bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yersinia pestis
YscG Protein Is a Syc-Like Chaperone That Directly Binds YscE
Authors: Day James B. , Guller Inna , Plano Gregory V. 
Published: July 2002

ABSTRACT

Pathogenic
Yersinia
species secrete virulence proteins, termed
Yersinia
outer proteins (Yops), upon contact with a eukaryotic cell. The secretion machinery is composed of 21
Yersinia
secretion (Ysc) proteins.
Yersinia pestis
mutants defective in expression of YscG or YscE were unable to export the Yops. YscG showed structural and limited amino-acid-sequence similarities to members of the specific Yop chaperone (Syc) family of proteins. YscG specifically recognized and bound YscE; however, unlike previously characterized Syc substrates, YscE was not exported from the cell. These data suggest that YscG functions as a chaperone for YscE.
Association of Protease Activity in
Vibrio cholerae
Vaccine Strains with Decreases in Transcellular Epithelial Resistance of Polarized T84 Intestinal Epithelial Cells
Authors: Mel Stephanie F. , Fullner Karla Jean , Wimer-Mackin Susan , Lencer Wayne I. , Mekalanos John J. 
Published: July 2002

ABSTRACT

Culture supernatants prepared from reactogenic strains of
Vibrio cholerae
cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.
Inhibition of Leukocyte Entry into the Brain by the Selectin Blocker Fucoidin Decreases Interleukin-1 (IL-1) Levels but Increases IL-8 Levels in Cerebrospinal Fluid during Experimental Pneumococcal Meningitis in Rabbits
Authors: Østergaard Christian , Yieng-Kow Runa Vavia , Benfield Thomas , Frimodt-Møller Niels , Espersen Frank , Lundgren Jens D. 
Published: July 2002

ABSTRACT

The polysaccharide fucoidin is a selectin blocker that inhibits leukocyte recruitment into the cerebrospinal fluid (CSF) during experimental pneumococcal meningitis. In the present study, the effect of fucoidin treatment on the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-8 into the CSF was investigated. Rabbits (
n
= 7) were treated intravenously with 10 mg of fucoidin/kg of body weight every second hour starting 4 h after intracisternal inoculation of ∼10
6
CFU of
Streptococcus pneumoniae
type 3 (untreated control group,
n
= 7). CSF samples were obtained every second hour during a 16-h study period. Treatment with fucoidin caused a consistent and significant decrease in CSF IL-1 levels (in picograms per milliliter) between 12 and 16 h (0 versus 170, 0 versus 526, and 60 versus 1,467, respectively;
P
< 0.02). A less consistent decrease in CSF TNF-α levels was observed in the fucoidin-treated group, but with no significant difference between the two groups (
P
> 0.05). In contrast, there was no attenuation in CSF IL-8 levels. Indeed, there was a significant increase in CSF IL-8 levels (in picograms per milliliter) in the fucoidin-treated group at 10 and 12 h (921 versus 574 and 1,397 versus 569, respectively;
P
< 0.09). In conclusion, our results suggest that blood-derived leukocytes mainly are responsible for the release of IL-1 and to some degree TNF-α into the CSF during pneumococcal meningitis, whereas IL-8 may be produced by local cells within the brain.
Type III Secretion-Dependent Cell Cycle Block Caused in HeLa Cells by Enteropathogenic
Escherichia coli
O103
Authors: Nougayrède Jean-Philippe , Boury Michèle , Tasca Christian , Marchès Olivier , Milon Alain , Oswald Eric , De Rycke Jean 
Published: July 2002

ABSTRACT

Rabbit enteropathogenic
Escherichia coli
(EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13

suc1

-agarose beads. This shows that Cdk1 is implicated in the G
2
/M arrest caused by EPEC strain E22.
Adoptive Transfer of CD4+T Cells Specific for Subunit A ofHelicobacter pyloriUrease ReducesH. pyloriStomach Colonization in Mice in the Absence of Interleukin-4 (IL-4)/IL-13 Receptor Signaling
Authors: Lucas Bernadette , Bumann Dirk , Walduck Anna , Koesling Jan , Develioglu Leyla , Meyer Thomas F. , Aebischer Toni 
Published: July 2002

