bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods
Authors: Martin-Laurent F. , Philippot L. , Hallet S. , Chaussod R. , Germon J. C. , Soulas G. , Catroux G.
Published: July 2002

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.
New Degenerate
Cytophaga
-
Flexibacter
-
Bacteroides
-Specific 16S Ribosomal DNA-Targeted Oligonucleotide Probes Reveal High Bacterial Diversity in River Taff Epilithon
Authors: O’Sullivan Louise A. , Weightman Andrew J. , Fry John C.
Published: July 2002

River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the
Cytophaga-Flexibacter-Bacteroides
division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the α subdivision of the division
Proteobacteria
(α-
Proteobacteria
), γ-
Proteobacteria
, gram-positive bacteria,
Cyanobacteria
, β-
Proteobacteria
, δ-
Proteobacteria
, and the
Prosthecobacter
group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups
Cytophaga
,
Sphingobacterium
,
Lewinella
, and
Cytophaga aurantiaca
. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.
Salmonella enterica
Serovar Typhimurium and
Escherichia coli
Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure
Authors: Natvig Erin E. , Ingham Steven C. , Ingham Barbara H. , Cooperband Leslie R. , Roper Teryl R.
Published: July 2002

Bovine manure, with or without added
Salmonella enterica
serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The
S. enterica
serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for
S. enterica
serovar Typhimurium and
Escherichia coli
(indigenous in manure). After the 1 March manure application,
S. enterica
serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application,
S. enterica
serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil,
S. enterica
serovar Typhimurium died at a similar rate (
P
≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with
S. enterica
serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20°C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of
S. enterica
serovar Typhimurium and
E. coli
in manure-fertilized soil. The number of indigenous
E. coli
in soil was never significantly lower (
P
< 0.05) than that of
S. enterica
serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne
S. enterica
serovar Typhimurium.
Immunoglobulin E Antileishmanial Antibody Response in Cutaneous Leishmaniasis
Authors: Sousa-Atta Maria L. B. , Salamé Gregório S. , D’Oliveira Argemiro , Almeida Roque P. , Atta Ajax M. , Carvalho Edgar M.
Published: July 2002

High levels of antileishmanial immunoglobulin E (IgE) antibodies are associated with disease activity in visceral leishmaniasis. Herein, we report our observations about the relationship between antileishmanial IgE antibodies and clinical aspects of cutaneous leishmaniasis. This study was carried out with 45 patients (29 male and 16 female), with ages ranging from 11 to 48 years. All subjects were from an area to which leishmaniasis is endemic, Corte de Pedra (Bahia, Brazil), and the duration of the illness was ≤30 days. The patients were classified as positive or negative for IgE serology in enzyme-linked immunosorbent assay with leishmanial antigens. IgE antibodies were detected in 18 patients (optical density, 0.421 ± 0.30; 95% confidence interval, 0.27 to 0.57), and only 3 (17%) had more than one ulcer. In this group the diameter of Montenegro’s reaction was 18 ± 12.2 mm. In the group with negative IgE serology, 11 of 27 patients (48%) presented two or more cutaneous ulcers, and the mean of the skin test result was 9 ± 6.9 mm. There was a positive correlation between IgE antibody levels and Montenegro’s reaction size and an inverse correlation between IgE antileishmanial antibodies and the number of skin ulcers. The presence of antileishmanial IgE antibodies in cutaneous leishmaniasis may be a result of immunoregulatory events with clinical implications.
Relevance of Dietary Lipids as Modulators of Immune Functions in Cells Infected with
Listeria monocytogenes
Authors: Puertollano María A. , de Pablo Manuel A. , Álvarez de Cienfuegos Gerardo
Published: July 2002

Nutritional status may have significant importance for the immune system, and particularly, unsaturated fatty acids may serve as modulators of immune functions. Clinical and epidemiological studies have demonstrated that fatty acids are involved in the reduction of the inflammatory processes that occur in diseases characterized by an overactivation of the immune system. At the same time, an increase in susceptibility to infection has also been reported. The importance of immune system modulation by dietary lipids in the presence of an intracellular bacterial pathogen, such as
Listeria monocytogenes
, was evaluated in the present study. BALB/c mice were divided into four groups which were each fed a low-fat (2.5% by weight) diet, an olive oil (OO; 20% by weight) diet, a fish oil (FO; 20% by weight) diet, or a hydrogenated coconut oil (HCO; 20% by weight) diet for 4 weeks. In each group, lymphocye proliferation was measured, and a reduction in the stimulation index was observed in the FO and HCO groups. Cytotoxicity exerted by
L. monocytogenes
was increased in the groups fed diets containing OO and FO after 6 h of incubation with the bacterium. An important increase in the production of reactive oxygen species was found in the groups fed the HCO diet after 12 h of incubation with
L. monocytogenes
. Finally, invasion and adhesion factors were not modified substantially by the action of dietary lipids, although these factors were reduced in cells from mice fed an FO diet. These results underline the importance of several dietary lipids as biological modulators of immune functions and their crucial role in the alteration of host natural resistance.
Elucidation of the Cross-Reactive Immunoglobulin M Response to Human Herpesviruses 6 and 7 on the Basis of Neutralizing Antibodies
Authors: Yoshida Mariko , Torigoe Sadayoshi , Yamada Masao
Published: July 2002

Human herpesvirus 6 (HHV-6) is closely related to HHV-7 in terms of genome organization and sequence. The cross-reactive responses between HHV-6 and HHV-7 have been reported by using immunofluorescent techniques. Recently we have shown that neutralizing (NT) antibody responses are specific to each virus and do not cross-react. We took advantage of this and used the NT antibody response to estimate the time of seroconversion to each virus and examined the pattern of humoral immune response, especially the immunoglobulin M (IgM) response, against each virus antigen in the natural course of infection with HHV-6 and HHV-7. In children who experienced HHV-6 infection first, followed by HHV-7 infection, the IgM response at the first HHV-6 infection was directed only against HHV-6, while no IgM response was directed against HHV-7 at the second HHV-7 infection. In contrast, in children who experienced HHV-7 infection first, followed by HHV-6 infection, the IgM response at the first HHV-7 infection was directed not only against HHV-7 but also against HHV-6. These data suggest that cross-reactive responses to heterologous viruses should be taken into consideration when making a diagnosis based on IgM antibody.
Polyclonal Antibodies to Glutathione
S
-Transferase- Verotoxin Subunit A Fusion Proteins Neutralize Verotoxins
Authors: Leung P. H. M. , Peiris J. S. M. , Ng W. W. S. , Yam W. C.
Published: July 2002

The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione
S
-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing
Escherichia coli
(VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% ± 7.9% and 3.6% ± 2.3%, respectively, and those of VT2 were 1.7% ± 2.3% and 82.5% ± 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.
Vaccination Strategies for Mucosal Immune Responses
Authors: Ogra Pearay L. , Faden Howard , Welliver Robert C.
Published: July 2002

Mucosal administration of vaccines is an important approach to the induction of appropriate immune responses to microbial and other environmental antigens in systemic sites and peripheral blood as well as in most external mucosal surfaces. The development of specific antibody- or T-cell-mediated immunologic responses and the induction of mucosally induced systemic immunologic hyporesponsiveness (oral or mucosal tolerance) depend on complex sets of immunologic events, including the nature of the antigenic stimulation of specialized lymphoid structures in the host, antigen-induced activation of different populations of regulatory T cells (Th1 versus Th2), and the expression of proinflammatory and immunoregulatory cytokines. Availability of mucosal vaccines will provide a painless approach to deliver large numbers of vaccine antigens for human immunization. Currently, an average infant will receive 20 to 25 percutaneous injections for vaccination against different childhood infections by 18 months of age. It should be possible to develop for human use effective, nonliving, recombinant, replicating, transgenic, and microbial vector- or plant-based mucosal vaccines to prevent infections. Based on the experience with many dietary antigens, it is also possible to manipulate the mucosal immune system to induce systemic tolerance against environmental, dietary, and possibly other autoantigens associated with allergic and autoimmune disorders. Mucosal immunity offers new strategies to induce protective immune responses against a variety of infectious agents. Such immunization may also provide new prophylactic or therapeutic avenues in the control of autoimmune diseases in humans.
Identification of a Fibronectin Binding Protein fromStreptococcus mutans
Authors: Chia Jean-San , Yeh Chiou-Yueh , Chen Jen-Yang
Published: July 2002

The interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein ofStreptococcus mutanswhich binds the ECM constituent fibronectin (Fn). Initially, we found thatS. mutanscould adsorb soluble Fn in plasma, but with lower efficiency thanStreptococcus pyogenes. In addition,S. mutanscould bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins fromS. mutansMT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence ofS. mutansto immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence ofS. mutansspecifically to Fn and endothelial cells in vitro. The characteristics ofS. mutansand FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.
Full Capacity of Recombinant
Escherichia coli
Heat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity
Authors: Batisson Isabelle , Der Vartanian Maurice , Gaillard-Martinie Brigitte , Contrepois Michel
Published: July 2002

We have successfully used the major subunit ClpG of
Escherichia coli
CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the
E. coli
human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the
E. coli
cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH
2
or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the
E. coli
cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of
E. coli
, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of
E. coli
for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.
Chronic
Helicobacter pylori
Infection Induces an Apoptosis-Resistant Phenotype Associated with Decreased Expression of p27
kip1
Authors: Shirin Haim , Sordillo Emilia Mia , Kolevska Tatiana K. , Hibshoosh Hanina , Kawabata Yuichi , Oh Sung H. , Kuebler Joachim F. , Delohery Thomas , Weghorst Christopher M. , Weinstein I. Bernard , Moss Steven F.
Published: July 2002

Helicobacter pylori
infection is associated with the development of gastric cancer. In short-term coculture with AGS gastric cells,
H. pylori
inhibits cell cycle progression and induces dose-dependent apoptosis. Based on the concept that an imbalance between proliferation and apoptosis may contribute to the emergence of gastric cancer, we chronically exposed AGS cells to
H. pylori
as a model of chronic exposure in humans. The AGS derivatives selected by this process were stably resistant not only to
H. pylori
-induced apoptosis but also to apoptosis induced by other enteric bacteria and by several toxic agents including radiation and cancer chemotherapy. Like the parental AGS cells, the derivatives underwent G
1
/S-phase cell cycle inhibition in response to
H. pylori
. The AGS derivatives displayed a marked decrease in cellular levels of the cell cycle control protein p27
kip1
. We found a similar decrease in epithelial cell p27
kip1
expression in gastric biopsy specimens from
H. pylori
-infected patients. These findings are consistent with observations that link decreases in the p27
kip1
level to increased susceptibility to cancer in mice with p27
kip1
deleted and to a poor prognosis of gastric cancer in humans. This is the first demonstration that bacterial infection can lead to apoptosis resistance and to cross-resistance to other inducers of apoptosis such as bacteria, chemotherapeutic agents, and radiation. The development of apoptosis resistance and downmodulation of p27
kip1
may contribute to the increased risk for gastric cancer observed in humans chronically exposed to
H. pylori
.
A 60-Kilodalton Immunodominant Glycoprotein Is Essential for Cell Wall Integrity and the Maintenance of Cell Shape in
Streptococcus mutans
Authors: Chia Jean-San , Chang Lan Yi , Shun Chia-Tung , Chang Ying-Ying , Chen Jen-Yang
Published: July 2002

