bluuebunny/crossref_metadata_2025_split huggingface dataset:
CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola
Authors: Cai Shichun , Zheng Xin , Dong Xiuzhu 
Published: July 2011

ABSTRACT

Previously, we found that exoglucanase Cel48A from
Cellulosilyticum ruminicola
H1 bound intensively to Avicel; however, no known carbohydrate-binding module (CBM) was observed in the protein. Bioinformatics suggested that a C-terminal fragment of 127 amino acids, named the
Cellulosilyticum
-specific paralogous module (CPM), could function in binding. CPM-appended proteins are all putative (hemi)cellulases from
Cellulosilyticum
spp. In the present work, we demonstrated that Cel48A without the CPM retained only exoglucanase activity and lost the Avicel-binding ability, while the isolated CPM exhibited a high affinity for Avicel. In addition, the CPM bound to chitin, but not to soluble polysaccharides, making it a type A CBM, which binds only insoluble polysaccharides. Phylogenetic analysis clustered the CPM and its homologs as a separate branch that was distantly related to CBM subfamilies 3a (28% identity), 3b (24% identity), and 3c (21% identity). Sequence alignment revealed distinct secondary structures of the new CBM 3 group, in particular, a conserved Pro66-Trp67 insert preceding strand β4′, a deletion preceding strand β6, and incomplete strands β8 and β9. An alanine scan for six aromatic and three nonaromatic amino acid residues (D66, P66, and R111) by site-directed mutagenesis determined that Phe62, Pro66, Trp67, Tyr68, Arg111, and Trp117 were the functional residues for binding. Among them, Phe62, Pro66, and Trp67 were the newly determined key sites in the CPM for binding. Three-dimensional homolog modeling revealed two types of substrate-binding sites, planar and groove, in the CPM. Thus, a new subfamily, CBM family 3d, is proposed.
Competence-Dependent Endogenous DNA Rearrangement and Uptake of Extracellular DNA Give a Natural Variant of Streptococcus mutans without Biofilm Formation
Authors: Narisawa Naoki , Kawarai Taketo , Suzuki Naomi , Sato Yutaka , Ochiai Kuniyasu , Ohnishi Makoto , Watanabe Haruo , Senpuku Hidenobu 
Published: July 2011

ABSTRACT

The production of water-insoluble glucan (WIG) enables
Streptococcus mutans
to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous
gtfB
and
gtfC
genes. Conversely, a single hybrid gene from the endogenous recombination of
gtfB
and
gtfC
is easily generated using RecA, resulting in
S. mutans
UA159 WIG
−
(rate of ∼1.0 × 10
−3
). The pneumococcus
recA
gene is regulated as a late competence gene.
comX
gene mutations did not lead to the appearance of WIG
−
cells. The biofilm collected from the flow cell had more WIG
−
cells than among the planktonic cells. Among the planktonic cells, WIG
−
cells appeared after 16 h and increased ∼10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG
−
cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG
+
and recipient WIG
−
cells, the recipient cells reverted to WIG
+
and acquired an intact
gtfBC
region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG
−
cells and that both are induced by the same signal transducer, the
com
system. Our findings may help in understanding how
S. mutans
can adapt to the oral environment and may explain the evolution of
S. mutans
.
Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia
Authors: Gill Jason J. , Summer Elizabeth J. , Russell William K. , Cologna Stephanie M. , Carlile Thomas M. , Fuller Alicia C. , Kitsopoulos Kate , Mebane Leslie M. , Parkinson Brandi N. , Sullivan David , Carmody Lisa A. , Gonzalez Carlos F. , LiPuma John J. , Young Ry 
Published: July 2011

ABSTRACTWithin theBurkholderia cepaciacomplex,B. cenocepaciais the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeuticB. cenocepaciapodophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.
UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and Their Prevalence in Bacteria
Authors: Coyne Michael J. , Fletcher C. Mark , Reinap Barbara , Comstock Laurie E. 
Published: July 2011

ABSTRACT

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of
Bacteroides fragilis
, designated BfUxs1 and BfUxs2.
Bfuxs1
is located in a conserved region of the
B. fragilis
genome, whereas
Bfuxs2
is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the
B. fragilis
NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in
Bacteroides
species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan.
Most Mutant OccR Proteins That Are Defective in Positive Control Hold Operator DNA in a Locked High-Angle Bend
Authors: Tsai Ching-Sung , Chen Chia-Sui , Winans Stephen C. 
Published: July 2011

ABSTRACT

OccR is a LysR-type transcriptional regulator of
Agrobacterium tumefaciens
that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the
occ
genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between
occQ
and
occR
in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the
occQ
promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.
Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter
Authors: Aktas Meriyem , Jost Kathinka A. , Fritz Christiane , Narberhaus Franz 
Published: July 2011

ABSTRACT

Agrobacterium tumefaciens
is a facultative phytopathogen that causes crown gall disease. For successful plant transformation
A. tumefaciens
requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in
A. tumefaciens
, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in
A. tumefaciens
. The ABC-type choline transporter is encoded by the chromosomally located
choXWV
operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [
14
C]choline uptake is severely reduced in
A. tumefaciens choX
mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [

K
D

] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine,

K
D

of ≈80 μM; betaine,

K
D

of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on
Sinorhizobium meliloti
ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding.
Role for Escherichia coli YidD in Membrane Protein Insertion
Authors: Yu Zhong , Lavèn Mariëlle , Klepsch Mirjam , de Gier Jan-Willem , Bitter Wilbert , van Ulsen Peter , Luirink Joen 
Published: July 2011

