bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Platelets and Platelet Microbicidal Protein Susceptibility on the Fate of
Staphylococcus aureus
in an In Vitro Model of Infective Endocarditis
Authors: Mercier Renee-Claude , Rybak Michael J. , Bayer Arnold S. , Yeaman Michael R.
Published: July 2002

Several lines of evidence indicate that platelets protect against endovascular infections such as infective endocarditis (IE). It is highly likely that a principal mechanism of this platelet host defense role is the release of platelet microbicidal proteins (PMPs) in response to agonists generated at sites of endovascular infection. We studied the ability of platelets to limit the colonization and proliferation of
Staphylococcus aureus
in an in vitro model of IE. Three isogenic
S. aureus
strains, differing in their in vitro susceptibility to thrombin-induced platelet microbicidal protein-1 (tPMP), were used: ISP479C (parental strain; highly susceptible to tPMP [tPMP
s
]); ISP479R (transposon mutant derived from ISP479; tPMP resistant [tPMP
r
]); or 757-5 (tPMP
r
transductant of the ISP479R genotype in the ISP479 parental background). Time-kill assays and in vitro IE models were used to examine the temporal relationship between thrombin-induced platelet activation and
S. aureus
killing. In time-kill studies, early platelet activation (30 min prior to bacterial exposure) correlated with a significant bactericidal effect against tPMP
s
ISP479C (
r
2
> 0.90,
P
< 0.02) but not against tPMP
r
strains, ISP479R or 757-5. In the IE model, thrombin activation significantly inhibited proliferation of ISP479C within simulated vegetations compared to strains ISP479R or 757-5 (
P
< 0.05). The latter differences were observed despite there being no detectable differences among the three
S. aureus
strains in initial colonization of simulated vegetations. Collectively, these data indicate that platelets limit intravegetation proliferation of tPMP
s
but not tPMP
r
S. aureus
. These findings underscore the likelihood that platelets play an important antimicrobial host defense role in preventing and/or limiting endovascular infections due to tPMP
s
pathogens.
Transcriptional Analysis ofp30Major Outer Membrane Multigene Family ofEhrlichiacanisin Dogs, Ticks, and Cell Culture at Different Temperatures
Authors: Unver Ahmet , Ohashi Norio , Tajima Tomoko , Stich Roger W. , Grover Debra , Rikihisa Yasuko
Published: July 2002

Ehrlichiacanis, an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis.E.canisimmunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs andRhipicephalussanguineusticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs inE.caniswere transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups ofR.sanguineusticks (adult males and females and nymphs) were separately infected withE.canisby feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower inE.caniscultivated in dog monocyte cell line DH82 at 25°C than inE.caniscultivated at 37°C. Analysis of differential expression ofp30multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis andE.canistransmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.
Use of Operon Fusions inMannheimia haemolyticaTo Identify Environmental andcis-Acting Regulators of Leukotoxin Transcription
Authors: Marciel Ann Marie , Highlander Sarah K.
Published: July 2002

The leukotoxin ofMannheimia haemolyticais an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight intoM. haemolyticapathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by PlapT, which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well aslktC::catchromosomal fusions, to study transcription initiation inM. haemolytica. Using these genetic tools, we have identifiedcis-acting sequences and environmental conditions that modulate transcription of the leukotoxin andlapTpromoters. By deletion analysis, promoters were shown to rely on sequences upstream of their −10 and −35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of Plkt. A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the PlktRNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. ThelktC::catoperon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.
Pulmonary Hypertension Syndrome in Broilers Caused byEnterococcus faecalis
Authors: Tankson J. D. , Thaxton J. P. , Vizzier-Thaxton Y.
Published: July 2002