We have demonstrated previously by Western blotting that in naturally sensitized humans, the serum or salivary antibody response to
Streptococcus mutans
was directed predominantly to a protein antigen with a size of approximately 60-kDa. To identify this immunodominant antigen, specific serum antibodies were eluted from immunoblots and five positive clones with inserts ranging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of
Streptococcus mutans
GS-5. Amino acid sequencing established the identity of this immunodominant antigen, a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associated general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide sequence. Discrepancy in the molecular mass was also observed in recombinant his-tagged IDG-60 (rIDG-60) expressed from
Escherichia coli
. Glycosylation, consisting of sialic acid, mannose galactose, and
N
-acetylgalactosamine, was detected by lectin binding to IDG-60 in cell wall extracts from
S. mutans
and rIDG-60 expressed in vivo or translated in vitro. Despite the presence of multiple Asn or Ser or Thr glycosylation sites, IDG-60 was resistant to the effect of
N
-glycosidase F and multiple
O
-glycosidase molecules but not to β-galactosidase. Insertional inactivation of the gene encoding IDG-60,
sagA
, resulted in a retarded growth rate, destabilization of the cell wall, and pleiomorphic cell shape with multifold ingrowth of cell wall. In addition, distinct from the parental GS-5 strain, the isogenic mutant GS-51 was unable to survive the challenge of low pH and high osmotic pressure or high temperature. Expression of the wild-type gene in trans within GS-51 from plasmid pDL277 complemented the growth defect and restored normal cell shape. These results suggested that IDG-60 is essential for maintaining the integrity of the cell wall and the uniformity of cell shape, both of which are indispensable for bacteria survival under stress conditions.
Molecular Characterization of Thermoinduced Immunogenic Proteins Q1p42 and Hsp15 of
Leptospira interrogans
Authors: Nally Jarlath E. , Artiushin Sergey , Timoney John F.
Published: July 2002

Leptospira interrogans
is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts. In vitro studies of organisms cultured at 30°C and shifted to 37°C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J. E. Nally, J. F. Timoney, and B. Stevenson, Infect. Immun. 69:400–404, 2001). In order to specifically identify antigenic proteins upregulated at 37°C, groups of three ponies were immunized with organisms shifted to 37°C for 5 to 7 days or with organisms maintained at 30°C. A lambda ZAP II genomic DNA library was screened with the pool of antisera to organisms shifted to 37°C. Clones reactive with this pool but unreactive with the pool of pony antisera to organisms cultured at 30°C were selected for further analysis. Sequence analysis of the first two clones identified open reading frames for proteins designated Qlp42 and Hsp15. Qlp42 is predicted to be an outer membrane lipoprotein. Its synthesis was upregulated when cultures were shifted from 30 to 37°C and downregulated when cultures were shifted from 37 to 30°C. Although the predicted molecular mass of Qlp42 is 39.8 kDa for the mature protein, Qlp42-specific equine antiserum was reactive with two bands of 30 and 29.5 kDa. Hsp15 is a stress response protein and a member of the Hsp20/α-crystallin family. PCR detected homologues of
qlp
42 and
hsp
15 in pathogenic serovars of
L. interrogans
but not in the nonpathogenic
Leptospira biflexa
. Enzyme-linked immunosorbent assays of antibody in convalescent sera from mares naturally infected with
L. interrogans
suggest that Qlp42 is expressed during leptospiral infection.
Anti-Class II Monoclonal Antibody-TargetedVibrio choleraeTcpA Pilin: Modulation of Serologic Response, Epitope Specificity, and Isotype
Authors: Wu Jia-Yan , Taylor Ronald K. , Wade William F.
Published: July 2002

Toxin-coregulated pilus (TCP) is a colonization factor required for cholera infection. It is not a strong immunogen when delivered in the context of whole cells, yet pilus subunits or TcpA derivative synthetic peptides induce protective responses. We examined the efficacy of immunizing mice with TCP conjugated to anti-class II monoclonal antibodies (MAb) with or without the addition of cholera toxin (CT) or anti-CD40 MAb to determine if the serologic response to TcpA could be manipulated. Anti-class II MAb-targeted TCP influenced the anti-TCP peptide serologic response with respect to titer and isotype. Responses to TcpA peptide 4 were induced with class II MAb-targeted TCP and not with nontargeted TCP. Class II MAb-targeting TcpA reduced the response to peptide 6 compared to the nontargeted TCP response. Class II MAb-targeted TcpA, if delivered with CT, enhanced the serologic response to TcpA peptides. The effectiveness of the combination of targeted TCP and CT was reduced if anti-CD40 MAb were included in the primary immunization. These data establish the need to understand the role of TCP presentation in the generation of B-cell epitopes in order to optimize TcpA-based cholera vaccines.
Coiling Phagocytosis ofBorrelia burgdorferiby Primary Human Macrophages Is Controlled by CDC42Hs and Rac1 and Involves Recruitment of Wiskott-Aldrich Syndrome Protein and Arp2/3 Complex
Authors: Linder Stefan , Heimerl Christiane , Fingerle Volker , Aepfelbacher Martin , Wilske Bettina
Published: July 2002

Lyme borreliosis is a multisystemic disorder primarily affecting the skin, nervous system, and joints. It is caused by the spirocheteBorrelia burgdorferisensu lato and is transmitted via ticks of theIxodidaefamily. Persistence of borreliae within macrophages has been implicated in the often chronic history of borreliosis. The uptake ofB. burgdorferiby professional phagocytes occurs predominantly by coiling phagocytosis, a host cell-driven process in which single pseudopods wrap around and engulf the spirochetes. In the present study, we investigated the molecular machinery and the signal transduction pathways controlling the formation of these unique uptake structures. We found that the phagocytosis of borreliae by primary human macrophages is accompanied by the formation of f-actin-rich structures, which in their morphological organization correspond well to the earlier described coiling pseudopods. Further experiments revealed that Wiskott-Aldrich Syndrome protein and Arp2/3 complex, major regulators of actin polymerization, are also recruited to these sites of actin accumulation. In addition, inhibition of an upstream regulator of Wiskott-Aldrich Syndrome protein, the Rho-family GTPase CDC42Hs, greatly inhibited the occurrence of borrelia-induced phagocytic uptake structures. Inhibition of Rac1, another Rho family GTPase, had a less-pronounced inhibitory effect, while blocking of Rho activity showed no discernible influence. These results suggest that basic mechanisms of actin polymerization that control other types of phagocytosis are also functional in the formation of the morphologically unique uptake structures in coiling phagocytosis. Our findings should enhance the understanding of the infection process ofB. burgdorferiand contribute to devising new strategies for countering Lyme disease.
Induction and Distribution of Intestinal Immune Responses after Administration of Recombinant Cholera Toxin B Subunit in the Ileal Pouches of Colectomized Patients
Authors: Kilhamn Jan , Brevinge Hans , Quiding-Järbrink Marianne , Svennerholm Ann-Mari , Jertborn Marianne
Published: July 2002

The induction and dissemination of mucosal immune responses to recombinant cholera toxin B subunit (rCTB) administered into the ileal pouches of patients, who had been colectomized because of ulcerative colitis, was analyzed. Biopsies from the duodenum and ileal pouch were collected, along with peripheral blood and ileostomy fluids. Two immunizations induced strong CTB-specific immunoglobulin A (IgA) antibody-secreting cell (ASC) responses in the duodenum in five of five patients, whereas weaker and less-frequent ASC responses were noted in the ileal pouch. Intestine-derived CTB-specific IgA ASCs were found in peripheral blood in three of the five patients. The vaccination also induced significant IgA antitoxin titer rises in ileostomy fluid in all of the patients. Increased production of gamma interferon in cell cultures from the ileal pouch was found in four of five patients after the vaccination. These results clearly indicate that rCTB administered into the distal ileum is capable of inducing B-cell responses in the “entire” small intestine and that homing of immunocompetent cells occurs preferentially to the duodenum.
Purification and Characterization of
Borrelia burgdorferi
from Feeding Nymphal Ticks (
Ixodes scapularis
)
Authors: Rathinavelu Sivaprakash , de Silva Aravinda M.
Published: July 2002

Here we describe a protocol for purifying
Borrelia burgdorferi
from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels of
B. burgdorferi
outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.
CD8+-T-Cell Response to Secreted and Nonsecreted Antigens Delivered by RecombinantListeria monocytogenesduring Secondary Infection
Authors: Tvinnereim Amy R. , Hamilton Sara E. , Harty John T.
Published: July 2002

Understanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens. We addressed the impact of antivector immunity, elicited by immunization with attenuatedactA-deficientListeria monocytogenes, on the CD8+-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinantL. monocytogenes. Challenges of immune mice withactA-deficient and with wild-type recombinantL. monocytogenesgenerated similar numbers of CD8+T cells specific for the NP118-126 epitope. High-dose immunization withactA-deficientL. monocytogenesresulted in substantial numbers of CD8+T cells specific for theL. monocytogenesLLO91-99 epitope in the effector and memory stages of the T-cell response. Challenge of these immune mice with recombinantL. monocytogenesresulted in rapid control of the infection and decreased CD8+-T-cell responses against both the secreted and nonsecreted form of the recombinant antigen compared to the response of naïve mice. In contrast, mice immunized with a low dose ofactA-deficientL. monocytogeneshad ∼10-fold fewer effector and memory T cells specific for LLO91-99 and a substantially higher CD8+-T-cell response against the recombinant antigen after challenge with recombinantL. monocytogenes. Although mice immunized with low-doseactA-deficientL. monocytogeneshad a substantial recall response to LLO91-99, which reached the same levels by 5 to 7 days postchallenge as that in high-dose-immunized mice, they exhibited decreased ability to controlL. monocytogenesreplication. Thus, the level of antivector immunity impacts the control of infection and efficiency of priming responses against new antigens introduced with the same vector.
A Complex Insertion Sequence Cluster at a Point of Interaction between the Linear Plasmid SCP1 and the Linear Chromosome of
Streptomyces coelicolor
A3(2)
Authors: Yamasaki Masayuki , Miyashita Kiyotaka , Cullum John , Kinashi Haruyasu
Published: July 2002

The giant linear plasmid SCP1 can integrate into the central region of the linear chromosome of
Streptomyces coelicolor
A3(2). Nucleotide sequence analysis around the target site for SCP1 integration in strain M145 identified a total of five copies of four insertion sequences (ISs) in a 6.5-kb DNA stretch. Three of the four (IS
468
, IS
469
, and IS
470
) are new IS elements, and the other is IS
466
. All of these elements contain one open reading frame which encodes a transposase-like protein. Two copies of IS
468
(IS
468
A and -B) are tandemly aligned at the left end of the cluster. Following these, IS
469
and IS
466
are located in a tail-to-tail orientation with 69.3% identity to each other. IS
470
is located at the right end of the cluster. The activities of IS
466
and IS
468
were demonstrated by transposition experiments and sequence comparison of several copies, respectively.
The Length of the Combined 3′ Untranslated Region and Poly(A) Tail Does Not Control Rates of Glyceraldehyde-3-Phosphate Dehydrogenase mRNA Translation in Three Species of Parasitic Protists
Authors: ter Kuile Benno H. , Sallés Fernando J.
Published: July 2002

Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3′ end has a role in regulating rates of translation in the parasitic protists
Trypanosoma brucei
,
Leishmania donovani
, and
Trichomonas vaginalis
. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3′ end under different growth conditions in all three species. Our results showed that the combined 3′ untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.
Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2
Authors: Rosche Thomas M. , Siddique Azeem , Larsen Michelle H. , Figurski David H.
Published: July 2002

Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci:
trfA
, the replication initiator gene, and
oriV
, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the
incC
gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of
incC
(IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for
incC
-mediated incompatibility is a KorB-binding site (O
B
). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the
incC
and
korB
genes individually has no obvious effect on
Escherichia coli
cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing
incC
,
korB
, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an
incC
-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2.
Pseudobactin Biogenesis in the Plant Growth-Promoting Rhizobacterium
Pseudomonas
Strain B10: Identification and Functional Analysis of the
l
-Ornithine

N
5

-Oxygenase (
psbA
) Gene
Authors: Ambrosi C. , Leoni L. , Putignani L. , Orsi N. , Visca P.
Published: July 2002

Pseudobactin
B10
, the fluorescent siderophore produced by the rhizobacterium
Pseudomonas
strain B10, contains the hydroxamate ligand
d
-

N
5

-hydroxyornithine (
d
-

N
5

-OH-Orn). We cloned the
l
-Orn

N
5

-oxygenase (
psbA
) gene from a genomic library of
Pseudomonas
strain B10 and demonstrated that PsbA is involved in the conversion of
l
-Orn to its

N
5

-OH derivative. PsbA shows significant similarity to microbial ω-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The
psbA
gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific
psbA
mutant of
Pseudomonas
strain B10 was biochemically complemented with the precursor
l
-

N
5

-OH-Orn, suggesting that
l
-Orn is hydroxylated before conversion to the
d
isomer. The
l
-Orn

