bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1,2-Dichloroethane by
Xanthobacter autotrophicus
Authors: Hunkeler D. , Aravena R.
Published: July 2002

Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by
Xanthobacter autotrophicus
GJ10 was investigated. A strong enrichment of
13
C in residual 1,2-DCA was observed, with a mean fractionation factor α ± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor α to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of
13
C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S
N
2 nucleophilic substitution. S
N
2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.
Does Enterohemorrhagic
Escherichia coli
O157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?
Authors: Makino Sou-Ichi , Kii Tsutomu , Asakura Hiroshi , Shirahata Toshikazu , Ikeda Tetsuya , Takeshi Kouichi , Itoh Kikuji
Published: July 2002

An outbreak caused by salted salmon roe contaminated with enterohemorrhagic
Escherichia coli
O157 occurred in Japan in 1998. Since about 0.75 to 1.5 viable cells were estimated to cause infection, we presumed that O157 might enter the viable but nonculturable (VNC) state in salted salmon roe and consequently that viable cell numbers might be underestimated. Although patient-originating O157 cells could not grow on agar plates after 72 h of incubation in 13% NaCl, they were resuscitated in yeast extract broth, and more than 90% of the cells were shown to be viable by fluorescent staining, suggesting that almost all of them could enter the VNC state in NaCl water. Roe-originating O157 was resistant to NaCl because it could grow on agar after 72 h of incubation in NaCl water, but about 20% of cells appeared to enter the VNC state. Therefore, germfree mice were infected with O157 to examine the resuscitation of cells in the VNC state and the retention of pathogenicity. O157 that originated in roe, but not patients, killed mice and was isolated from the intestine. However, these isolates had become sensitive to NaCl. O157 cells of roe origin incubated in normal media also killed mice and were isolated from the intestine, but they also became transiently NaCl sensitive. We therefore propose that bacterial cells might enter the VNC state under conditions of stress, such as those encountered in vivo or in high salt concentrations, and then revive when those conditions have eased. If so, the VNC state in food is potentially dangerous from a public health viewpoint and may have to be considered at the time of food inspection. Finally, the establishment of a simple recovery system for VNC cells should be established.
Characterization of AbiR, a Novel Multicomponent Abortive Infection Mechanism Encoded by Plasmid pKR223 of
Lactococcus lactis
subsp.
lactis
KR2
Authors: Twomey Denis P. , De Urraza Patricio J. , McKay Larry L. , O'Sullivan Daniel J.
Published: July 2002

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated
Lla
KR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the
Lla
KR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, “early”-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.
Detection and Enumeration of Methanotrophs in Acidic
Sphagnum
Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for
Methylocella palustris
Authors: Dedysh Svetlana N. , Derakshani Manigee , Liesack Werner
Published: July 2002

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph
Methylocella palustris
using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the
Methylosinus
/
Methylocystis
group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a
Sphagnum
peat sample of pH 4.2.
M. palustris
was enumerated at greater than 10
6
cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of
M. palustris
. The cell counts with probe MA-221 suggested that only 10
4
type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10
6
type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of
Methylosinus
and
Methylocystis
, whereas probe MA-221, originally described as group specific, does not detect a large proportion of
Methylocystis
strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10
6
cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that
M. palustris
and a defined population of
Methylocystis
spp. were the predominant methanotrophs detectable by FISH in an acidic
Sphagnum
peat bog.
Epidemiology of
Rhodococcus equi
Strains on Thoroughbred Horse Farms
Authors: Morton Anna C. , Begg Angela P. , Anderson Garry A. , Takai Shinji , Lämmler Christoph , Browning Glenn F.
Published: July 2002

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of
Rhodococcus equi
, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of
R. equi
which carry the
vapA
gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.
Complete Denitration of Nitroglycerin by Bacteria Isolated from a Washwater Soakaway
Authors: Marshall Samantha J. , White Graham F.
Published: July 2002

Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology asPseudomonas putida, anArthrobacterspecies, aKlebsiellaspecies, and aRhodococcusspecies. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of theArthrobacterstrain, which achieved removal of only the first nitro group within the time course of the experiment. TheKlebsiellastrain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that theRhodococcusspecies was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures ofPenicillium corylophilum Dierckxsupplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.
Role of Serotype-Specific Polysaccharide in the Resistance ofStreptococcus mutansto Phagocytosis by Human Polymorphonuclear Leukocytes
Authors: Tsuda Hiromasa , Yamashita Yoshihisa , Toyoshima Kuniaki , Yamaguchi Noboru , Oho Takahiko , Nakano Yoshio , Nagata Kengo , Koga Toshihiko
Published: July 2002

