bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Selenite- and Tellurite-Inducible Gene in
Escherichia coli
Authors: Guzzo Julie , Dubow Michael S.
Published: July 2002

Selenium is both an essential and a toxic trace element, and the range of concentrations between the two is extremely narrow. Although tellurium is not essential and is only rarely found in the environment, it is considered to be extremely toxic. Several hypotheses have been proposed to account for the toxic effects of selenite and tellurite. However, these potential mechanisms have yet to be fully substantiated. Through screening of an
Escherichia coli luxAB
transcriptional gene fusion library, we identified a clone whose luminescence increased in the presence of increasing concentrations of sodium selenite or sodium tellurite. Cloning and sequencing of the
luxAB
junction revealed that the fusion had occurred in a previously uncharacterized open reading frame, termed
o393
or
yhfC
, which we have now designated
gutS
, for gene up-regulated by tellurite and selenite. Transcription from
gutS
in the presence of selenite or tellurite was confirmed by RNA dot blot analysis. In vivo expression of the GutS polypeptide, using the pET expression system, revealed a polypeptide of approximately 43 kDa, in good agreement with its predicted molecular mass. Although the function of GutS remains to be elucidated, homology searches as well as protein motif and secondary-structure analyses have provided clues which may implicate GutS in transport in response to selenite and tellurite.
Natural Mediators in the Oxidation of Polycyclic Aromatic Hydrocarbons by Laccase Mediator Systems
Authors: Johannes Christian , Majcherczyk Andrzej
Published: July 2002

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from
Trametes versicolor
and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.
16S rRNA Gene-Based Detection of Tetrachloroethene-DechlorinatingDesulfuromonasandDehalococcoidesSpecies
Authors: Löffler Frank E. , Sun Qing , Li Jieran , Tiedje James M.
Published: July 2002

Members of the generaDesulfuromonasandDehalococcoidesreductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of theDehalococcoidesgroup (comprisingDehalococcoides ethenogenesandDehalococcoidessp. strain FL2) and the acetate-oxidizing, PCE-dechlorinatingDesulfuromonasgroup (comprisingDesulfuromonassp. strain BB1 andDesulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with theDesulfuromonasdechlorinator-targeted primer pair was 1 × 103BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and theDehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with theDehalococcoides-targeted primers, and another contaminated aquifer tested positive with theDesulfuromonasdechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other knownDesulfuromonasspecies. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizingDesulfuromonasand hydrogenotrophicDehalococcoidesspecies in samples yielding positive PCR signals with the specific primers.
Use of an Enzyme-Linked Lectinsorbent Assay To Monitor the Shift in Polysaccharide Composition in Bacterial Biofilms
Authors: Leriche V. , Sibille P. , Carpentier B.
Published: July 2002

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices:
d
-glucose or
d
-mannose for ConA and
N
-acetyl-
d
-glucosamine or
N
-acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for
Stenotrophomonas maltophilia
and to a lesser extent
Staphylococcus sciuri
, the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.
Method for Host-Independent Detection of Generalized Transducing Bacteriophages in Natural Habitats
Authors: Sander Michaela , Schmieger Horst
Published: July 2002

Despite an increasing interest in horizontal gene transfer in bacteria, the role of generalized transduction in this process has not been well investigated yet. Certainly one of the reasons is that only a small fraction of general transducing bacteriophages have been characterized, because many bacterial hosts needed for propagation and identification are not culturable or are simply unknown. A method for host-independent detection of transducing bacteriophages was developed. Phage-encapsulated DNA was used as a template for PCR amplification of 16S ribosomal DNA using primers specific for the 16S rRNA genes of most eubacteria. Sequencing of the cloned amplification products permits the identification of the host bacteria. The
Salmonella
phage P22 was used as an example. Applying this method to a sample of the supernatant of the mixed liquor in the aeration tank of an activated sludge treatment works revealed the presence of transducing phages infecting several bacterial species for which such phages have not yet been described. This method is suitable for estimating the contribution of generalized transduction to horizontal gene transfer in different habitats.
Low-Pressure UV Inactivation and DNA Repair Potential of
Cryptosporidium parvum
Oocysts
Authors: Shin Gwy-Am , Linden Karl G. , Arrowood Michael J. , Sobsey Mark D.
Published: July 2002

Because
Cryptosporidium parvum
oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified
C. parvum
oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV.
C. parvum
infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm
2
(=30 J/m
2
), the reduction reached the cell culture assay detection limit of ∼3 log
10
. At UV doses of 1.2 and 3 mJ/cm
2
, the log
10
reductions of
C. parvum
oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that
C. parvum
oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.
Serological Evaluation of Thin-Layer Immunoassay–Enzyme-Linked Immunosorbent Assay for Antibody Detection in Human Trichinellosis
Authors: Gómez-Priego Alberto , Crecencio-Rosales Lidia , de-la-Rosa Jorge-Luis
Published: July 2002

A new immunoenzymatic test, named the thin-layer immunoassay–enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from
Trichinella spiralis
muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (
n
= 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 μl each of antigen (7 μg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis.
Significant Role of Interleukin-8 in Pathogenesis of Pulmonary Disease Due to
Mycoplasma pneumoniae
Infection
Authors: Narita Mitsuo , Tanaka Hiroshi , Yamada Satoshi , Abe Shosaku , Ariga Tadashi , Sakiyama Yukio
Published: July 2002

We found elevated levels of interleukin-8 in pleural fluid samples from patients with pleural effusion and with a sustained fibrotic change of the lung due to
Mycoplasma pneumoniae
infection. This result suggests a critical role of interleukin-8 in the pathogenesis of a certain type of pulmonary disease caused by
M. pneumoniae
.
Opsonophagocytosis of Fluorescent Polystyrene Beads Coupled to
Neisseria meningitidis
Serogroup A, C, Y, or W135 Polysaccharide Correlates with Serum Bactericidal Activity
Authors: Martinez Joseph , Pilishvili Tamara , Barnard Suzanne , Caba Joseph , Spear Willie , Romero-Steiner Sandra , Carlone George M.
Published: July 2002

We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to
Neisseria meningitidis
serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean
r
= 0.96;
P
< 0.001). OPA could be used in meningococcal vaccine evaluation.
Importance of M-Protein C Terminus as Substrate Antigen for Serodetection of Equine Arteritis Virus Infection
Authors: Jeronimo Célia , Archambault Denis
Published: July 2002

Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombinant glutathione
S
-transferase-M fusion protein. Various deletion mutant clones, which covered the entire sequence of the M protein, were then generated by inverse PCR and expressed in
Escherichia coli
to examine, by a Western blot assay, the antigenic reactivity of the clone-derived truncated M proteins with sera from horses either experimentally or naturally infected with EAV. Deletion of the hydrophobic N-terminal 87 aa did not abolish immune reactivity of the protein with serum antibodies to EAV, thereby demonstrating the antigenicity of the C-terminal region (aa 88 to 162) of the M protein. Further truncations of the M-protein C-terminal domain defined particular linear epitope-containing amino acid sequence regions. However, only the M-protein C-terminal region was readily recognized by all EAV-specific horse antisera tested in this study. Based on these findings, only the M-protein C-terminal polypeptide composed of aa 88 to 162 is necessary to identify horse serum antibodies specific to the EAV M protein. Thus, this polypeptide might be useful for serodetection of EAV-infected animals.
Cytotoxic Cell Vacuolating Activity from
Vibrio cholerae
Hemolysin
Authors: Coelho Ana , Andrade João R. C. , Vicente Ana Carolina P. , Dirita Victor J.
Published: July 2002

A
Vibrio cholerae
cytotoxin, designated VcVac, was found to cause vacuolation in Vero cells. It was originally detected in the pathogenic O1 Amazonia variant of
V. cholerae
and later shown to be produced in environmental strains and some El Tor strains. Comparison of VcVac production in various strains suggested that hemolysin was responsible for the vacuolating phenotype. Genetic experiments established a firm correlation between vacuolation and hemolysin production. The mammalian cell vacuolating activity of the
V. cholerae
hemolysin is a new property of this protein and points to a previously unknown type of interaction between
V. cholerae
and its host.
The Cytotoxic Enterotoxin ofAeromonas hydrophilaInduces Proinflammatory Cytokine Production and Activates Arachidonic Acid Metabolism in Macrophages
Authors: Chopra A. K. , Xu X.-J. , Ribardo D. , Gonzalez M. , Kuhl K. , Peterson J. W. , Houston C. W.
Published: July 2002