N
5

-hydroxylase-defective mutants of
Pseudomonas
strain B10 and
Pseudomonas aeruginosa
PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen
Erwinia carotovora
in iron-poor medium. The extent of in vitro inhibition of
E. carotovora
was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms.
Exfoliatin-Producing Strains Define a Fourth
agr
Specificity Group in
Staphylococcus aureus
Authors: Jarraud S. , Lyon G. J. , Figueiredo A. M. S. , Gérard Lina , Vandenesch F. , Etienne J. , Muir T. W. , Novick R. P.
Published: July 2002

The staphylococcal virulon is activated by the density-sensing
agr
system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by
agrD
. A central segment of the
agr
locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of
Staphylococcus aureus
isolates into three different
agr
specificity groups and to the division of non-
aureus
staphylococci into a number of others. The AIPs cross-inhibit the
agr
responses between groups. We have previously shown that most menstrual toxic shock strains belong to
agr
specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth
agr
specificity group among
S. aureus
strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the
agr
response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the
agr
autoinduction threshold.
Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ς
S
-Dependent Transcription in
Pseudomonas putida
Authors: Ojangu Eve-Ly , Tover Andres , Teras Riho , Kivisaar Maia
Published: July 2002

The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ς
S
. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ς
S
(Eς
S
) recognizes a number of promoters which are also recognized by that containing ς
70
(Eς
70
). We have previously shown that transposon Tn
4652
can activate silent genes in starving
Pseudomonas putida
cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς
70
-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and
rpoS
-deficient mutant
P. putida
, the effect of ς
S
on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ς
S
-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ς
S
-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ς
S
-specific transcription in concert with the sequence downstream from the −10 box.
On the Functional Interchangeability, Oxidant versus Reductant, of Members of the Thioredoxin Superfamily
Authors: Debarbieux Laurent , Beckwith Jon
Published: July 2002

Escherichia coli
thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Åslund, and J. Beckwith, EMBO J. 17:5543–5550, 1998). We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L. Debarbieux and J. Beckwith, Proc. Natl. Acad. Sci. USA 95:10751–10756, 1998). Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it. While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself. Further, we confirm the findings of Jonda et al. (S. Jonda, M. Huber-Wunderlich, R. Glockshuber, and E. Mössner, EMBO J. 18:3271–3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex. Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant.
Stress Triggers a Process That Limits Activation of the
Bacillus subtilis
Stress Transcription Factor ς
B
Authors: Scott Janelle M. , Mitchell Theresa , Haldenwang W. G.
Published: July 2002

Stress-induced activation of the
Bacillus subtilis
transcription factor ς
B
is transitory. To determine whether the process that limits ς
B
activation is itself triggered by stress,
B. subtilis
strains in which the stress pathway was artificially activated by the induced expression of a positive regulatory protein (RsbT) were exposed to ethanol stress and were monitored for the persistence of ς
B
activity. Without ethanol treatment, the induced cultures displayed continuously high ς
B
activity. Ethanol treatment restricted ongoing ς
B
activity, but only in strains with intact
rsbX
and -
S
genes. The loss of other gene products (RsbR and Obg) known to participate in the stress activation pathway had little influence in blocking the ethanol effect. The data argue that stress upregulates the activity of the RsbX-S regulatory pair to restrict ς
B
induction following stress.
The
Bacillus subtilis
HBsu Protein Modifies the Effects of α/β-Type, Small Acid-Soluble Spore Proteins on DNA
Authors: Ross Margery A. , Setlow Peter
Published: July 2002

HBsu, the
Bacillus subtilis
homolog of the
Escherichia coli
HU proteins and the major chromosomal protein in vegetative cells of
B. subtilis
, is present at similar levels in vegetative cells and spores (∼5 × 10
4
monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the α/β-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with α/β-type SASP on the nucleoid, suggesting that HBsu could modulate α/β-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered α/β-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing α/β-type SASP-mediated positive supercoiling, and greatly ameliorated the α/β-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the α/β-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of α/β-type SASP on the properties of DNA in the developing and dormant spore.
Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin β-Ketoacyl-Acyl Carrier Protein Synthase
Authors: He Min , Varoglu Mustafa , Sherman David H.
Published: July 2002

A three-dimensional model of the
Streptomyces coelicolor
actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related
Escherichia coli
fatty acid synthase condensing enzyme β-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production.
3
H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.