A field strain ofEnterococcus faecaliswas administered to broiler chicks at doses of 0, 3 × 106, 1.5 × 107, and 2 × 107bacteria/bird either intra-abdominally or intravenously. In trials 1 to 3, birds were reared communally in a broiler house on pine shaving litter. In trial 4, challenged and control birds were maintained in separate isolation rooms in metal cages with raised wire floors. Challenged birds exhibited a characteristic cavity or depression in the external wall of the right ventricle. A subjective scoring system was devised to quantify challenge effects by assigning each heart a score of 1 to 4. The average number of birds, over all trials and over all dose levels, exhibiting the ventricular cavity was 93%. This value in controls was 5%. The average heart score for challenged birds was 3.1, and that for controls was 0.20. Heart scores of challenged and control chicks were not different in birds reared communally or in separate isolation rooms. Additionally, both routes of administration were equally effective. Results suggest that challenge withE. faecaliscaused pulmonary hypertension.
High-Level Expression of NRAMP1 in Peripheral Blood Cells and Tuberculous Granulomas fromMycobacterium bovis-Infected Bovines
Authors: Estrada-Chávez Ciro , Pereira-Suárez Ana L. , Meraz Marco A. , Arriaga Camila , Garcı́a-Carrancá Alejandro , Sánchez-Rodriguez Cristina , Mancilla Raúl
Published: July 2002

By Western blotting, we demonstrate high-level expression of NRAMP1 proteins in peripheral blood cells and granulomas ofMycobacterium bovis-infected bovines. Immunohistochemistry of granulomatous lesions showed heavily labeled epithelioid macrophages and Langhans cells. These data suggest thatM. bovisinfection enhances NRAMP1 expression and that active tuberculosis can occur despite this response.
Pneumococcal Conjugate Vaccines Overcome Splenic Dependency of Antibody Response to Pneumococcal Polysaccharides
Authors: Breukels Mijke A. , Zandvoort Andre , van den Dobbelsteen Germie P. J. M. , van den Muijsenberg Adrie , Lodewijk Monique E. , Beurret Michel , Klok Pieter A. , Timens Wim , Rijkers Ger T.
Published: July 2002

Protection against infections with
Streptococcus pneumoniae
depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.
Rapid, Transient Phosphatidylserine Externalization Induced in Host Cells by Infection withChlamydiaspp
Authors: Goth Samuel R. , Stephens Richard S.
Published: July 2002

Chlamydiaorganisms are obligate intracellular bacterial pathogens responsible for a range of human diseases. Persistent infection or reinfection withChlamydia trachomatisleads to scarring of ocular or genital tissues, andChlamydia pneumoniaeinfection is associated with the development of atherosclerosis. We demonstrate thatC. trachomatisandC. pneumoniaeinfection in vitro elicits the externalization of the lipid phosphatidylserine on the surface of human epithelial, endothelial, granulocytic, and monocytic cells. Phosphatidylserine externalization is associated with cellular development, differentiation, and death. Infection-induced phosphatidylserine externalization was immediate, transient, calcium dependent, and infectious dose dependent and was unaffected by a broad-spectrum caspase inhibitor.Chlamydia-infected cells accelerated plasma clotting and increased the macrophage phagocytosis of infected cells that was phosphatidylserine dependent. The rapid externalization of phosphatidylserine by infected cells may be an important factor in the pathogenesis of chlamydial infections.
Modulation of Cytokine Release in Ex Vivo-Stimulated Blood from Borreliosis Patients
Authors: Diterich Isabel , Härter Luc , Hassler Dieter , Wendel Albrecht , Hartung Thomas
Published: July 2002