An aerolysin-related cytotoxic enterotoxin (Act) ofAeromonas hydrophilapossesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice. The role of Act in the virulence of the organism has been demonstrated. In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E2[PGE2]). Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1β (IL-1β) and IL-6 in the murine macrophage cell line RAW264.7. Act also activated transcription of the gene encoding inducible nitric oxide synthase. Act evoked the production of PGE2coupled to the cyclooxygenase-2 (COX-2) pathway. AA is a substrate for PGE2, and Act produced AA from phospholipids by inducing group V secretory phospholipase A2. We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages. cAMP, along with PGE2, could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury. After Act treatment of RAW cells, we detected an increased translocation of NF-κB and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays. Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines. The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-κB and CREB. This is the first report of the detailed mechanisms of action of Act fromA. hydrophila.
Interdependency of Interleukin-10 and Interleukin-12 in Regulation of T-Cell Differentiation and Effector Function of Monocytes in Response to Stimulation withCryptococcus neoformans
Authors: Retini Cinzia , Kozel Thomas R. , Pietrella Donatella , Monari Claudia , Bistoni Francesco , Vecchiarelli Anna
Published: July 2002

We previously demonstrated that the principal component of capsular material ofCryptococcus neoformans, glucuronoxylomannan (GXM), induces interleukin-10 (IL-10) secretion from human monocytes. Here we report that encapsulation of the yeast with GXM is able to down-regulate interleukin-12 (IL-12) production by monocytes that would normally occur in the absence of encapsulation. This phenomenon appeared to be the result of inhibition of the phagocytic process by encapsulation with GXM as well as of negative signals such as IL-10 secretion produced by interaction of GXM with leukocytes. Decreased secretion of IL-12 correlated with decreased release of gamma interferon (IFN-γ) from T cells, suggesting a role for encapsulation with GXM in hindering a T helper type 1 (Th1) response. This is supported by the ability of encapsulation with GXM to limit increased expression of B7-1 costimulatory molecules that otherwise might limit IL-10 secretion. Endogenous IL-10 played a critical role in modulatory activity associated with encapsulation with GXM. Blocking IL-10 with monoclonal antibody to IL-10 resulted in increased (i) IL-12 secretion, (ii) IFN-γ release from T cells, and (iii) killing ofC. neoformansby monocytes. These results suggest that encapsulation with GXM limits development of a protective Th1-type response, an inhibitory process in which IL-10 plays a critical role. Scavengers of GXM and/or IL-10 could be useful in a protective Th1-type response in patients with cryptococcosis.
Critical Role for Signal Transducer and Activator of Transcription Factor 6 in Mediating Intestinal Muscle Hypercontractility and Worm Expulsion inTrichinella spiralis-Infected Mice
Authors: Khan W. I. , Vallance B. A. , Blennerhassett P. A. , Deng Y. , Verdu E. F. , Matthaei K. I. , Collins S. M.
Published: July 2002

Intestinal nematode infections in rats or mice are accompanied by intestinal muscle hyper contractility that may contribute to parasite expulsion from the gut. Previous studies demonstrated that both the expulsion of nematode parasites and the associated muscle hyper contractility are dependent on CD4+T helper cells. Nevertheless, the precise immunological mechanism underlying changes in intestinal muscle function remains to be determined. In this study, we investigated the role of interleukin 4 (IL-4) and signal transducer and activator of transcription factor 6 (STAT6) in the development of intestinal muscle hypercontractility and worm expulsion by infecting IL-4 and STAT6-deficient mice withTrichinella spiralis. Worm expulsion was almost normal in IL-4-deficient mice but substantially delayed in STAT6-deficient mice. Consistent with delayed worm expulsion, we also observed a marked attenuation of carbachol-induced muscle contraction in STAT6-deficient mice but only a moderate decrease in muscle hypercontractility in IL-4-deficient mice. In addition, we also observed severe impairment of T helper type 2 cytokine responses and intestinal mucosal mastocytosis in STAT6-deficient mice, although some degree of intestinal tissue eosinophilia was evident in these animals. These results are consistent with the hypothesis that STAT6-dependent changes in intestinal muscle function contribute to host protection in nematode infection.
Induction of Lysogenic Bacteriophage and Phage-Associated Toxin from Group A Streptococci during Coculture with Human Pharyngeal Cells
Authors: Broudy Thomas B. , Pancholi Vijaykumar , Fischetti Vincent A.
Published: July 2002

We found that when group A streptococci are cocultured with human pharyngeal cells, they upregulate and secrete a 25-kDa toxin, determined to be the bacteriophage-encoded streptococcal pyrogenic exotoxin C (SpeC). This prompted us to determine if the bacteriophage themselves are induced during coculture conditions. We found that bacteriophage induction does occur, resulting in the release of ∼10
5
phage particles during the 3-h coculture. Furthermore, we show that the bacteriophage induction event is mediated by a pharyngeal cell soluble factor for which we provide an initial characterization.
Gamma Interferon Prevents the Inhibitory Effects of Oxidative Stress on Host Responses toEscherichia coliInfection
Authors: Parmely Michael J. , Wang Fuan , Wright Douglas
Published: July 2002

Oxidative stress occurs in animals challenged with bacterial endotoxin and can affect the expression of important host inflammatory genes. However, much less is known about the effects of oxidative stress on responses to gram-negative bacteria. The current study compared the effects of redox imbalance on hepatic responses of mice toEscherichia colibacteria versus purified endotoxic lipopolysaccharide (LPS). Oxidative stress induced by glutathione depletion virtually eliminated hepatic tumor necrosis factor alpha responses to bothE. coliand LPS. Inducible NO synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) expression was also markedly inhibited by glutathione depletion in LPS-challenged mice, but was unaffected inE. coli-infected animals. Three findings suggested that gamma interferon (IFN-γ) production explained the differences between LPS and bacterial challenge. Glutathione depletion completely inhibited the IFN-γ response to LPS, but only partially inhibited IFN-γ production in infected mice. Exogenous IFN-γ restored iNOS and ICAM-1 responses to LPS in stressed mice. Conversely, IFN-γ-deficient, glutathione-depleted mice showed a marked decrease in iNOS and ICAM-1 expression when challenged withE. coli. These findings indicate that both the nature of the microbial challenge and the production of IFN-γ can be important in determining the effects of redox imbalance during gram-negative bacterial infections.
Interaction between Host Complement and Mosquito-Midgut-StagePlasmodium berghei
Authors: Margos Gabriele , Navarette Sandra , Butcher Geoff , Davies Alex , Willers Christine , Sinden Robert E. , Lachmann Peter J.
Published: July 2002

After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement onPlasmodiumdevelopmental stages in the mosquito blood meal using the rodent malaria parasitePlasmodium bergheiand rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion ofP. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages ofP. bergheisurvived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.
Exploitation of Interleukin-8-Induced Neutrophil Chemotaxis by the Agent of Human Granulocytic Ehrlichiosis
Authors: Akkoyunlu Mustafa , Malawista Stephen E. , Anguita Juan , Fikrig Erol
Published: July 2002

The agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium with a tropism for neutrophils; however, the mechanisms of bacterial dissemination are not yet understood. Interleukin-8 (IL-8) is a chemokine that induces neutrophil migration to sites of infection for host defense against pathogens. We now show that HGE bacteria, and the HGE-44 protein, induce IL-8 secretion in a promyelocytic (HL-60) cell line that has been differentiated along the neutrophil lineage with retinoic acid and in neutrophils. Infected HL-60 cells also demonstrate upregulation of CXCR2, an IL-8 receptor, but not CXCR1. Human neutrophils migrate towardsEhrlichiasp.-infected cells in a chemotaxis chamber assay, and this movement can be blocked with antibodies to IL-8. Finally, immunocompetent and severe combined immunodeficient mice administered CXCR2 antisera, and CXCR2−/−mice that lack the human IL-8 receptor homologue, are much less susceptible to granulocytic ehrlichiosis than are control animals. These results demonstrate that HGE bacteria induce IL-8 production by host cells and, paradoxically, appear to exploit this chemokine to enhance infection.
The Efficiency of the Translocation ofMycobacterium tuberculosisacross a Bilayer of Epithelial and Endothelial Cells as a Model of the Alveolar Wall Is a Consequence of Transport within Mononuclear Phagocytes and Invasion of Alveolar Epithelial Cells
Authors: Bermudez Luiz E. , Sangari Felix J. , Kolonoski Peter , Petrofsky Mary , Goodman Joseph
Published: July 2002

