bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unconventional Mode of Attachment of the
Ruminococcus flavefaciens
Cellulosome to the Cell Surface
Authors: Rincon Marco T. , Čepeljnik Tadej , Martin Jennifer C. , Lamed Raphael , Barak Yoav , Bayer Edward A. , Flint Harry J. 
Published: November 2005

ABSTRACT

Sequence extension of the scaffoldin gene cluster from
Ruminococcus flavefaciens
revealed a new gene (
scaE
) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in
Escherichia coli
and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulose-bound
Ruminococcus flavefaciens
protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in
R. flavefaciens
differs from those reported thus far for any other cellulosome system.
Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid Biosynthesis, Leads to Mycobacterial Cell Lysis
Authors: Bhatt Apoorva , Kremer Laurent , Dai Annie Z. , Sacchettini James C. , Jacobs William R. 
Published: November 2005

ABSTRACT

Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two β-ketoacyl-acyl carrier protein synthases, KasA and KasB, in
Mycobacterium smegmatis
. Using specialized transduction for allelic exchange, null
kasB
mutants, but not
kasA
mutants, could be generated in
Mycobacterium smegmatis
, suggesting that unlike
kasB
,
kasA
is essential. To confirm the essentiality of
kasA
, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (
c
onditional
e
xpression
s
pecialized
t
ransduction
e
ssentiality
t
est), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null
inhA
and
kasA
mutants. We studied the effects of depletion of KasA in
M. smegmatis
using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of α and epoxy mycolate biosynthesis and to accumulation of α′-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a “crumpled” appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.
Roles of the
Escherichia coli
RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In Vivo
Authors: Maul Robert W. , Sutton Mark D. 
Published: November 2005

ABSTRACT

The
Escherichia coli
β sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the
dnaN159
allele, which encodes a mutant form of the β clamp (β159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in
E. coli
, we have further characterized effects of the
dnaN159
allele on polymerase usage. Three of the five
E.coli
DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the
dinB
-encoded polymerase IV is sufficient to result in conditional lethality of the
dnaN159
strain. In contrast, chronically activated RecA protein, expressed from the
recA730
allele, is lethal to the
dnaN159
strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., Δ
recR
), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., Δ
polB
or Δ
umuDC
). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the
dnaN159
strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.
Bacillus subtilis
Cysteine Synthetase Is a Global Regulator of the Expression of Genes Involved in Sulfur Assimilation
Authors: Albanesi Daniela , Mansilla Maria Cecilia , Schujman Gustavo E. , de Mendoza Diego 
Published: November 2005

ABSTRACT

The synthesis of
l
-cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In
Bacillus subtilis
the
cysH
operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the
cysK
gene encoding an
O
-acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the
cysH
operon. Using DNA microarrays we found that, in addition to
cysH
, almost all of the genes required for sulfate assimilation are constitutively expressed in
cysK
mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.
Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence ofCorynebacterium glutamicum
Authors: Marienhagen Jan , Kennerknecht Nicole , Sahm Hermann , Eggeling Lothar 
Published: November 2005

ABSTRACTTwenty putative aminotransferase (AT) proteins ofCorynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others withl-glutamate,l-aspartate, andl-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference)l-glutamate > 2-aminobutyrate >l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizesl-alanine to aminate 2-oxo-isovalerate, thel-valine precursor, and 2-oxo-butyrate. A second AT active with thel-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs forl-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.
The Twin-Arginine Translocation Pathway of
Mycobacterium smegmatis
Is Functional and Required for the Export of Mycobacterial β-Lactamases
Authors: McDonough Justin A. , Hacker Kari E. , Flores Anthony R. , Pavelka Martin S. , Braunstein Miriam 
Published: November 2005

ABSTRACT

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of
M. tuberculosis
and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of Δ
tatA
and Δ
tatC
deletion mutants of
M. smegmatis
, which demonstrated that
tatA
and
tatC
encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active β-lactamases of
M. smegmatis
(BlaS) and
M. tuberculosis
(BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of
M. tuberculosis
phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active ′BlaC. Thus, ′BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.
Evidence for Acquisition ofLegionellaType IV Secretion Substrates via Interdomain Horizontal Gene Transfer
Authors: de Felipe Karim Suwwan , Pampou Sergey , Jovanovic Oliver S. , Pericone Christopher D. , Ye Senna F. , Kalachikov Sergey , Shuman Howard A. 
Published: November 2005

ABSTRACTIntracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the γ-proteobacteriumLegionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells.L. pneumophilatranslocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of theL. pneumophilaPhiladelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G+C biases compared to otherL. pneumophilagenes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that inL. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.
Mapping of a YscY Binding Domain within the LcrH Chaperone That Is Required for Regulation of
Yersinia
Type III Secretion
Authors: Bröms Jeanette E. , Edqvist Petra J. , Carlsson Katrin E. , Forsberg Åke , Francis Matthew S. 
Published: November 2005

ABSTRACT

Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of
Yersinia pseudotuberculosis
. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native
Pseudomonas aeruginosa
or when produced in
Yersinia.
In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the
Yersinia
type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of
P. aeruginosa.
YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of
Yersinia
and Pcr3 of
P. aeruginosa.
Still, Pcr4 was unable to complement a Δ
yscY
null mutant defective for type III secretion and
yop-
regulatory control in
Yersinia
, despite the ability of YscY to function in
P. aeruginosa.
Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to
Yersinia
type III secretion.
CapE, a 47-Amino-Acid Peptide, Is Necessary for
Bacillus anthracis
Polyglutamate Capsule Synthesis
Authors: Candela Thomas , Mock Michèle , Fouet Agnès 
Published: November 2005

ABSTRACT

Polyglutamate is found in various bacteria, but displays different functions depending on the species and their environment. Here, we describe a minimal polyglutamate synthesis system in
Bacillus anthracis
. In addition to the three genes previously described as sufficient for polyglutamate synthesis, this system includes a small open reading frame,
capE
, belonging to the
cap
operon. The polyglutamate system's requirement for the five
cap
genes, for capsulation and anchoring, was assayed in nonpolar mutants. The
capA
,
capB
,
capC
, and
capE
genes are all necessary and are sufficient for polyglutamate synthesis by
B. anthracis. capD
is required for polyglutamate anchoring to the peptidoglycan. The 47-amino-acid peptide encoded by
capE
is localized in the
B. anthracis
membrane. It is not a regulator and it is required for polyglutamate synthesis, suggesting that it has a structural role in polyglutamate synthesis. CapE appears to interact with CapA.
Bacillus subtilis ywtC
is similar to
capE
and we named it
pgsE
. Genes similar to
capE
or
pgsE
were found in
B. subtilis
natto,
Bacillus licheniformis
, and
Staphylococcus epidermidis
, species that produce polyglutamate. All the bacterial polyglutamate synthesis systems analyzed show a similar genetic organization and, we suggest, the same protein requirements.
Characterization of
Mycobacterium tuberculosis
Rv3676 (CRP
Mt
), a Cyclic AMP Receptor Protein-Like DNA Binding Protein
Authors: Bai Guangchun , McCue Lee Ann , McDonough Kathleen A. 
Published: November 2005

ABSTRACT

Little is known about cyclic AMP (cAMP) function in
Mycobacterium tuberculosis
, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins.
M. tuberculosis
Rv3676, which we have designated CRP
Mt
, is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP
Mt
's interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP
Mt
DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for
Escherichia coli
. CRP
Mt
binding sites were identified in a total of 73 promoter regions regulating 114 genes in the
M. tuberculosis
genome, which are being explored as a regulon. Specific CRP
Mt
binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP
Mt
. cAMP enhanced CRP
Mt
's ability to bind DNA and caused allosteric alterations in CRP
Mt
conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in
M. tuberculosis
, suggesting a role for cAMP signal transduction in
M. tuberculosis
and implicating CRP
Mt
as a cAMP-responsive global regulator.
Alternate Sigma Factor RpoS Is Required for the In Vivo-Specific Repression ofBorrelia burgdorferiPlasmid lp54-BorneospAandlp6.6Genes
Authors: Caimano Melissa J. , Eggers Christian H. , Gonzalez Cynthia A. , Radolf Justin D. 
Published: November 2005

ABSTRACTWhile numerous positively regulated loci have been characterized during the enzootic cycle ofBorrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependentB. burgdorferiloci,ospAandlp6.6. The downregulation ofospAandIp6.6appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with theospAandIp6.6promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.
Tryptophan Catabolism: Identification and Characterization of a New Degradative Pathway
Authors: Colabroy Keri L. , Begley Tadhg P. 
Published: November 2005

ABSTRACT

A new tryptophan catabolic pathway is characterized from
Burkholderia cepacia
J2315. In this pathway, tryptophan is converted to 2-amino-3-carboxymuconate semialdehyde, which is enzymatically degraded to pyruvate and acetate via the intermediates 2-aminomuconate and 4-oxalocrotonate. This pathway differs from the proposed mammalian pathway which converts 2-aminomuconate to 2-ketoadipate and, ultimately, glutaryl-coenzyme A.
Rac3-Mediated Transformation Requires Multiple Effector Pathways
Authors: Keller Patricia J. , Gable Christyn M. , Wing Michele R. , Cox Adrienne D. 
Published: November 2005