To clarify the role of cell surface components ofStreptococcus mutansin resistance to phagocytosis by human polymorphonuclear leukocytes (PMNs), several isogenic mutants ofS. mutansdefective in cell surface components were studied with a luminol-enhanced chemiluminescence (CL) assay, a killing assay, and a transmission electron microscope. The CL responses of human PMNs to mutant Xc11 defective in a major cell surface antigen, PAc, and mutant Xc16 defective in two surface glucosyltransferases (GTF-I and GTF-SI) were the same as the response to the wild-type strain, Xc. In contrast, mutant Xc24R, which was defective in serotype c-specific polysaccharide, induced a markedly higher CL response than the other strains. The killing assay showed that human PMNs killed more Xc24R than the parent strain and the other mutants. The transmission electron microscopic observation indicated that Xc24R cells were more internalized by human PMNs than the parental strain Xc. These results may be reflected by the fact that strain Xc24R was more phagocytosed than strain Xc. The CL response of human PMNs to a mutant defective in polysaccharide serotype e or f was similar to the response to Xc24R. Furthermore, mutants defective in serotype-specific polysaccharide were markedly more hydrophobic than the wild-type strains and the other mutants, suggesting that the hydrophilic nature of polysaccharides may protect the bacterium from phagocytosis. We conclude that the serotype-specific polysaccharide, but not the cell surface proteins on the cell surface ofS. mutans, may play an important role in the resistance to phagocytosis.
Depressed CD40 Ligand Expression Contributes to Reduced Gamma Interferon Production in Human Tuberculosis
Authors: Samten Buka , Thomas Elaine K. , Gong Jianhua , Barnes Peter F.
Published: July 2002

Expression of CD40 ligand (CD40L) correlated directly withMycobacterium tuberculosis-stimulated gamma interferon (IFN-γ) production by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy tuberculin reactors. The CD40L agonist increasedM. tuberculosis-induced IFN-γ production by PBMC, and anti-CD40 or anti-CD40L antibodies reduced IFN-γ production. CD40L expression on PBMC was reduced by exposure to B cells and to soluble factors fromM. tuberculosis-infected monocytes. These findings suggest that CD40L dysregulation contributes to reduced IFN-γ production in human tuberculosis.
Expression of Intimin γ from Enterohemorrhagic
Escherichia coli
in
Citrobacter rodentium
Authors: Hartland Elizabeth L. , Huter Veronika , Higgins Lisa M. , Goncalves Nathalie S. , Dougan Gordon , Phillips Alan D. , MacDonald Thomas T. , Frankel Gad
Published: July 2002

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated α, β, γ, δ, and ɛ, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of
Citrobacter rodentium
, which normally displays intimin β, to express intimin γ from enterohemorrhagic
Escherichia coli
. We show that intimin γ binds to the translocated intimin receptor (Tir) from
C. rodentium
and has the ability to produce attaching and effacing lesions on HEp-2 cells. However,
C. rodentium
expressing intimin γ could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.
DNA-Based Immunization withTrypanosoma cruziComplement Regulatory Protein Elicits Complement Lytic Antibodies and Confers Protection againstTrypanosoma cruziInfection
Authors: Sepulveda Pilar , Hontebeyrie Mireille , Liegeard Pascal , Mascilli Alexia , Norris Karen A.
Published: July 2002

A complement regulatory protein (CRP) ofTrypanosoma cruziwas evaluated as a vaccine candidate in a murine model of experimentalT. cruziinfection. Recombinant CRP derived from anEscherichia coliexpression system and a plasmid encoding the full-lengthcrpstructural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with thecrpDNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge withT. cruzitrypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruziDNA vaccine.
Role of Fc Gamma Receptors in Triggering Host Cell Activation and Cytokine Release byBorrelia burgdorferi
Authors: Talkington Jeffrey , Nickell Steven P.
Published: July 2002