The mechanism(s) by whichMycobacterium tuberculosiscrosses the alveolar wall to establish infection in the lung is not well known. In an attempt to better understand the mechanism of translocation and create a model to study the different stages of bacterial crossing through the alveolar wall, we established a two-layer transwell system.M. tuberculosisH37Rv was evaluated regarding the ability to cross and disrupt the membrane.M. tuberculosisinvaded A549 type II alveolar cells with an efficiency of 2 to 3% of the initial inoculum, although it was not efficient in invading endothelial cells. However, bacteria that invaded A549 cells were subsequently able to be taken up by endothelial cells with an efficiency of 5 to 6% of the inoculum. When incubated with a bicellular transwell monolayer (epithelial and endothelial cells),M. tuberculosistranslocated into the lower chamber with efficiency (3 to 4%).M. tuberculosiswas also able to efficiently translocate across the bicellular layer when inside monocytes. Infected monocytes crossed the barrier with greater efficiency when A549 alveolar cells were infected withM. tuberculosisthan when A549 cells were not infected. We identified two potential mechanisms by whichM. tuberculosisgains access to deeper tissues, by translocating across epithelial cells and by traveling into the blood vessels within monocytes.
Functional Variation of the Antigen I/II Surface Protein in
Streptococcus mutans
and
Streptococcus intermedius
Authors: Petersen F. C. , Assev S. , van der Mei H. C. , Busscher H. J. , Scheie A. A.
Published: July 2002

Although
Streptococcus intermedius
and
Streptococcus mutans
are regarded as members of the commensal microflora of the body,
S. intermedius
is often associated with deep-seated purulent infections, whereas
S. mutans
is frequently associated with dental caries. In this study, we investigated the roles of the
S. mutans
and
S. intermedius
antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in
S. mutans
, but not in
S. intermedius
, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the
S. mutans
and
S. intermedius
antigen I/II. Adhesion of
S. mutans
or
S. intermedius
to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the
S. intermedius
mutant. The
S. mutans
isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of
S. mutans
and
S. intermedius
to host components and in determining cell surface properties.
Association of a Determinant on Mouse Chromosome 18 with Experimental SeverePlasmodium bergheiMalaria
Authors: Nagayasu Eiji , Nagakura Koichi , Akaki Mayumi , Tamiya Gen , Makino Satoshi , Nakano Yamaji , Kimura Minoru , Aikawa Masamichi
Published: July 2002

Experimental severe malaria (ESM; also known as experimental cerebral malaria) is an acute lethal syndrome caused by infection withPlasmodium bergheiANKA and associated with coma and other neurological manifestations in mice. Various inbred strains of mice exhibit differences in susceptibility to the development of ESM. For example, C57BL/6 mice are highly susceptible and DBA/2 mice are relatively resistant. We report here the results of a genomewide scan for host genomic regions that control resistance to ESM in DBA/2 mice using an F2intercross population of susceptible and resistant strains. A region of mid-chromosome 18 was found to be a major determinant of resistance to ESM.
Pressure Regulation of Soluble Cytochromes
c
in a Deep-Sea Piezophilic Bacterium,
Shewanella violacea
Authors: Yamada Mitsunori , Nakasone Kaoru , Tamegai Hideyuki , Kato Chiaki , Usami Ron , Horikoshi Koki
Published: July 2002

Two
c
-type cytochromes from the soluble fraction of a deep-sea moderately piezophilic bacterium,
Shewanella violacea
, were purified and characterized, and the genes coding for these cytochromes were cloned and sequenced. One of the cytochromes, designated cytochrome
c
A
, was found to have a molecular mass of approximately 8.3 kDa, and it contained one heme
c
per molecule. The other, designated cytochrome
c
B
, was found to have a molecular mass of approximately 23 kDa, and it contained two heme
c
molecules per protein molecule. The amount of cytochrome
c
B
expressed in cells grown at high hydrostatic pressure (50 MPa) was less than that in cells grown at atmospheric pressure, whereas cytochrome
c
A
was constitutively expressed under all pressure conditions examined. The results of Northern blotting analysis were consistent with the above-mentioned observations and suggested that the pressure regulation of cytochrome
c
B
gene expression occurred at the transcriptional level. These results suggest that the components of the respiratory chain of moderately piezophilic
S. violacea
could be exchanged according to the growth pressure conditions.
Characterization of a
Bordetella pertussis
Diaminopimelate (DAP) Biosynthesis Locus Identifies
dapC
, a Novel Gene Coding for an
N
-Succinyl-
l,l
-DAP Aminotransferase
Authors: Fuchs Thilo M. , Schneider Boris , Krumbach Karin , Eggeling Lothar , Gross Roy
Published: July 2002