In lipopolysaccharide-stimulated blood from 71 late-stage borreliosis patients, the ex vivo cytokine release capacity of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) was reduced to 28% ± 5% and to 31% ± 5% (P≤ 0.001), respectively, compared to that of 24 healthy controls. White blood cell counts were normal in both groups. To investigate direct interactions between the pathogen and the immune cells, blood from healthy controls was exposed in vitro to live or heat-killedBorreliaor toBorrelialysate. Compared to the pattern induced by bacterial endotoxins, a reduced release of TNF-α and IFN-γ and an enhanced secretion of interleukin-10 and granulocyte colony-stimulating factor was found. In blood from 10 borreliosis patients stimulated withBorrelialysate, TNF-α formation was decreased to 31% ± 14% and IFN-γ formation was decreased to 8% ± 3% (P≤ 0.001) compared to the cytokine response of blood from healthy controls (n= 24). We propose to consider anti-inflammatory changes in the blood cytokine response capacity elicited byBorreliaas a condition that might favor the persistence of the spirochete.
Clostridium difficile
Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins
Authors: Nusrat A. , von Eichel-Streiber C. , Turner J. R. , Verkade P. , Madara J. L. , Parkos C. A.
Published: July 2002

The anaerobic bacterium
Clostridium difficile
is the etiologic agent of pseudomembranous colitis.
C. difficile
toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021–16026, 1998). We utilized purified reference toxins of
C. difficile
, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting “raft-like” membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.
Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against
Streptococcus pneumoniae
Infection
Authors: Wizemann Theresa M. , Heinrichs Jon H. , Adamou John E. , Erwin Alice L. , Kunsch Charles , Choi Gil H. , Barash Steven C. , Rosen Craig A. , Masure H. Robert , Tuomanen Elaine , Gayle Anthony , Brewah Yambasu A. , Walsh William , Barren Philip , Lathigra Raju , Hanson Mark , Langermann Solomon , Johnson Syd , Koenig Scott
Published: July 2002

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen
Streptococcus pneumoniae
, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated
S. pneumoniae
infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against
S. pneumoniae
.
Passive Antibody toBartonella henselaeProtects against Clinical Disease following Homologous Challenge but Does Not Prevent Bacteremia in Cats
Authors: O'Reilly Kathy L. , Parr Katy A. , Brown Tracy P. , Tedder-Ferguson Belinda , Scholl Daniel T.
Published: July 2002

We challenged cats transfused with anti-Bartonellaserum and kittens born to antibody-positive queens withBartonella henselaeto determine the contribution of antibodies to the control ofB. henselaein cats. In both experiments, antibody-positive cats were protected from clinical disease but passive antibody to the homologous strain ofB. henselaedid not prevent bacteremia.
Monocytes Augment Bacterial Species- and Strain-Dependent Induction of Tissue Factor Activity in Bacterium-Infected Human Vascular Endothelial Cells
Authors: Veltrop M. H. A. M. , Thompson J. , Beekhuizen H.
Published: July 2002

In bacterial endocarditis (BE), intravascular infection withStaphylococcus aureus,Streptococcus sanguis,orStaphylococcus epidermidiscan lead to formation of a fibrin clot on the inner surface of the heart and cause heart dysfunction. The events that start the coagulation in the early stage of the disease are largely unknown. We have recently shown that human endothelial cells (EC) upon binding and internalization ofS. aureus, but notS. sanguisorS. epidermidis, express tissue factor (TF)-dependent procoagulant activity (TFA). The present study shows that infection of EC with these three pathogens induces surface expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and monocyte adhesion. Subsequent coculture of these cells synergistically enhanced TFA, which was exclusively dependent on TF molecules that were expressed on EC during coculture. TFA induction required direct contact between monocytes and bacterium-infected EC, but the signals for this response were not generated by the binding of monocytes through their β2- or α4-integrins to ICAM-1 or VCAM-1, respectively, on infected EC. The mechanism by which monocytes induce TFA in bacterium-infected EC was partly mediated by the proinflammatory cytokine interleukin-1 produced by the cells during coculture. Endogenous tumor necrosis factor alpha was not involved. This modulating effect of monocytes on species- and strain-dependent TFA of bacterium-infected EC supports our hypothesis that in an early stage in the pathogenesis of BE, as well as other intravascular infections that lead to detrimental fibrin formation, the coagulation cascade can be activated on the surfaces of EC as a consequence of specific interactions between pathogenic bacteria, EC, and monocytes.
Pneumolysin Potentiates Production of Prostaglandin E
2
and Leukotriene B
4
by Human Neutrophils
Authors: Cockeran Riana , Steel Helen C. , Mitchell Timothy J. , Feldman Charles , Anderson Ronald
Published: July 2002

Exposure to pneumolysin (8.37 and 41.75 ng/ml) caused a calcium-dependent increase in the generation of prostaglandin E
2
and leukotriene B
4
by both resting and chemoattractant-activated human neutrophils in vitro. These interactions of pneumolysin with neutrophils may result in dysregulation of inflammatory responses during pneumococcal infection.
Evolutionary Relationships among Virulence-Associated Histidine Kinases
Authors: Brinkman Fiona S. L. , Macfarlane Emma L. A. , Warrener Paul , Hancock Robert E. W.
Published: July 2002

A strong relationship between virulence-associated sensor histidine kinases of fungi and those in
Streptomyces coelicolor
was observed, and phylogenetic analysis suggested that bacterium-to-eukaryote horizontal gene transfer had occurred between ancestors of these organisms. Phylogenetic analysis also identified a group of histidine kinases orthologous to the
Streptomyces
proteins that includes
Pseudomonas aeruginosa
GacS. We provide evidence that GacS is important for swarming motility, lipase production, and virulence in mice and had evolved to have partial functional overlaps with PhoQ, a less-related virulence-associated histidine kinase.
Kinetics ofBartonella birtlesiiInfection in Experimentally Infected Mice and Pathogenic Effect on Reproductive Functions
Authors: Boulouis Henri J. , Barrat Francine , Bermond Delphine , Bernex Florence , Thibault Danièle , Heller Rémy , Fontaine Jean-Jacques , Piémont Yves , Chomel Bruno B.
Published: July 2002

The kinetics of infection and the pathogenic effects on the reproductive function of laboratory mice infected withBartonella birtlesiirecovered from anApodemusspecies are described.B. birtlesiiinfection, as determined by bacteremia, occurred in BALB/c mice inoculated intravenously. Inoculation with a low-dose inoculum (1.5 × 103CFU) induced bacteremia in only 75% of the mice compared to all of the mice inoculated with higher doses (≥1.5 × 104). Mice became bacteremic for at least 5 weeks (range, 5 to 8 weeks) with a peak ranging from 2 × 103to 105CFU/ml of blood. The bacteremia level was significantly higher in virgin females than in males but the duration of bacteremia was similar. In mice infected before pregnancy (n= 20), fetal loss was evaluated by enumerating resorption and fetal death on day 18 of gestation. The fetal death and resorption percentage of infected mice was 36.3% versus 14.5% for controls (P< 0.0001). Fetal suffering was evaluated by weighing viable fetuses. The weight of viable fetuses was significantly lower for infected mice than for uninfected mice (P< 0.0002). Transplacental transmission ofBartonellawas demonstrated since 76% of the fetal resorptions tested was culture positive forB. birtlesii. The histopathological analysis of the placentas of infected mice showed vascular lesions in the maternal placenta, which could explain the reproductive disorders observed. BALB/c mice appeared to be a useful model for studyingBartonellainfection. This study provides the first evidence of reproductive disorders in mice experimentally infected with aBartonellastrain originating from a wild rodent.
Specific Expression ofAnaplasma marginaleMajor Surface Protein 2 Salivary Gland Variants Occurs in the Midgut and Is an Early Event during Tick Transmission
Authors: Löhr Christiane V. , Rurangirwa Fred R. , McElwain Terry F. , Stiller David , Palmer Guy H.
Published: July 2002