Borrelia burgdorferi, the spirochetal bacterium that causes human Lyme disease, encodes numerous lipoproteins which have the capacity to trigger the release of proinflammatory cytokines from a variety of host cell types, and it is generally believed that these cytokines contribute to the disease process in vivo. We previously reported that low-passage-number infectiousB. burgdorferispirochetes express a novel lipidation-independent activity which induces secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) by the mouse MC/9 mast cell line. Using RNase protection assays, we determined that mast cells exposed in vitro to low-passage-number, but not high-passage-number,B. burgdorferispirochetes show increased expression of additional mRNAs representing several chemokines, including macrophage-inflammatory protein 1α (MIP-1α), MIP-1β, and TCA3, as well as the proinflammatory cytokine interleukin-6. Furthermore, mast cell TNF-α secretion can be inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin and also by preincubation with purified mouse immunoglobulin G1 (IgG1) and IgG2a, but not mouse IgG3, and by a mouse Fc gamma receptor II and III (FcγRII/III)-specific rat monoclonal antibody, suggesting the likely involvement of host FcγRIII inB. burgdorferi-mediated signaling. A role for passively adsorbed rabbit or bovine IgG or serum components inB. burgdorferi-mediated FcγR signaling was excluded in control experiments. These studies confirm that low-passage-numberB. burgdorferispirochetes express a novel activity which upregulates the expression of a variety of host cell chemokine and cytokine genes, and they also establish a novel antibody-independent role for FcγRs in transduction of activation signals by bacterial products.
Minor Nucleotide Substitutions in the
omp31
Gene of
Brucella ovis
Result in Antigenic Differences in the Major Outer Membrane Protein That It Encodes Compared to Those of the Other
Brucella
Species
Authors: Vizcaı́no Nieves , Kittelberger Reinhold , Cloeckaert Axel , Marı́n Clara M. , Fernández-Lago Luis
Published: July 2002

The gene coding for the major outer membrane protein Omp31 was sequenced in five
Brucella
species and their biovars. Although the
omp31
genes appeared to be highly conserved in the genus
Brucella
, nine nucleotide substitutions were detected in the gene of
Brucella ovis
compared to that of
Brucella melitensis
. As shown by differential binding properties of monoclonal antibodies (MAbs) to the two
Brucella
species, these nucleotide substitutions result in different antigenic properties of Omp31. The antigenic differences were also evidenced when sera from
B. ovis
-infected rams were tested by Western blotting with the recombinant
B. melitensis
or
B. ovis
Omp31 proteins. Twelve available sera reacted with recombinant
B. ovis
Omp31, but only four of them reacted with recombinant
B. melitensis
Omp31. These results validate previous evidence for the potential of Omp31 as a diagnostic antigen for
B. ovis
infection in rams and demonstrate that
B. ovis
Omp31, instead of
B. melitensis
Omp31, should be used to evaluate this point. The antigenic differences between the
B. melitensis
and
B. ovis
Omp31 proteins should also be taken into account when Omp31 is evaluated as a candidate for the development of subcellular vaccines against
B. ovis
infection. No reactivity against recombinant
B. melitensis
Omp31 was detected, by Western blotting, with sera from
B. melitensis
-infected sheep. Accordingly, Omp31 does not seem to be a good diagnostic antigen for
B. melitensis
infections in sheep. Two immunodominant regions were identified on the
B. ovis
Omp31 protein by using recombinant DNA techniques and specific MAbs. Sera from
B. ovis
-infected rams that reacted with the recombinant protein were tested by Western blotting against one of these immunodominant regions shown to be exposed at the bacterial surface. Only 4 of the 12 sera reacted, but with strong intensity.
Induction of Epithelial Cell Death Including Apoptosis by Enteropathogenic
Escherichia coli
Expressing Bundle-Forming Pili
Authors: Abul-Milh Maan , Wu Ying , Lau Bedy , Lingwood Clifford A. , Foster Debora Barnett
Published: July 2002

Infection with enteropathogenic
Escherichia coli
(EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world. The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation. We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells. BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis. The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a
bfp
-carrying plasmid. Strains which did not express BFP induced significantly less cell death, including a
bfpA
disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island. These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine.
Analysis of
Escherichia coli
Strains Causing Bacteriuria during Pregnancy: Selection for Strains That Do Not Express Type 1 Fimbriae
Authors: Graham J. C. , Leathart J. B. S. , Keegan S. J. , Pearson J. , Bint A. , Gally D. L.
Published: July 2002