Abstract
Our initial characterization of Rac3, a close relative of the small GTPase Rac1, established its ability to promote membrane ruffling, transformation, and activation of c-jun transcriptional activity. The finding that Rac3 is transforming, and its similarity to Rac1, a protein that has a well-established connection to many processes important for cancer progression, prompted further investigation into Rac3 transformation. We used effector domain mutants (EDMs) to explore the relationship among Rac signaling, transformation, and effector usage. All Rac3 EDMs tested (N26D, F37L, Y40C, and N43D) retained the ability to promote membrane ruffling and focus formation. In contrast, only the N43D mutant promoted anchorage independence. This differs from Rac1, where both N26D and N43D mutants were impaired in both types of transformation. To learn more about the signaling pathways involved, we did luciferase reporter assays and glutathione S-transferase pull-down assays for effector binding. We found evidence for a functional link between activation of phospholipase Cβ2 by Rac3 and signaling to the serum response factor (SRF). Surprisingly, we also found that Rac3 binds poorly to the known Rac1 effectors mixed lineage kinases 2 and 3 (MLK2 and MLK3). Transcription of cyclin D1 was the only pathway that correlated with growth in soft agar. Our experiments show that activation of membrane ruffling and transcriptional activation of c-jun, SRF, or E2F are not sufficient to promote anchorage-independent growth mediated by Rac3. Instead, multiple effector pathways are required for Rac3 transformation, and these overlap partially but not completely with those used by Rac1.
Quantification of Cellular Proliferation in Tumor and Normal Tissues of Patients with Breast Cancer by [18F]Fluorothymidine-Positron Emission Tomography Imaging: Evaluation of Analytical Methods
Authors: Kenny Laura M. , Vigushin David M. , Al-Nahhas Adil , Osman Safiye , Luthra Sajinder K. , Shousha Sami , Coombes R. Charles , Aboagye Eric O. 
Published: November 2005

AbstractThere is an unmet need to develop imaging methods for the early and objective assessment of breast tumors to therapy. 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT)–positron emission tomography represents a new approach to imaging thymidine kinase activity, and hence, cellular proliferation. We compared graphical, spectral, and semiquantitative analytic methodologies for quantifying [18F]FLT kinetics in tumor and normal tissue of patients with locally advanced and metastatic breast cancer. The resultant kinetic parameters were correlated with the Ki-67 labeling index from tumor biopsies. [18F]FLT accumulation was detected in primary tumor, nodal disease, and lung metastasis. In large tumors, there was substantial heterogeneity in regional radiotracer uptake, reflecting heterogeneity in cellular proliferation; radiotracer uptake in primary tumors also differed from that of metastases. [18F]FLT was metabolized in patients to a single metabolite [18F]FLT-glucuronide. Unmetabolized [18F]FLT accounted for 71.54 ± 1.50% of plasma radioactivity by 90 minutes. The rate constant for the metabolite-corrected net irreversible uptake of [18F]FLT (Ki) ranged from 0.6 to 10.4 × 10−4 and from 0 to 0.6 × 10−4 mL plasma cleared/s/mL tissue in tumor (29 regions, 15 patients) and normal tissues, respectively. Tumor Ki and fractional retention of radiotracer determined by spectral analysis correlated with Ki-67 labeling index (r = 0.92, P &lt; 0.0001 and r = 0.92, P &lt; 0.0001, respectively). These correlations were superior to those determined by semiquantitative methods. We conclude that [18F]FLT-positron emission tomography is a promising clinical tool for imaging cellular proliferation in breast cancer, and is most predictive when analyzed by graphical and spectral methods.
Renal Cancer Treatment with Low Levels of Mixed Chimerism Induced by Nonmyeloablative Regimen Using Cyclophosphamide in Mice
Authors: Harano Masahiko , Eto Masatoshi , Iwai Toshiro , Tatsugami Katsunori , Kiyoshima Keijiro , Kamiryo Yoriyuki , Tsuneyoshi Masazumi , Yoshikai Yasunobu , Naito Seiji 
Published: November 2005

AbstractRecently, much attention has been paid to nonmyeloablative allogeneic stem cell transplantation for the treatment of metastatic renal cancer. Mature donor T cells cause graft-versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor activity associated with this treatment. Hence, the segregation of the graft-versus-tumor activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Here, we show a modified cyclophosphamide-induced tolerance system for the treatment of murine renal cell carcinoma, RENCA, by shifting the equal balance between graft-versus-host and host-versus-graft reactions toward graft-versus-host reaction with donor lymphocyte infusion. Our results clearly show the antitumor activity against RENCA with only low levels of mixed chimerism in the periphery and the in vivo and in vitro acquired immunity against RENCA even when mixed chimerism is almost undetectable. Because the withdrawal of mixed chimerism reduces the risk of GVHD, the antitumor activity is thus sequentially segregated from the initial GVHD in our model. We believe that this is the first unique model system of nonmyeloablative allogeneic hemopoietic cell transplantation to ever be reported for the treatment of renal cancer.