Infectivity ofAnaplasmaspp. develops when infected ticks feed on a mammalian host (transmission feed). SpecificAnaplasma marginalemajor surface protein 2 (MSP2) variants are selected for within the tick and are expressed within the salivary glands. The aims of this study were to determine when and where MSP2 variant selection occurs in the tick, how MSP2 expression is regulated in salivary glands of transmission-feeding ticks, and whether the number ofA. marginaleorganisms per salivary gland is significantly increased during transmission feeding. The South Idaho strain ofA. marginalewas used, as MSP2 expression is restricted to two variants, SGV1 and SGV2, inDermacentor andersoni.Using Western blot, real-time PCR, and DNA sequencing analyses it was shown that restriction and expression of MSP2 occurs early in the midgut within the first 48 h of the blood meal, when ticks acquire infection.A. marginaleis present in the tick salivary glands before transmission feeding is initiated, but themsp2mRNA and MSP2 protein levels perA. marginaleorganism increase only minimally and transiently in salivary glands of transmission-feeding ticks compared to that of unfed ticks.A. marginalenumbers per tick increase gradually in salivary glands of both transmission-fed and unfed ticks. It is concluded that MSP2 variant selection is an early event in the tick and that MSP2 variants SGV1 and SGV2 are expressed both in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict temporal correlation between MSP2 expression and the development of infectivity.
Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of
Mycoplasma agalactiae
, AvgC, with the Use of Synthetic Peptides
Authors: Santona Antonella , Carta Franco , Fraghí Peppinetta , Turrini Franco
Published: July 2002

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of
Mycoplasma agalactiae
was studied and found to correspond to the AvgC protein encoded by the
avgC
gene. The
avg
genes of
M. agalactiae
, which encode four variable surface lipoproteins, display a significant homology to the
vsp
(variable membrane surface lipoproteins) genes of the bovine pathogen
Mycoplasma bovis
at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against
M. agalactiae
infection.
Antigenic Variation ofEhrlichia chaffeensisResulting from Differential Expression of the 28-Kilodalton Protein Gene Family
Authors: Long S. Wesley , Zhang Xiao-Feng , Qi Hai , Standaert Steven , Walker David H. , Yu Xue-Jie
Published: July 2002

The transcriptional activity and allele variation of the 28-kDa outer membrane protein gene (p28) ofEhrlichia chaffeensiswere analyzed to determine the mechanism of the antigenic variation of the 28-kDa outer membrane proteins. Reverse transcriptase PCR amplification of mRNA indicated that 16 of the 22 members of thep28multigene family were transcribed. Amino acid sequence analysis indicated that the p28-19 protein was produced in vitro in the Arkansas strain. Thep28-19gene and its promoter region were sequenced and compared in 12 clinical isolates ofE. chaffeensisto determine allele variation. The variation of thep28-19gene among the isolates is limited to three types represented by strains Arkansas, 91HE17, and Sapulpa, respectively. These results indicate that the majority of thep28genes are active genes and that antigenic variation of theE. chaffeensis28-kDa proteins may result from differential expression of thep28gene family members rather than gene conversion.
NikR Mediates Nickel-Responsive Transcriptional Induction of Urease Expression inHelicobacter pylori
Authors: van Vliet Arnoud H. M. , Poppelaars Sophie W. , Davies Beverly J. , Stoof Jeroen , Bereswill Stefan , Kist Manfred , Penn Charles W. , Kuipers Ernst J. , Kusters Johannes G.
Published: July 2002