Escherichia coli
isolates from patients with bacteriuria of pregnancy were compared by PCR with isolates from patients with community-acquired cystitis for the presence of established virulence determinants. The strains from patients with bacteriuria of pregnancy were less likely to carry genes for P-family, S-family, and F1C adhesins, cytotoxic necrotizing factor 1, and aerobactin, but virtually all of the strains carried the genes for type 1 fimbriae. Standard mannose-sensitive agglutination of yeast cells showed that only 15 of 42 bacteriuria strains (36%) expressed type 1 fimbriae compared with 32 of 42 strains from community-acquired symptomatic infections (76%) (
P
< 0.01). This difference was confirmed by analysis of all isolates for an allele of the type 1 fimbrial regulatory region (
fim
switch), which negates type 1 fimbrial expression by preventing the
fim
switch from being inverted to the on phase. This allele,
fimS49
, was found in 8 of 47 bacteriuria strains from pregnant women (17.0%) compared with 2 of 60 strains isolated from patients with cystitis (3.3%) (
P
< 0.05). Determination of the phase switch orientation in vivo by analysis of freshly collected infected urine from patients with bacteriuria showed that the
fim
switch was detectable in the off orientation in 17 of 23 urine samples analyzed (74%). These data indicate that type 1 fimbriae are not necessary to maintain the majority of
E. coli
bacteriurias in pregnant women since there appears to be selection against their expression in this particular group. This is in contrast to the considered role of this adhesin in community-acquired symptomatic infections. The lack of type 1 fimbria expression is likely to contribute to the asymptomatic nature of bacteriuria in pregnant women, although approximately one-third of the bacteriuria isolates do possess key virulence determinants. If left untreated, this subset of isolates pose the greatest threat to the health of the mother and unborn child.
Transcription of Candidate Virulence Genes of
Haemophilus ducreyi
during Infection of Human Volunteers
Authors: Throm Robert E. , Spinola Stanley M.
Published: July 2002

Haemophilus ducreyi
expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (
momp, ftpA, losB, lst, cdtC
, and
hhdB
) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with
H. ducreyi
were subjected to reverse transcription-PCR. Transcripts corresponding to
momp, ftpA, losB, lst, cdtB
, and
hhdA
were expressed in vivo. In addition, transcripts for other putative virulence determinants such as
ompA2, tdhA, lspA1
, and
lspA2
were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.
Major Histocompatibility Complex Class II-Independent Generation of Neutralizing Antibodies against T-Cell-DependentBorrelia burgdorferiAntigens Presented by Dendritic Cells: Regulation by NK and γδ T Cells
Authors: Mbow M. Lamine , Zeidner Nordin , Gilmore Robert D. , Dolan Marc , Piesman Joseph , Titus Richard G.
Published: July 2002

We previously showed that adoptive transfer ofBorrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized withB. burgdorferi-pulsed MHC class II-deficient (MHC class II−/−) DCs mounted a humoral response against protective antigens, includingB. burgdorferiouter surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by γδ T cells. In contrast, anti-OspA Ab production required the presence of αβ T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II−/−DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as γδ and NK cells, can influence the generation of a protective humoral response againstB. burgdorferiantigens.
Potential Virulence Role of theLegionella pneumophila ptsPOrtholog
Authors: Higa Futoshi , Edelstein Paul H.
Published: July 2002

We previously identified theLegionella pneumophila ptsP(phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties ofL. pneumophila ptsPand its complete sequence. TheL. pneumophila ptsPwas 2,295 bases in length. Its deduced amino acid sequence had high similarity withptsPorthologs ofPseudomonas aeruginosa, Azotobacter vinelandii, andEscherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of theL. pneumophila ptsPmutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. TheL. pneumophila ptsPmutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. TheL. pneumophila ptsPmutant expressed DotA and IcmX in apparently normal amounts, suggesting that theptsPmutation did not affectdotAandicmXregulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show thatL. pneumophila ptsPis required for full in vivo virulence ofL. pneumophila, most probably by affecting intracellular growth.
Dissociated Linkage of Cytokine-Inducing Activity and Cytotoxicity to Different Domains of Listeriolysin O from
Listeria monocytogenes
Authors: Kohda Chikara , Kawamura Ikuo , Baba Hisashi , Nomura Takamasa , Ito Yutaka , Kimoto Terumi , Watanabe Isao , Mitsuyama Masao
Published: July 2002

