pictures selected from a standard catalogue were used as controls. Undergraduate students (n = 212, 18% smokers) evaluated the emotional content of each picture in two affective dimensions: hedonic valence and arousal. Participants were not provided with the sources of distinction between control and warning pictures.Results:The judgements of hedonic content of the warning pictures ranged from neutral to very unpleasant. None was classified as highly arousing. Smokers judged warning pictures representing people smoking significantly more pleasant than pictures without smoking scenes, and significantly more so than non-smokers. No significant differences between smokers and non-smokers were found for warning pictures without these smoking scenes.Conclusion:Previous studies have shown that the most threatening and arousing pictures prompt the greatest evidence of defensive activation. Emotional ratings of Brazilian warning pictures described them as unpleasant but moderately arousing. To intensify avoidance of the packages, future graphic warnings should therefore generate more arousal. The ratings for the Brazilian warning pictures indicated that, except for those depicting people smoking, judgements by smokers and non-smokers were similar, suggesting a potential applicability in both prevention and cessation. Smoking cues, however, should be avoided.