bluuebunny/crossref_metadata_2025_split huggingface dataset:
Episomal Expression of Specific Sense and Antisense mRNAs inLeishmania amazonensis: Modulation of gp63 Level in Promastigotes and Their Infection of Macrophages In Vitro
Authors: Chen De-Qiao , Kolli Bala Krishna , Yadava Nagendra , Lu Hong Gang , Gilman-Sachs Alice , Peterson Daniel A. , Chang Kwang-Poo 
Published: May 2009

ABSTRACTThe major surface glycoprotein (gp63) ofLeishmania amazonensisis a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into aLeishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.
Identification of Genes Involved in Biofilm Formation and Respiration via Mini-
Himar
Transposon Mutagenesis of
Geobacter sulfurreducens
Authors: Rollefson Janet B. , Levar Caleb E. , Bond Daniel R. 
Published: April 2009

ABSTRACT

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe
Geobacter sulfurreducens
requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in
Geobacter
spp. and identify novel genes involved in this process.
Escherichia coli
mating strains and plasmids were used to develop a conjugation protocol and deliver mini-
Himar
transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in
Geobacter
spp.
Characterization of the
Escherichia coli
ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System
Authors: Lien Hsiang-Yun , Shy Ru-Shan , Peng Sheng-Shiang , Wu Yuei-Long , Weng Yu-Ting , Chen Hsuan-He , Su Pin-Chih , Ng Wei-Fu , Chen Yu-Chun , Chang Pei-Yi , Wu Whei-Fen 
Published: April 2009

ABSTRACT

In
Escherichia coli
, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and
cpsB
::
lacZ
assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.
Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria
Authors: Wischgoll Simon , Taubert Martin , Peters Franziska , Jehmlich Nico , von Bergen Martin , Boll Matthias 
Published: April 2009

ABSTRACT

In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO
2
. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms
Geobacter metallireducens
(GDH
Geo
) (Fe[III] reducing) and
Desulfococcus multivorans
(GDH
Des
) (sulfate reducing). GDH
Geo
was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced
bamM
gene. The gene coding for GDH
Des
was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDH
Geo
catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDH
Des
catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDH
Geo
, the

K
m

was 30 ± 2 μM and the
V
max
was 3.2 ± 0.2 μmol min
−1
mg
−1
, and for GDH
Des
, the

K
m

was 52 ± 5 μM and the
V
max
was 11 ± 1 μmol min
−1
mg
−1
. GDH
Des
but not GDH
Geo
was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDH
Geo
but are missing in GDH
Des
. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.
Subcellular Localization of TatAd of
Bacillus subtilis
Depends on the Presence of TatCd or TatCy
Authors: Ridder Anja N. J. A. , de Jong Esther J. , Jongbloed Jan D. H. , Kuipers Oscar P. 
Published: April 2009

ABSTRACT

The gram-positive bacterium
Bacillus subtilis
contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in
B. subtilis
is controlled by TatC.
The Quorum Sensing-Dependent Gene
katG
of
Burkholderia glumae
Is Important for Protection from Visible Light
Authors: Chun Heejin , Choi Okhee , Goo Eunhye , Kim Nayeon , Kim Hongsup , Kang Yongsung , Kim Jinwoo , Moon Jae Sun , Hwang Ingyu 
Published: April 2009

ABSTRACT

Quorum sensing (QS) plays important roles in the pathogenicity of
Burkholderia glumae
, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in
B. glumae
. The QS-defective mutant of
B. glumae
exhibited less catalase activity than wild-type
B. glumae
. A β-glucuronidase assay of a
katG
::Tn
3-gusA78
reporter fusion protein revealed that
katG
expression is under the control of QS. Furthermore,
katG
expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates
katG
expression. The
katG
mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 μmol
−1
m
−2
, the survival rate of the
katG
mutant was 10
5
-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the
katG
mutant.
In Vivo Sensitivity of Blue-Light-Dependent Signaling Mediated by AppA/PpsR or PrrB/PrrA in
Rhodobacter sphaeroides
Authors: Metz Sebastian , Jäger Andreas , Klug Gabriele 
Published: April 2009

ABSTRACT

Formation of photosynthesis complexes in
Rhodobacter sphaeroides
is regulated in a redox- and light-dependent manner by the AppA/PpsR and PrrB/PrrA systems. While on the one hand, blue light is sensed by the flavin adenine dinucleotide-binding BLUF domain of AppA, on the other, light is absorbed by bacteriochlorophyll signals through PrrB/PrrA. We show that much smaller quantities initiate the AppA-mediated response to blue light than the bacteriochlorophyll-mediated response.
ResDE-Dependent Regulation of Enterotoxin Gene Expression in
Bacillus cereus
: Evidence for Multiple Modes of Binding for ResD and Interaction with Fnr
Authors: Esbelin Julia , Armengaud Jean , Zigha Assia , Duport Catherine 
Published: April 2009

ABSTRACT

In the food-borne pathogen
Bacillus cereus
F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of
hbl
and
nhe
transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (
p
) of the enterotoxin regulator genes
resDE
,
fnr
, and
plcR
and the enterotoxin structural genes
nhe
and
hbl
, but with different affinities. Binding of ResD to
pplcR
,
pnhe
, and
phbl
was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and
presDE
and
pfnr
. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates
B. cereus
virulence.
Molecular Dissection of the
secA2
Locus of Group B Streptococcus Reveals that Glycosylation of the Srr1 LPXTG Protein Is Required for Full Virulence
Authors: Mistou Michel-Yves , Dramsi Shaynoor , Brega Sara , Poyart Claire , Trieu-Cuot Patrick 
Published: April 2009

ABSTRACT

In streptococci, the
secA2
locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The
secA2
locus of
Streptococcus agalactiae
strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.
Active Transcription of rRNA Operons Condenses the Nucleoid inEscherichia coli: Examining the Effect of Transcription on Nucleoid Structure in the Absence of Transertion
Authors: Cabrera Julio E. , Cagliero Cedric , Quan Selwyn , Squires Catherine L. , Jin Ding Jun 
Published: April 2009

ABSTRACTInEscherichia colithe genome must be compacted ∼1,000-fold to be contained in a cellular structure termed the nucleoid. It is proposed that the structure of the nucleoid is determined by a balance of multiple compaction forces and one major expansion force. The latter is mediated by transertion, a coupling of transcription, translation, and translocation of nascent membrane proteins and/or exported proteins. In supporting this notion, it has been shown consistently that inhibition of transertion by the translation inhibitor chloramphenicol results in nucleoid condensation due to the compaction forces that remain active in the cell. Our previous study showed that during optimal growth, RNA polymerase is concentrated into transcription foci or “factories,” analogous to the eukaryotic nucleolus, indicating that transcription and RNA polymerase distribution affect the nucleoid structure. However, the interpretation of the role of transcription in the structure of the nucleoid is complicated by the fact that transcription is implicated in both compacting forces and the expansion force. In this work, we used a new approach to further examine the effect of transcription, specifically from rRNA operons, on the structure of the nucleoid, when the major expansion force was eliminated. Our results showed that transcription is necessary for the chloramphenicol-induced nucleoid compaction. Further, an active transcription from multiple rRNA operons in chromosome is critical for the compaction of nucleoid induced by inhibition of translation. All together, our data demonstrated that transcription of rRNA operons is a key mechanism affecting genome compaction and nucleoid structure.
Autoproteolysis of YscU of
Yersinia pseudotuberculosis
Is Important for Regulation of Expression and Secretion of Yop Proteins
Authors: Björnfot Ann-Catrin , Lavander Moa , Forsberg Åke , Wolf-Watz Hans 
Published: April 2009

ABSTRACT

YscU of
Yersinia
can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU
CC
. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-
cis
-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca
2+
conditions) and calcium-depleted medium (−Ca
2+
conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca
2+
-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca
2+
conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca
2+
conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.
Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in
Streptococcus sanguinis
Authors: Das Sankar , Kanamoto Taisei , Ge Xiuchun , Xu Ping , Unoki Takeshi , Munro Cindy L. , Kitten Todd 
Published: April 2009

ABSTRACT

Streptococcus sanguinis
is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in
S. sanguinis
strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (
lgt
) and signal peptidase II (
lspA
). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in
lgt
,
lspA
, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of
ssaB
, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [
3
H]palmitate labeling and Western blot analysis confirmed that the
lgt
mutant failed to acylate lipoproteins, that the
lspA
mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.
Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in
Shewanella oneidensis
MR-1
Authors: Charania M. A. , Brockman K. L. , Zhang Y. , Banerjee A. , Pinchuk G. E. , Fredrickson J. K. , Beliaev A. S. , Saffarini D. A. 
Published: April 2009

ABSTRACT

Unlike other bacteria that use FNR to regulate anaerobic respiration,
Shewanella oneidensis
MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes,
cyaA
,
cyaB
, and
cyaC
, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an
Escherichia coli cya
mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either
cyaA
or
cyaB
did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of
cyaC
caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a
crp
mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both
cyaA
and
cyaC
were deleted. Microarray analysis of gene expression in the
crp
and
cyaC
mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the
cyaC
mutant but not in the
crp
mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.
Community-Based Peer-Led Diabetes Self-management
Authors: Lorig Kate , Ritter Philip L. , Villa Frank J. , Armas Jean 
Published: May 2009

Purpose The purpose of this study is to determine the effectiveness of a community-based diabetes self-management program comparing treatment participants to a randomized usual-care control group at 6 months. Methods A total of 345 adults with type 2 diabetes but no criteria for high A1C were randomized to a usual-care control group or 6-week community-based, peer-led diabetes self-management program (DSMP). Randomized participants were compared at 6 months. The DSMP intervention participants were followed for an additional 6 months (12 months total). A1C and body mass index were measured at baseline, 6 months, and 12 months. All other data were collected by self-administered questionnaires. Results At 6 months, DSMP participants did not demonstrate improvements in A1C as compared with controls. Baseline A1C was much lower than in similar trials. Participants did have significant improvements in depression, symptoms of hypoglycemia, communication with physicians, healthy eating, and reading food labels (P < .01). They also had significant improvements in patient activation and self-efficacy. At 12 months, DSMP intervention participants continued to demonstrate improvements in depression, communication with physicians, healthy eating, patient activation, and self-efficacy (P < .01). There were no significant changes in utilization measures. Conclusions These findings suggest that people with diabetes without elevated A1C can benefit from a community-based, peer-led diabetes program. Given the large number of people with diabetes and lack of low-cost diabetes education, the DSMP deserves consideration for implementation.