Listeriolysin O (LLO), a cholesterol-binding cytolysin of
Listeria monocytogenes
, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-γ)-inducing activities. rLLO529 exhibited both IFN-γ-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-γ production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-γ-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-γ production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-γ-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.
Regulatory Role of Interleukin-10 in Experimental Group B Streptococcal Arthritis
Authors: Puliti Manuela , von Hunolstein Christina , Verwaerde Claudie , Bistoni Francesco , Orefici Graziella , Tissi Luciana
Published: July 2002

Intravenous inoculation of CD-1 mice with 107CFU of type IV group BStreptococcus(GBS) results in a high incidence of diffuse septic arthritis,associated with high levels of systemic and local production of interleukin-1β (IL-1β) and IL-6. In this study, the role of the anti-inflammatory cytokine IL-10 in the evolution of GBS systemic infection and arthritis was evaluated. IL-10 production was evident in sera and joints of GBS-infected mice. Neutralization of endogenous IL-10 by administration of anti-IL-10 antibodies (1 mg/mouse) at the time of infection resulted in worsening of articular lesions and 60% mortality associated with early sustained production of IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α). The effect of IL-10 supplementation was assessed by administering IL-10 (100, 200, or 400 ng/mouse) once a day for 5 days, starting 1 h after infection. Treatment with IL-10 had a beneficial effect on GBS arthritis, and there was a clear-cut dose dependence. The decrease in pathology was associated with a significant reduction in IL-6, IL-1β, and TNF-α production. Histological findings showed limited periarticular inflammation and a few-cell influx in the articular cavity of IL-10-treated mice, confirming clinical observations. In conclusion, this study provides further information concerning the role of IL-10 in regulating the immune response and inflammation and calls attention to the potential therapeutic use of IL-10 in GBS arthritis.
Transformation of
Rickettsia prowazekii
to Erythromycin Resistance Encoded by the
Escherichia coli ereB
Gene
Authors: Rachek Lyudmila I. , Hines Andria , Tucker Aimee M. , Winkler Herbert H. , Wood David O.
Published: July 2002

Rickettsia prowazekii
, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an
R. prowazekii rpoB
chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in
R. prowazekii
of the
Escherichia coli ereB
gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in
R. prowazekii
. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.
HARO7
Encodes Chorismate Mutase of the Methylotrophic Yeast
Hansenula polymorpha
and Is Derepressed upon Methanol Utilization
Authors: Krappmann Sven , Pries Ralph , Gellissen Gerd , Hiller Mark , Braus Gerhard H.
Published: July 2002

The
HARO7
gene of the methylotrophic, thermotolerant yeast
Hansenula polymorpha
was cloned by functional complementation.
HARO7
encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC
5.4.99.5
) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The
HARO7
gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (
k
cat
= 319.1 s
−1
,

n
H

= 1.56, [
S
]
0.5
= 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of
Saccharomyces cerevisiae
. Using the Cre-
loxP
recombination system, we constructed mutant strains carrying a disrupted
HARO7
gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb
HARO7
mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on
HARO7
expression.
Sequence Variations within PrfA DNA Binding Sites and Effects on
Listeria monocytogenes
Virulence Gene Expression
Authors: Williams James R. , Thayyullathil Chetna , Freitag Nancy E.
Published: July 2002

Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to
Listeria monocytogenes
virulence gene expression. Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as
hly
and
mpl
. Other virulence genes, such as
actA
and
plcB
, may depend upon additional factors for full regulation of gene expression.
Accumulation of the PhaP Phasin of
Ralstonia eutropha
Is Dependent on Production of Polyhydroxybutyrate in Cells
Authors: York Gregory M. , Junker Björn H. , Stubbe JoAnne , Sinskey Anthony J.
Published: July 2002

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of
Ralstonia eutropha. R. eutropha phaC
PHA synthase and
phaP
phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a
phaP
-
gfp
translational fusion in place of the wild-type alleles of
phaC
and
phaP
. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the
phaP
-
gfp
fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that
R. eutropha
has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.
Global Analysis of the General Stress Response of
Bacillus subtilis
Authors: Petersohn Anja , Brigulla Matthias , Haas Stefan , Hoheisel Jörg D. , Völker Uwe , Hecker Michael
Published: July 2002

Gene arrays containing all currently known open reading frames of
Bacillus subtilis
were used to examine the general stress response of
Bacillus
. By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as ς
B
-dependent general stress genes. The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor ς
B
. At least 24 of these 125 ς
B
-dependent genes seemed to be subject to a second, ς
B
-independent stress induction mechanism. Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the ς
B
regulon indicates that even this approach has not yet elucidated the entire regulon. Most of the ς
B
-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their classification as ς
B
-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance. A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress. The first results of this program, showing the diminished salt resistance of
yjbC
and
yjbD
mutants compared to that of the wild type, are presented. Only a few new ς
B
-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase. In addition to analysis of the ς
B
-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a ς
B
-independent manner is presented. Perhaps the most interesting of the ς
B
-independent stress phenomena was the induction of the extracytoplasmic function sigma factor ς
W
and its entire regulon by salt shock.
Redox Signal Transduction by the ArcB Sensor Kinase of
Haemophilus influenzae
Lacking the PAS Domain
Authors: Georgellis Dimitris , Kwon Ohsuk , Lin Edmund C. C. , Wong Sandy M. , Akerley Brian J.
Published: July 2002

The Arc (anoxic redox control) two-component signal transduction system of
Escherichia coli
, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the
arcA
and
arcB
genes of
Haemophilus influenzae
specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of
H. influenzae
mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in
E. coli
, indicating a high degree of functional conservation of the signal transduction system. The
H. influenzae
ArcB, however, lacks the PAS domain present in the region of
E. coli
ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of
H. influenzae
mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.
Biochemical Analysis of Interactions between Outer Membrane Proteins That Contribute to Starch Utilization by
Bacteroides thetaiotaomicron
Authors: Cho Kyu Hong , Salyers Abigail A.
Published: July 2002

An early step in the utilization of starch by
Bacteroides thetaiotaomicron
is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of
B. thetaiotaomicron
that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (μ
max
, 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex.
Ability for Anaerobic Growth Is Not Sufficient for Development of the Petite Phenotype in
Saccharomyces kluyveri
Authors: Mo/ller Kasper , Olsson Lisbeth , Piškur Jure
Published: July 2002

Saccharomyces cerevisiae
is a petite-phenotype-positive (“petite-positive”) yeast, which can successfully grow in the absence of oxygen. On the other hand,
Kluyveromyces lactis
as well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that
Saccharomyces kluyveri
can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view,
S. kluyveri
is apparently more closely related to
S. cerevisiae
than to
K. lactis
. These observations suggest that the progenitor of the modern
Saccharomyces
and
Kluyveromyces
yeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the
K. lactis
and
S. kluyveri-S. cerevisiae
lineages, the latter developed the ability to grow anaerobically. However, while the
S. kluyveri
lineage has remained petite negative, the lineage leading to the modern
Saccharomyces
sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.
Characterization of In Vitro Interactions between a Truncated TonB Protein fromEscherichia coliand the Outer Membrane Receptors FhuA and FepA
Authors: Moeck Gregory S. , Letellier Lucienne
Published: July 2002

High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB structure and function, we expressed a recombinant form ofEscherichia coliTonB lacking the N-terminal cytoplasmic membrane anchor. This protein (H6-′TonB;Mr, 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation experiments indicated that the H6-′TonB preparation was almost monodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography. The frictional ratio (2.0) suggested that H6-′TonB adopts a highly asymmetrical form with an axial ratio of 15. H6-′TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified H6-′TonB was found to be stable and thus shows promise for high-resolution structural studies.
Hypoxic Response ofMycobacterium tuberculosisStudied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins
Authors: Rosenkrands Ida , Slayden Richard A. , Crawford Janne , Aagaard Claus , Barry Clifton E. , Andersen Peter
Published: July 2002

The events involved in the establishment of a latent infection withMycobacterium tuberculosisare not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight intoM. tuberculosisprotein expression in in vitro latency models where oxygen is depleted. The response ofM. tuberculosisto low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates ofM. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). InM. tuberculosisculture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response inM. tuberculosisand potentially provide important insights into the physiology of the latent bacilli.
Bacillus subtilis
Tolerance of Moderate Concentrations of Rifampin Involves the σ
B
-Dependent General and Multiple Stress Response
Authors: Bandow Julia Elisabeth , Brötz Heike , Hecker Michael
Published: July 2002

Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of
Bacillus subtilis
led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment.
l
-[
35
S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The σ
B
-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the β subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the σ
D
regulon were also induced. The rifampin-triggered growth arrest was extended in a
sigB
mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the σ
B
signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The
sigB
mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the
sigB
mutant by a factor of 100. We conclude that the general stress response of
B. subtilis
is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.