bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein
Authors: Matsunaga James , Young Tracy A. , Barnett Jeanne K. , Barnett Dean , Bolin Carole A. , Haake David A. 
Published: July 2002

ABSTRACTLeptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulentLeptospira kirschneriserovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that inEscherichia coliexpressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel ofLeptospirastrains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45is a peripheral membrane protein. Finally, we found that P31LipL45levels increased asLeptospiraentered the stationary phase, indicating that P31LipL45levels were regulated. Hamsters infected withL. kirschneriformed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed byL. kirschnerithat colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
Role of the Dermonecrotic Toxin of
Bordetella bronchiseptica
in the Pathogenesis of Respiratory Disease in Swine
Authors: Brockmeier Susan L. , Register Karen B. , Magyar Tibor , Lax Alistair J. , Pullinger Gillian D. , Kunkle Robert A. 
Published: July 2002

ABSTRACT

Bordetella bronchiseptica
is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic
dnt
mutants to their virulent DNT
+
parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the
dnt
mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the
dnt
mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the
dnt
mutant strains. Therefore, production of DNT by
B. bronchiseptica
is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.
Modulation of Immune Responses toMycobacterium bovisin Cattle Depleted of WC1+γδ T Cells
Authors: Kennedy Hilary E. , Welsh Michael D. , Bryson David G. , Cassidy Joseph P. , Forster Fiona I. , Howard Christopher J. , Collins Robert A. , Pollock John M. 
Published: July 2002

ABSTRACTIt is accepted that cell-mediated immune responses predominate in mycobacterial infections. Many studies have shown that CD4+T cells produce Th1 cytokines, such as gamma interferon (IFN-γ), in response to mycobacterial antigens and that the cytolytic activity of CD8+cells toward infected macrophages is important. However, the extent and manner in which γδ T cells participate in this response remain unclear. In ruminants, γδ T cells comprise a major proportion of the peripheral blood mononuclear cell population. We have previously shown that WC1+γδ T cells are involved early inMycobacterium bovisinfection of cattle, but their specific functions are not well understood. Here we describe an in vivo model of bovine tuberculosis in which the WC1+γδ T cells were depleted from the peripheral circulation and respiratory tract, by infusion of WC1+-specific monoclonal antibody, prior to infection. While no effects on disease pathology were observed in this experiment, results indicate that WC1+γδ T cells, which become significantly activated (CD25+) in the circulation of control calves from 21 days postinfection, may play a role in modulating the developing immune response toM. bovis. WC1+-depleted animals exhibited decreased antigen-specific lymphocyte proliferative response, an increased antigen-specific production of interleukin-4, and a lack of specific immunoglobulin G2 antibody. This suggests that WC1+γδ TCR+cells contribute, either directly or indirectly, toward the Th1 bias of the immune response in bovine tuberculosis—a hypothesis supported by the decreased innate production of IFN-γ, which was observed in WC1+-depleted calves.
Stachylysin May Be a Cause of Hemorrhaging in Humans Exposed to
Stachybotrys chartarum
Authors: Vesper Stephen J. , Vesper Mary Jo 
Published: July 2002

ABSTRACT

Stachybotrys chartarum
is a toxigenic fungus that has been associated with human health concerns such as nasal bleeding in adults and pulmonary hemosiderosis (PH) in infants. Seven of eight strains of
S. chartarum
isolated from homes of infants with PH in Cleveland, Ohio, and the strain from the lung of an infant with PH in Texas produced stachylysin in tryptic soy broth (TSB), whereas only one out of eight strains isolated from control homes produced stachylysin. However, all strains produced stachylysin when grown on TSB with 0.7% sheep's blood. When stachylysin was injected into
Lumbricus terrestis
, the erythrocruorin hemoglobin (absorbance peaks at 280 and 415 nm) was released, resulting in a lethal effect. These results support the hypothesis that stachylysin may be one agent responsible for hemorrhaging in humans.
Role of Nitric Oxide in Host Defense in Murine Salmonellosis as a Function of Its Antibacterial and Antiapoptotic Activities
Authors: Alam Mohammad Samiul , Akaike Takaaki , Okamoto Shinichiro , Kubota Tatsuo , Yoshitake Jun , Sawa Tomohiro , Miyamoto Yoichi , Tamura Fumio , Maeda Hiroshi 
Published: July 2002

ABSTRACTHost defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulentSalmonella entericaserovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD50s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 × 104CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD50, 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD50, 6 × 102CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute toSalmonellapathogenicity. These results indicate that NO has significant host defense functions inSalmonellainfections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.
Cryptic and Exposed Invariable Regions of VlsE, the Variable Surface Antigen ofBorrelia burgdorferisl
Authors: Liang Fang Ting , Nowling Jena M. , Philipp Mario T. 
Published: July 2002

ABSTRACTBorrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies ofB. burgdorferisensu lato. IR6is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR2to IR5) were investigated. Antisera to synthetic peptides based on each of the IR2to IR5sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR2, IR4, and IR5but not to IR3, indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.
The N-Terminal Region of the
Escherichia coli
WecA (Rfe) Protein, Containing Three Predicted Transmembrane Helices, Is Required for Function but Not for Membrane Insertion
Authors: Amer Amal O. , Valvano Miguel A. 
Published: July 2002

ABSTRACT

The correct site for translation initiation for
Escherichia coli
WecA (Rfe), presumably involved in catalyzing the transfer of
N
-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.
Escherichia coli
Responses to a Single DNA Adduct
Authors: Pandya Gagan A. , Yang In-Young , Grollman Arthur P. , Moriya Masaaki 
Published: July 2002

ABSTRACT

To study the mechanisms by which
Escherichia coli
modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various
E. coli
pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1,
N
6
-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional
recA
,
recF
,
recO
, and
recR
genes, suggesting the mechanism to be daughter strand gap repair; (iii) the
ruvABC
genes or the
recG
gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.
Mutagenesis and Functional Characterization of the
glnB
,
glnA
, and
nifA
Genes from the Photosynthetic Bacterium
Rhodospirillum rubrum
Authors: Zhang Yaoping , Pohlmann Edward L. , Ludden Paul W. , Roberts Gary P. 
Published: July 2002

ABSTRACT

Nitrogen fixation is tightly regulated in
Rhodospirillum rubrum
at two different levels: transcriptional regulation of
nif
expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase–dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of
glnB
,
glnA
, and
nifA
mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect
glnB
(structural gene for P
II
) were constructed. While P
II
-Y51F showed a lower nitrogenase activity than that of wild type, a P
II
deletion mutant showed very little
nif
expression. This effect of P
II
on
nif
expression is apparently the result of a requirement of P
II
for NifA activation, whose activity is regulated by NH
4
+
in
R. rubrum
. The modification of glutamine synthetase (GS) in these
glnB
mutants appears to be similar to that seen in wild type, suggesting that a paralog of P
II
might exist in
R. rubrum
and regulate the modification of GS. P
II
also appears to be involved in the regulation of DRAT activity, since an altered response to NH
4
+
was found in a mutant expressing P
II
-Y51F. The adenylylation of GS plays no significant role in
nif
expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH
4
+
and darkness treatments.
Kinetics and Substrate Specificity of Membrane-Reconstituted Peptide Transporter DtpT of
Lactococcus lactis
Authors: Fang Gang , Konings Wil N. , Poolman Bert 
Published: July 2002

ABSTRACT

The peptide transport protein DtpT of
Lactococcus lactis
was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[
14
C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, ω-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the
l
configuration, and
trans
-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT
1
and PepT
2
shows that the bacterial transporter is more restrictive in its substrate recognition.
Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family
Authors: Daugherty Matthew , Vonstein Veronika , Overbeek Ross , Osterman Andrei 
Published: July 2002

ABSTRACTShikimate kinase (EC2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi‖1500322 inMethanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC2.7.1.39) of the GHMP-kinase superfamily. The latter functionality inM. jannaschiiis assigned to another gene (gi‖1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed inEscherichia coliand purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases:Km,shikimate= 414 ± 33 μM,Km,ATP= 48 ± 4 μM, andkcat= 57 ± 2 s−1for the predicted shikimate kinase andKm,homoserine= 188 ± 37 μM,Km,ATP= 101 ± 7 μM, andkcat= 28 ± 1 s−1for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.
Regulation of
Staphylococcus aureus
Type 5 and Type 8 Capsular Polysaccharides by CO
2
Authors: Herbert Silvia , Newell Steven W. , Lee Chia , Wieland Karsten-Peter , Dassy Bruno , Fournier Jean-Michel , Wolz Christiane , Döring Gerd 
Published: July 2002

ABSTRACT

Staphylococcus aureus
expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO
2
. Here we show that CO
2
reduces CP5 expression at the transcriptional level and that CO
2
regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO
2
caused a significant decrease in CP8 expression in four
S. aureus
strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine
S. aureus
strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of
cap5
and
cap8
. To test whether these sequence variations are responsible for the different responses to CO
2
, promoter regions from selected strains were fused to the reporter gene
xyl
E in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO
2
in all derivatives of strain Newman and was always positively regulated by CO
2
in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO
2
response.
trans
-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive
S. aureus
strains.
Lowering
S
-Adenosylmethionine Levels in
Escherichia coli
Modulates C-to-T Transition Mutations
Authors: Macintyre Georgina , Atwood C. Victoria , Cupples Claire G. 
Published: July 2002

ABSTRACT

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor,
S
-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM.
Escherichia coli
SAM levels were lowered by reducing SAM synthetase activity via the introduction of a
metK84
allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. The
metK84
strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P
BAD
promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.
Identification of the Ω4514 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
That Is Transcribed In Vitro by the Major Vegetative RNA Polymerase
Authors: Hao Tong , Biran Dvora , Velicer Gregory J. , Kroos Lee 
Published: July 2002

ABSTRACT

Ω4514 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
genome that fuses
lacZ
expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and σ
A
RNA polymerase, the major form of RNA polymerase in growing
M. xanthus
, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Ω4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn
5 lac
Ω4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An
orf1
disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of
orf2
nor insertion Ω4514 in
orf3
caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an
orf1
mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Ω4514 is the first example of a developmentally regulated
M. xanthus
operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.