ABSTRACTProtection in the murine model ofHelicobacter pyloriinfection may be mediated by CD4+T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterizedH. pyloriurease-specific CD4+T cells from BALB/c mice immunized withSalmonella entericaserovar Typhimurium expressingH. pyloriurease (subunits A and B). The CD4+T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-γ), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-γ and IL-10. Adoptive transfer of the UreA-specific CD4+T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+T cells into IL-4 receptor α chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis ofH. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.
Mycobacterium bovis BCG-Induced Granuloma Formation Depends on Gamma Interferon and CD40 Ligand but Does Not Require CD28
Authors: Hogan Laura H. , Markofski Wes , Bock Anja , Barger Brittany , Morrissey James D. , Sandor Matyas 
Published: July 2002

ABSTRACTProgressive granuloma formation is a hallmark of chronic mycobacterial infection. Granulomas are localized, protective inflammatory reactions initiated by CD4+T cells, which contribute to control of bacterial growth and blockade of bacterial dissemination. In order to understand the costimulatory requirements that allow CD4+T cells to directly or indirectly induce granulomas, we studied granuloma formation after 6 weeks inMycobacterium bovisBCG-infected CD28- and CD40 ligand (CD40L)-deficient mice and compared it to granuloma formation in infected wild-type inbred mice and infected cytokine-deficient mice. We characterized granulomas morphologically in liver sections, analyzed granuloma infiltrating cells by flow cytometry, and measured cytokine production by cultured granuloma cells. CD28-deficient mice have no defect at the local inflammatory site, inasmuch as they form protective granulomas and control bacterial growth. However, there are fewer activated T cells in the spleen compared to infected wild-type animals, and quantitative differences in the cellular composition of the granuloma are observed by flow cytometry. In CD40L-deficient mice, the granuloma phenotype is very similar to the phenotype in gamma interferon (IFN-γ)-deficient mice. Both IFN-γ-deficient and CD40L-deficient mice form granulomas which prevent bacterial dissemination, but control of bacterial growth is significantly impaired. The relative proportion of CD4+T cells in granulomas from both CD28−/−and CD40L−/−mice is significantly decreased compared with wild-type animals. Both models demonstrate that the phenotype and activation stage of systemic T cells do not always correlate with the phenotype and activation stage of the localized granulomatous response.
Complete DNA Sequence and Comparative Analysis of the 50-Kilobase Virulence Plasmid of
Salmonella enterica
Serovar Choleraesuis
Authors: Haneda Takeshi , Okada Nobuhiko , Nakazawa Noriko , Kawakami Takatoshi , Danbara Hirofumi 
Published: July 2002

ABSTRACT

The complete nucleotide sequence of pKDSC50, a large virulence plasmid from
Salmonella enterica
serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the
spv
operon, which is composed of genes essential for systemic infection by nontyphoidal
Salmonella
. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the
spvRABCD
genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the
pef
(plasmid-encoded fimbria) operon and
rck
(resistance to complement killing) gene, which has been identified on the virulence plasmid of
S. enterica
serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic
Escherichia coli
O157:H7 and the enteropathogenic
E. coli
(EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of
Salmonella
serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.
Conformational Nature of the
Borrelia burgdorferi
Decorin Binding Protein A Epitopes That Elicit Protective Antibodies
Authors: Ulbrandt Nancy D. , Cassatt David R. , Patel Nita K. , Roberts William C. , Bachy Christine M. , Fazenbaker Christine A. , Hanson Mark S. 
Published: July 2002

ABSTRACT

Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental
Borrelia burgdorferi
infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from
B. burgdorferi
strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous
B. garinii
and
B. afzelii
strains. Additionally, nonsecreted recombinant forms of DbpA
N40
were found to be inferior to secreted lipoprotein DbpA
N40
in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.
Mutational Analysis of the
tra
Locus of the Broad-Host-Range
Streptomyces
Plasmid pIJ101
Authors: Pettis Gregg S. , Cohen Stanley N. 
Published: July 2002

ABSTRACT

The
tra
gene of
Streptomyces lividans
plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the
tra
gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211–6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.
Heterologous NNR-Mediated Nitric Oxide Signaling in
Escherichia coli
Authors: Hutchings Matthew I. , Shearer Neil , Wastell Sarah , van Spanning Rob J. M. , Spiro Stephen 
Published: July 2002

ABSTRACT

The transcription factor NNR from
Paracoccus denitrificans
was expressed in a strain of
Escherichia coli
carrying a plasmid-borne fusion of the
melR
promoter to
lacZ
, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO
+
donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the
E. coli
reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a “positive control” phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
Purification and Characterization of an Iron Superoxide Dismutase and a Catalase from the Sulfate-Reducing Bacterium
Desulfovibrio gigas
Authors: Dos Santos Wagner G. , Pacheco Isabel , Liu Ming-Yih , Teixeira Miguel , Xavier António V. , LeGall Jean 
Published: July 2002

ABSTRACT

The iron-containing superoxide dismutase (FeSOD; EC
1.15.1.1
) and catalase (EC
1.11.1.6
) enzymes constitutively expressed by the strictly anaerobic bacterium
Desulfovibrio gigas
were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms,
D. gigas
FeSOD is sensitive to H
2
O
2
and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand,
D. gigas
catalase has an estimated molecular mass of 186 ± 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H
2
O
2
and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (
g

y
,
x

= 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.
Towards Single-Copy Gene Expression Systems Making Gene Cloning Physiologically Relevant: Lambda InCh, a Simple
Escherichia coli
Plasmid-Chromosome Shuttle System
Authors: Boyd Dana , Weiss David S. , Chen Joseph C. , Beckwith Jon 
Published: July 2002

ABSTRACT

We describe a simple system for reversible, stable integration of plasmid-borne genes into the
Escherichia coli
chromosome. Most ordinary
E. coli
strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used. A single genetic element, a lambda phage, is the only specialized vector required. The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted.
SpoIIB Localizes to Active Sites of Septal Biogenesis and Spatially Regulates Septal Thinning during Engulfment in
Bacillus subtilis
Authors: Perez Ana R. , Abanes-De Mello Angelica , Pogliano Kit 
Published: July 2002

ABSTRACT

A key step in the
Bacillus subtilis
spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that
spoIIB
mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in
spoIID
,
spoIIM
, and
spoIIP
mutants. However, unlike the sporangia of those three mutants,
spoIIB
mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in
spoIIB
mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.
The CcrM DNA Methyltransferase of
Agrobacterium tumefaciens
Is Essential, and Its Activity Is Cell Cycle Regulated
Authors: Kahng Lyn Sue , Shapiro Lucy 
Published: July 2002

ABSTRACT

DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in
Escherichia coli
and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of
Agrobacterium tumefaciens
was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the −35 position; this motif was footprinted by purified
Caulobacter crescentus
CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of
Agrobacterium
cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling
ccrM
expression.
umuDC-dnaQ
Interaction and Its Implications for Cell Cycle Regulation and SOS Mutagenesis in
Escherichia coli
Authors: Sutton Mark D. , Murli Sumati , Opperman Timothy , Klein Carly , Walker Graham C. 
Published: July 2002

ABSTRACT

The
Escherichia coli
SOS-regulated
umuDC
gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD′ proteins, both encoded by the
umuD
gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the
umuDC
gene products. Here we show that overproduction of the ɛ proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the
umuDC
gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD′ and the C-terminal domain of ɛ.