The important human pathogenHelicobacter pylorirequires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity ofH. pyloriurease were previously demonstrated to be induced by nickel supplementation of growth media. Here it is demonstrated that the HP1338 protein, an ortholog of theEscherichia colinickel regulatory protein NikR, mediates nickel-responsive induction of urease expression inH. pylori. Mutation of the HP1338 gene (nikR) ofH. pyloristrain 26695 resulted in significant growth inhibition of thenikRmutant in the presence of supplementation with NiCl2at ≥100 μM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl2. Mutation ofnikRdid not affect urease subunit expression or urease enzyme activity in unsupplemented growth media. However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-typeH. pyloriwas absent in theH. pylori nikRmutant. A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in theureApromoter, as demonstrated by using a genomicureA::lacZreporter gene fusion. In conclusion, theH. pyloriNikR protein and a 19-bp operator sequence in theureApromoter are both essential for nickel-responsive induction of urease expression inH. pylori.
Protective Immunity Conferred by AttenuatedaroADerivatives ofPasteurella multocidaB:2 Strains in a Mouse Model of Hemorrhagic Septicemia
Authors: Tabatabaei Mohammad , Liu Zhiqi , Finucane Anna , Parton Roger , Coote John
Published: July 2002

Hemorrhagic septicemia (HS) is a fatal systemic disease of cattle and buffaloes. In South Asia HS is caused by infection withPasteurella multocidaserotype B:2. Some control is achieved with alum-precipitated or oil-adjuvanted killed whole-cell vaccines injected subcutaneously, but these vaccines provide only short-term immunity and require annual administration for effective use. Live attenuated vaccines have the advantage of a natural route of entry into the host, but for live strains to be used as vaccines, the mode of attenuation should be well defined. We constructedaroAattenuated derivatives of twoP. multocidaserotype B:2 strains by allelic exchange of the nativearoAsequence witharoAsequences disrupted with a kanamycin resistance cassette or with marker-freearoAsequences containing an internal deletion. These strains were confirmed to bearoAmutants by PCR and Southern blot analysis, enzyme assay, and lack of growth on minimal medium. ThearoAderivatives were highly attenuated for virulence in a mouse model of HS. Mouse challenge experiments showed that intraperitoneal or intranasal vaccination of anaroAstrain completely protected mice against challenge with a high dose (>1,000 50% lethal doses) of either the parent strain or the other wild-type B:2 strain. The spread of the parent and thearoAderivatives to different organs was compared when the organisms were inoculated by different routes.
A Dispensable Role for Forespore-Specific Gene Expression in Engulfment of the Forespore during Sporulation of
Bacillus subtilis
Authors: Sun Ya-Lin , Sharp Marc D. , Pogliano Kit
Published: July 2002

During the stage of engulfment in the
Bacillus subtilis
spore formation pathway, the larger mother cell engulfs the smaller forespore. We have tested the role of forespore-specific gene expression in engulfment using two separate approaches. First, using an assay that unambiguously detects sporangia that have completed engulfment, we found that a mutant lacking the only forespore-expressed engulfment protein identified thus far, SpoIIQ, is able to efficiently complete engulfment under certain sporulation conditions. However, we have found that the mutant is defective, under all conditions, in the expression of the late-forespore-specific transcription factor ς
G
; thus, SpoIIQ is essential for spore production. Second, to determine if engulfment could proceed in the absence of forespore-specific gene expression, we made use of a strain in which activation of the mother cell-specific sigma factor ς
E
was uncoupled from forespore-specific gene expression. Remarkably, engulfment occurred in the complete absence of ς
F
-directed gene expression under the same conditions permissive for engulfment in the absence of SpoIIQ. Our results demonstrate that forespore-specific gene expression is not essential for engulfment, suggesting that the machinery used to move the membranes around the forespore is within the mother cell.
Analysis of the SOS Response in
Salmonella enterica
Serovar Typhimurium Using RNA Fingerprinting by Arbitrarily Primed PCR
Authors: Benson Nicholas R. , Wong Rita M.-Y. , McClelland Michael
Published: July 2002