ABSTRACT

YidC has an essential but poorly defined function in membrane protein insertion and folding in bacteria. The
yidC
gene is located in a gene cluster that is highly conserved in Gram-negative bacteria, the gene order being
rpmH
,
rnpA
,
yidD
,
yidC
, and
trmE
. Here, we show that
Escherichia coli yidD
, which overlaps with
rnpA
and is only 2 bp upstream of
yidC
, is expressed and localizes to the inner membrane, probably through an amphipathic helix. Inactivation of
yidD
had no discernible effect on cell growth and viability. However, compared to control cells, Δ
yidD
cells were affected in the insertion and processing of three YidC-dependent inner membrane proteins. Furthermore,
in vitro
cross-linking showed that YidD is in proximity of a nascent inner membrane protein during its localization in the Sec-YidC translocon, suggesting that YidD might be involved in the insertion process.
FliZ Acts as a Repressor of the
ydiV
Gene, Which Encodes an Anti-FlhD
4
C
2
Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium
Authors: Wada Takeo , Tanabe Yasushi , Kutsukake Kazuhiro 
Published: July 2011

ABSTRACT

YdiV acts as an anti-FlhD
4
C
2
factor, which negatively regulates the class 2 flagellar operons in poor medium in
Salmonella enterica
serovar Typhimurium. On the other hand, one of the class 2 flagellar genes,
fliZ
, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the
ydiV
mutant background. Transcription of the
ydiV
gene was shown to increase in the
fliZ
mutant. Purified FliZ protein was shown
in vitro
to bind to the promoter region of the
nlpC
gene, which is located just upstream of the
ydiV
gene, and to repress its transcription. These results indicate that FliZ is a repressor of the
nlpC-ydiV
operon and activates the class 2 operons by repressing
ydiV
expression. Therefore, the
fliZ
and
ydiV
genes form a regulatory loop.
Rsp Inhibits Attachment and Biofilm Formation by Repressing
fnbA
in Staphylococcus aureus MW2
Authors: Lei Mei G. , Cue David , Roux Christelle M. , Dunman Paul M. , Lee Chia Y. 
Published: July 2011

ABSTRACT

Biofilms contribute to virulence of
Staphylococcus aureus
. Formation of biofilms is multifactorial, involving polysaccharide, protein, and DNA components, which are controlled by various regulators. Here we report that deletion of the
rsp
gene resulted in an increase in biofilm formation in strain MW2, suggesting that Rsp is a repressor of biofilm formation. Using SDS-PAGE, we found that Rsp profoundly affected cell surface and secreted proteins. The
rsp
gene was transcribed monocistronically, and the transcripts were most abundant at the exponential growth phase. Microarray analyses revealed that Rsp represses 75 genes, including 9 genes encoding cell wall-anchored proteins, and activates 22 genes, including 5 genes encoding secreted proteases. Among these genes,
fnbA
,
fnbB
,
sasG
, and
spa
(which encode cell wall-anchored proteins) and
splABCD
(which encode secreted proteases) have been implicated in biofilm formation. To deconvolute Rsp's contribution to biofilm formation, we analyzed deletion mutants of these genes either in the wild-type or in the
rsp
mutant background. We found that
fnbA
deletion in the
rsp
mutant restored biofilm formation to the wild-type level, indicating that FnbA plays a major role in Rsp regulation of biofilm formation. Further studies revealed that Rsp inhibited biofilm formation at the stage of primary attachment through repressing
fnbA
. Rsp belongs to the AraC/XylS family of regulatory proteins. We expressed the putative Rsp DNA binding domain (Rsp
DBD
) in
Escherichia coli
and showed that Rsp
DBD
was able to specifically bind to a short DNA fragment containing the
fnbA
promoter, suggesting that Rsp represses
fnbA
expression by direct DNA binding.
Interaction of Mycobacterium tuberculosis Elongation Factor Tu with GTP Is Regulated by Phosphorylation
Authors: Sajid Andaleeb , Arora Gunjan , Gupta Meetu , Singhal Anshika , Chakraborty Kausik , Nandicoori Vinay Kumar , Singh Yogendra 
Published: July 2011

ABSTRACT

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the
Mycobacterium tuberculosis
phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of
M. tuberculosis
Ef-Tu (
Mtb
Ef-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in
Mycobacterium smegmatis
indeed reduced the level of protein synthesis.
Mtb
Ef-Tu was found to be phosphorylated by PknB on multiple sites, including Thr
118
, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated
Mtb
Ef-Tu but not on the phosphorylated protein. Our results show that the modulation of the
Mtb
Ef-Tu–GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.
Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii
Authors: Hicks Linda D. , Warrier Indu , Raghavan Rahul , Minnick Michael F. 
Published: July 2011

ABSTRACT

The 23S rRNA gene of
Coxiella burnetii
, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in
Coxiella
's biology, we determined the intrinsic stability time periods (
in vitro
half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA.
In vivo
half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an
Escherichia coli
model. Intron RNAs were quantified in synchronous cultures of
C. burnetii
and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the
Coxiella
intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The
in vitro
efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant
Coxiella
RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator
in vivo
.
Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA
Authors: Allen Courtni E. , Schmitt Michael P. 
Published: July 2011

ABSTRACT

The human pathogen
Corynebacterium diphtheriae
utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in
C. diphtheriae
involves the
dtxR
- and iron-regulated
hmu
hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione
S
-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various
Corynebacterium
species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in
C. diphtheriae
in which hemin is initially obtained from Hb by HtaA and then transferred between surface-anchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.
Mutational Analysis of the Control Cable That Mediates Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor
Authors: Kitanovic Smiljka , Ames Peter , Parkinson John S. 
Published: July 2011

ABSTRACT

During transmembrane signaling by
Escherichia coli
Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment.