bluuebunny/crossref_metadata_2025_split huggingface dataset:
The First 45 Amino Acids of SopA Are Necessary for InvB Binding and SPI-1 Secretion
Authors: Higashide Wendy , Zhou Daoguo 
Published: March 2006

ABSTRACT

Salmonella enterica
serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE
78-240
, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of
Salmonella
SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA
1-45
, but not SopA
1-44
, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.
Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by
Enterococcus faecalis
Authors: van Merode Annet E. J. , van der Mei Henny C. , Busscher Henk J. , Krom Bastiaan P. 
Published: March 2006

ABSTRACT

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms.
Enterococcus faecalis
strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of
E. faecalis
strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (
esp
) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.
Biosynthesis of the N-Linked Glycan inCampylobacter jejuniand Addition onto Protein through Block Transfer
Authors: Kelly John , Jarrell Harold , Millar Lorna , Tessier Luc , Fiori Laura M. , Lau Peter C. , Allan Brenda , Szymanski Christine M. 
Published: March 2006

ABSTRACTIn eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated thatCampylobacter jejuniis the first member of theBacteriato possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitateC. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation inBacteriaand provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.
Dual Control of Quorum Sensing by Two TraM-Type Antiactivators in
Agrobacterium tumefaciens
Octopine Strain A6
Authors: Wang Chao , Zhang Hai-Bao , Chen Guozhou , Chen Lingling , Zhang Lian-Hui 
Published: March 2006

ABSTRACT

Agrobacterium tumefaciens
wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the
traM
gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated
traM
was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second
traM
homologue. The homologue, designated
traM2
, has 64% and 65% identities with
traM
at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the
tra
gene promoters. Deletion of
traM2
in strain A6 harboring the mutated
traM
confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.
The Transcriptional Regulator VqmA Increases Expression of the Quorum-Sensing Activator HapR in
Vibrio cholerae
Authors: Liu Zhi , Hsiao Ansel , Joelsson Adam , Zhu Jun 
Published: March 2006

ABSTRACT

Vibrio cholerae
is the causative agent of the severe diarrheal disease cholera. A number of environmental stimuli regulate virulence gene expression in
V. cholerae
, including quorum-sensing signals. At high cell densities, quorum sensing in
V. cholerae
invokes a series of signal transduction pathways in order to activate the expression of the master regulator HapR, which then represses the virulence regulon and biofilm-related genes and activates protease production. In this study, we identified a transcriptional regulator, VqmA (VCA1078), that activates
hapR
expression at low cell densities. Under in vitro inducing conditions, constitutive expression of VqmA represses the virulence regulon in a HapR-dependent manner. VqmA increases
hapR
transcription as measured by the activity of the
hapR-lacZ
reporter, and it increases HapR production as measured by Western blotting. Using a heterogenous
luxCDABE
cosmid, we found that VqmA stimulates quorum-sensing regulation at lower cell densities and that this stimulation bypasses the known LuxO-small-RNA regulatory circuits. Furthermore, we showed that VqmA regulates
hapR
transcription directly by binding to its promoter region and that expression of
vqmA
is cell density dependent and autoregulated. The physiological role of VqmA is also discussed.
Role of
oxyR
in the Oral Anaerobe
Porphyromonas gingivalis
Authors: Diaz Patricia I. , Slakeski Nada , Reynolds Eric C. , Morona Renato , Rogers Anthony H. , Kolenbrander Paul E. 
Published: March 2006

ABSTRACT

Porphyromonas gingivalis
is an anaerobic microorganism that inhabits the oral cavity, where oxidative stress represents a constant challenge. A putative transcriptional regulator associated with oxidative stress, an
oxyR
homologue, is known from the
P. gingivalis
W83 genome sequence. We used microarrays to characterize the response of
P. gingivalis
to H
2
O
2
and examine the role of
oxyR
in the regulation of this response. Most organisms in which
oxyR
has been investigated are facultative anaerobes or aerobes. In contrast to the OxyR-regulated response of these microorganisms to H
2
O
2
, the main feature of the response in
P. gingivalis
was a concerted up-regulation of insertion sequence elements related to IS
1
transposases. Common OxyR-regulated genes such as
dps
and
ahpFC
were not positively regulated in
P. gingivalis
in response to H
2
O
2
. However, their expression was dependent on the presence of a functional OxyR, as revealed by microarray comparison of an
oxyR
mutant to the wild type. Phenotypic characterization of the
oxyR
mutant showed that OxyR plays a role in both the resistance to H
2
O
2
and the aerotolerance of
P. gingivalis. Escherichia coli
and other bacteria with more complex respiratory requirements use OxyR for regulating resistance to H
2
O
2
and use a separate regulator for aerotolerance. In
P. gingivalis
, the presence of a single protein combining the two functions might be related to the comparatively smaller genome size of this anaerobic microorganism. In conclusion, these results suggest that OxyR does not act as a sensor of H
2
O
2
in
P. gingivalis
but constitutively activates transcription of oxidative-stress-related genes under anaerobic growth.
Effect ofNeisseria meningitidisFur Mutations on Global Control of Gene Transcription
Authors: Delany Isabel , Grifantini Renata , Bartolini Erika , Rappuoli Rino , Scarlato Vincenzo 
Published: March 2006

ABSTRACTThe ferric uptake regulator Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection. In this study we used comparative transcriptome analysis to define the role of the Fur protein in the global control of gene transcription and iron regulation inNeisseria meningitidis. By using the Fur-null mutant and its complemented derivative, we identified 83 genes whose transcription is controlled by Fur. We report that Fur may control differential expression of these genes by binding directly to their promoters or through indirect mechanisms. In addition, mutation of thefurgene resulted in the induction of the heat shock response, and transcription of these genes does not respond to iron limitation. Furthermore, analysis of the iron starvation stimulon in the Fur-null mutant provided evidences of iron-responsive regulation that is independent of Fur. We began to dissect the regulatory networks of Fur and the heat shock (stress) response inN. meningitidis, and the observed interlink between the two circuits is discussed.
The Antimicrobial Activity of Marinocine, Synthesized by
Marinomonas mediterranea
, Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity
Authors: Lucas-Elío Patricia , Gómez Daniel , Solano Francisco , Sanchez-Amat Antonio 
Published: March 2006

ABSTRACT

Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium
Marinomonas mediterranea
. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing
l
-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of
Escherichia coli
strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by
Pseudoalteromonas tunicata
, and some hypothetical proteins. The gene coding for marinocine has been named
lodA
, standing for lysine oxidase, and it seems to form part of an operon with a second gene,
lodB
, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of
lodA
mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned.
Organization of FliN Subunits in the Flagellar Motor of
Escherichia coli
Authors: Paul Koushik , Blair David F. 
Published: March 2006

ABSTRACT

FliN is a major constituent of the C ring in the flagellar basal body of many bacteria. It is present in >100 copies per flagellum and together with FliM and FliG forms the switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise switching. FliN is essential for flagellar assembly and switching, but its precise functions are unknown. The C-terminal part of the protein is best conserved and most important for function; a crystal structure of this C-terminal domain of FliN from
Thermotoga maritima
revealed a saddle-shaped dimer formed mainly from β strands (P. N. Brown, M. A. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair, J. Bacteriol.
187:
2890-2902, 2005). Equilibrium sedimentation studies showed that FliN can form stable tetramers and that a FliM
1
FliN
4
complex is also stable. Here, we have examined the organization of FliN subunits by using targeted cross-linking. Cys residues were introduced at various positions in FliN, singly or in pairs, and disulfide cross-linking was induced by oxidation. Efficient cross-linking was observed for certain positions near the ends of the dimer and for some positions in the structurally uncharacterized N-terminal domain. Certain combinations of two Cys replacements gave a high yield of cross-linked tetramer. The results support a model in which FliN is organized in doughnut-shaped tetramers, stabilized in part by contacts involving the N-terminal domain. Electron microscopic reconstructions show a bulge at the bottom of the C-ring whose size and shape are a close match for the hypothesized FliN tetramer.
Kinetic Characterization of the Monofunctional Glycosyltransferase from
Staphylococcus aureus
Authors: Terrak Mohammed , Nguyen-Distèche Martine 
Published: March 2006

ABSTRACT

The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-
N
-acetylglucosaminyl-
N
-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the
Staphylococcus aureus
MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of ∼5,800 M
−1
s
−1
and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn
2+
. The properties of
S. aureus
MGT are distinct from those of
S. aureus
PBP2 and
Escherichia coli
MGT, but they are similar to those of
E. coli
PBP1b. We examined the role of the conserved Glu100 of
S. aureus
MGT (equivalent to the proposed catalytic Glu233 of
E. coli
PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in
S. aureus
MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.
Comparative Genomic Analysis of Three Strains of
Ehrlichia ruminantium
Reveals an Active Process of Genome Size Plasticity
Authors: Frutos Roger , Viari Alain , Ferraz Conchita , Morgat Anne , Eychenié Sophie , Kandassamy Yane , Chantal Isabelle , Bensaid Albert , Coissac Eric , Vachiery Nathalie , Demaille Jacques , Martinez Dominique 
Published: March 2006

ABSTRACT

Ehrlichia ruminantium
is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of
E. ruminantium
of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other
Rickettsiales
(
Anaplasma
,
Wolbachia
, and
Rickettsia
spp.). Gene order is highly conserved between the
E. ruminantium
strains and with
A. marginale
. In contrast, there is very little conservation of gene order with members of the
Rickettsiaceae
. However, gene order may be locally conserved, as illustrated by the
tuf
operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe
/
Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover,
E. ruminantium
displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to
E. ruminantium
and is not observed in the other
Rickettsiales
.
Role ofmurFin Cell Wall Biosynthesis: Isolation and Characterization of amurFConditional Mutant ofStaphylococcus aureus
Authors: Sobral R. G. , Ludovice A. M. , de Lencastre H. , Tomasz A. 
Published: March 2006

ABSTRACTTheStaphylococcus aureus murFgene was placed under the control of a promoter inducible by IPTG (isopropyl-β-d-thiogalactopyranoside). It was demonstrated thatmurFis an essential gene; it is cotranscribed withddlAand growth rate, level of beta-lactam antibiotic resistance, and rates of transcription of themecAandpbpBgenes paralleled the rates of transcription ofmurF. At suboptimal concentrations of the inducer, a UDP-linked muramyl tripeptide accumulated in the cytoplasm in parallel with the decline in the amounts of the normal pentapeptide cell wall precursor. The abnormal tripeptide component incorporated into the cell wall as a monomeric muropeptide, accompanied by a decrease in the oligomerization degree of the peptidoglycan. However, incorporation of the tripeptide into the cell wall was limited to a relatively low threshold value. Further reduction of the amounts of pentapeptide cell wall precursor caused a gradual decrease in the cellular amounts of peptidoglycan, the production of a thinner peripheral cell wall, aberrant septae, and an overall increase in the diameter of the cells. The observations suggest that the role ofmurFexceeds its primary function in peptidoglycan biosynthesis and may also be involved in the control of cell division.
Genomic Organization and Molecular Characterization of
Clostridium difficile
Bacteriophage ΦCD119
Authors: Govind Revathi , Fralick Joe A. , Rolfe Rial D. 
Published: March 2006

ABSTRACT

In this study, we have isolated a temperate phage (ΦCD119) from a pathogenic
Clostridium difficile
strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the
Myoviridae
family of the tailed phages and the order
Caudovirales
. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The ΦCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ΦCD119 attachment site
attP
lies in a noncoding region close to the putative integrase (
int
) gene. We have identified the phage integration site on the
C. difficile
chromosome (
attB
) located in a noncoding region just upstream of gene
gltP
, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a
C. difficile
phage.
Cross-Regulation of the
Bacillus subtilis glnRA
and
tnrA
Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA
Authors: Zalieckas Jill M. , Wray Lewis V. , Fisher Susan H. 
Published: March 2006

ABSTRACT

Two
Bacillus subtilis
transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the
tnrA
gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which lies immediately upstream of the −35 region of the
tnrA
promoter is required for regulation of
tnrA
expression by both GlnR and TnrA. Expression of the
glnRA
operon, which contains two GlnR/TnrA binding sites (
glnRAo1
and
glnRAo2
) in its promoter region, is repressed by both GlnR and TnrA. The
glnRAo2
site, which overlaps the −35 region of the
glnRA
promoter, was shown to be required for regulation by both GlnR and TnrA, while the
glnRAo1
site which lies upstream of the −35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to
tnrA
and
glnRA
promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the
tnrA
promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the
glnRA
promoter region were greater than 3 μM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.