We report an analysis of a sample of the SOS response of
Salmonella enterica
serovar Typhimurium using the differential display of RNA fingerprinting gels of arbitrarily primed PCR products. The SOS response was induced by the addition of mitomycin C to an exponentially growing culture of serovar Typhimurium, and the RNA population was sampled during the following 2 h. These experiments revealed 21 differentially expressed PCR fragments representing mRNA transcripts. These 21 fragments correspond to 20 distinct genes. All of these transcripts were positively regulated, with the observed induction starting 10 to 120 min after addition of mitomycin C. Fifteen of the 21 transcripts have no homologue in the public sequence data banks and are therefore classified as novel. The remaining six transcripts corresponded to the
recE
,
stpA
,
sulA
, and
umuC
genes, and to a gene encoding a hypothetical protein in the
Escherichia coli lysU-cadA
intergenic region; the
recE
gene was represented twice by nonoverlapping fragments. In order to determine if the induction of these 20 transcripts constitutes part of a classical SOS regulon, we assessed the induction of these genes in a
recA
mutant. With one exception, the increased expression of these genes in response to mitomycin C was dependent on the presence of a functional
recA
allele. The exception was fivefold induced in the absence of a functional RecA protein, suggesting another layer of regulation in response to mitomycin C, in addition to the RecA-LexA pathway of SOS induction. Our data reveal several genes belonging to operons known to be directly involved in pathogenesis. In addition, we have found several phage-like sequences, some of which may be landmarks of pathogenicity determinants. On the basis of these observations, we propose that the general use of DNA-damaging agents coupled with differential gene expression analysis may be a useful and easy method for identifying pathogenicity determinants in diverse organisms.
Enterococcus faecalis
V583 Contains a Cytochrome
bd
-Type Respiratory Oxidase
Authors: Winstedt Lena , Frankenberg Lena , Hederstedt Lars , von Wachenfeldt Claes
Published: July 2002

We have cloned an
Enterococcus faecalis
gene cluster,
cydABCD
, which when expressed in
Bacillus subtilis
results in a functional cytochrome
bd
terminal oxidase. Our results indicate that
E. faecalis
V583 cells have the capacity of aerobic respiration when grown in the presence of heme.
The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle inSaccharomyces cerevisiae
Authors: Bakker Barbara M. , Bro Christoffer , Kötter Peter , Luttik Marijke A. H. , van Dijken Johannes P. , Pronk Jack T.
Published: July 2002

NDI1is the unique gene encoding the internal mitochondrial NADH dehydrogenase ofSaccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly,NDI1is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion ofADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, anndi1Δ mutant was capable of respiratory growth under these conditions. However, when bothADH3andNDI1were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of thendi1Δ mutant. In anaerobic batch cultures, the maximum specific growth rate of theadh3Δ mutant (0.22 h−1) was substantially reduced compared to that of the wild-type strain (0.33 h−1). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in theadh3Δ ndi1Δ mutant, contributing to residual redox-shuttle activity in this strain.
Overexpression of Protease-Deficient DegP
S210A
Rescues the Lethal Phenotype of
Escherichia coli
OmpF Assembly Mutants in a
degP
Background
Authors: Misra Rajeev , CastilloKeller Maria , Deng Ming
Published: July 2002

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a
degP
null background was eliminated when a protease-deficient DegP
S210A
protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP
S210A
was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP
S210A
also fractionated. These results suggested that the DegP
S210A
-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.
Alternative Transcription Factor ς
B
Is Involved in Regulation of Biofilm Expression in a
Staphylococcus aureus
Mucosal Isolate
Authors: Rachid Shwan , Ohlsen Knut , Wallner Ursula , Hacker Jörg , Hecker Michael , Ziebuhr Wilma
Published: July 2002

Osmotic stress was found to induce biofilm formation in a
Staphylococcus aureus
mucosal isolate. Inactivation of a global regulator of the bacterial stress response, the alternative transcription factor ς
B
, resulted in a biofilm-negative phenotype and loss of salt-induced biofilm production. Complementation of the mutant strain with an expression plasmid encoding ς
B
completely restored the wild-type phenotype. The combined data suggest a critical role of ς
B
in
S. aureus
biofilm regulation under environmental stress conditions.
Minimal and Contributing Sequence Determinants of the
cis
-Acting Locus of Transfer (
clt
) of Streptomycete Plasmid pIJ101 Occur within an Intrinsically Curved Plasmid Region
Authors: Ducote Matthew J. , Prakash Shubha , Pettis Gregg S.
Published: July 2002

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101
tra
gene, as well as a
cis
-acting plasmid function known as
clt
. Here we show that the minimal pIJ101
clt
locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3′ end of the
korB
regulatory gene. Evidence that sequences extending beyond the minimal locus and into the
korB
open reading frame influence
clt
transfer function and demonstration that
clt-korB
sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.
Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from
Escherichia coli
Authors: Kim Geun Joong , Lee Dong Eun , Kim Hak-Sung
Published: July 2002

A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes from
Escherichia coli
, flanked by related genes, were identified and characterized. From the genome sequence of
E. coli
, the
allB
gene and a putative open reading frame, tentatively designated as a
hyuA
(for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to
allB
, high-level expression of
hyuA
in
E. coli
of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures.
allB
was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame,
hyuA
, located near 64.4 min on the physical map and known as a UUG start, coded for
d
-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily,
E. coli
phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5′ position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in
E. coli
.
Physical Map of the Chromosome of the Apple Proliferation Phytoplasma
Authors: Lauer Ulrich , Seemüller Erich
Published: July 2002

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with
Bss
HII,
Sma
I,
Mlu
I, and
Apa
I restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the
fus
and
tuf
genes, and three other genes were placed on the map. Genome sizes and
Bss
HII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.
Multiple Alleles ofTreponema pallidumRepeat Gene D inTreponema pallidumIsolates
Authors: Centurion-Lara Arturo , Sun Eileen S. , Barrett Lynn K. , Castro Christa , Lukehart Sheila A. , Van Voorhis Wesley C.
Published: July 2002

Two newtprDalleles have been identified inTreponema pallidum:tprD2is found in 7 of 12T. pallidumsubsp.pallidumisolates and 7 of 8 non-pallidumisolates, andtprD3is found in oneT. pallidumsubsp.pertenueisolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating thattprD2is expressed during infection.
NadN and
e
(P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in
Haemophilus influenzae
Authors: Kemmer Gabriele , Reilly Thomas J. , Schmidt-Brauns Joachim , Zlotnik Gary W. , Green Bruce A. , Fiske Michael J. , Herbert Mark , Kraiß Anita , Schlör Stefan , Smith Arnold , Reidl Joachim
Published: July 2002

Haemophilus influenzae
has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein
e
(P4), which has phosphatase activity and is encoded by
hel
, and a periplasmic NAD nucleotidase, encoded by
nadN
. It has also been observed that the latter gene product is essential for
H. influenzae
growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5′-nucleotidase activity. The
e
(P4) protein is also shown to have NMN 5′-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of
nadN
or deletion and site-directed mutants of
hel
had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A
hel
and
nadN
double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.
Autotrophic CO2Fixation byChloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle
Authors: Herter Sylvia , Farfsing Jan , Gad'On Nasser , Rieder Christoph , Eisenreich Wolfgang , Bacher Adelbert , Fuchs Georg
Published: July 2002

In the facultative autotrophic organismChloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO2molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-13C2]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C1units derived therefrom and not into other cell compounds.
In Vivo Synthesis of the Periplasmic Domain of TonB Inhibits Transport through the FecA and FhuA Iron Siderophore Transporters of
Escherichia coli
Authors: Howard S. Peter , Herrmann Christina , Stratilo Chad W. , Braun V.
Published: July 2002

The siderophore transport activities of the two outer membrane proteins FhuA and FecA of
Escherichia coli
require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the
fecABCDE
transport genes by FecA, infection by phage φ80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuAΔ5-160 was also inhibited by TonB(33-239), although FhuAΔ5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.