The functional complementation of two
Escherichia coli
strains defective in the succinylase pathway of
meso
-diaminopimelate (
meso
-DAP) biosynthesis with a
Bordetella pertussis
gene library resulted in the isolation of a putative
dap
operon containing three open reading frames (ORFs). In line with the successful complementation of the
E. coli dapD
and
dapE
mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of
E. coli
and many other bacteria which exhibit tetrahydrodipicolinate succinylase and
N
-succinyl-
l,l
-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5′-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with
N
-succinyl-
l,l
-DAP aminotransferase activity converting
N
-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to
N
-succinyl-
l,l
-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of
B. pertussis
. Apart from the pyridoxal-5′-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with
N
-succinyl-
l,l
-DAP aminotransferase activity, ArgD of
E. coli
.
Physical Morphology and Surface Properties of Unsaturated
Pseudomonas putida
Biofilms
Authors: Auerbach Ilene D. , Sorensen Cody , Hansma Helen G. , Holden Patricia A.
Published: July 2002

Unsaturated biofilms of
Pseudomonas putida
, i.e., biofilms grown in humid air, were analyzed by atomic force microscopy to determine surface morphology, roughness, and adhesion forces in the outer and basal cell layers of fresh and desiccated biofilms. Desiccated biofilms were equilibrated with a 75.5% relative humidity atmosphere, which is far below the relative humidity of 98 to 99% at which these biofilms were cultured. In sharp contrast to the effects of drying on biofilms grown in fluid, we observed that drying caused little change in morphology, roughness, or adhesion forces in these unsaturated biofilms. Surface roughness for moist and dry biofilms increased approximately linearly with increasing scan sizes. This indicated that the divides between bacteria contributed more to overall roughness than did extracellular polymeric substances (EPS) on individual bacteria. The EPS formed higher-order structures we termed mesostructures. These mesostructures are much larger than the discrete polymers of glycolipids and proteins that have been previously characterized on the outer surface of these gram-negative bacteria.
Mycobacterium smegmatis
Has Two Pyrazinamidase Enzymes, PncA and PzaA
Authors: Guo Ming , Sun Zhonghe , Zhang Ying
Published: July 2002

The
Mycobacterium smegmatis pncA
gene, encoding nicotinamidase/pyrazinamidase, was identified. While it was similar to counterparts from other mycobacteria, the
M. smegmatis
PncA had little homology to the other
M. smegmatis
pyrazinamidase/nicotinamidase, encoded by the
pzaA
gene. Transformation of
Mycobacterium bovis
strain BCG with
M. smegmatis pncA
or
pzaA
conferred susceptibility to pyrazinamide.
Mapping of the
Cryptococcus neoformans MAT
α Locus: Presence of Mating Type-Specific Mitogen-Activated Protein Kinase Cascade Homologs
Authors: Karos M. , Chang Y. C. , McClelland C. M. , Clarke D. L. , Fu J. , Wickes B. L. , Kwon-Chung K. J.
Published: July 2002

In this study we investigated the relationship between the
MAT
α locus of
Cryptococcus neoformans
and several
MAT
α-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including
STE12
α,
STE11
α, and
STE20
α. To resolve the location of the genes, we screened a cosmid library of the
MAT
α strain B-4500 (JEC21), which was chosen for the
C. neoformans
genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire
MAT
α locus. It was found that
STE12
α,
STE11
α, and
STE20
α are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of
MF
α, the mating type α-pheromone gene, a
MAT
α-specific myosin gene, and a pheromone receptor (
CPR
α) were identified within the locus. We created a physical map, based on the restriction enzyme
Bam
HI, and identified both borders of the
MAT
α locus. The
MAT
α locus of
C. neoformans
is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.
Measuring Virulence Factor Expression by the Pathogenic Bacterium
Photorhabdus luminescens
in Culture and during Insect Infection
Authors: Daborn Phillip J. , Waterfield Nicholas , Blight Mark A. , Ffrench-Constant Richard H.
Published: July 2002

During insect infection
Photorhabdus luminescens
emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at ≈48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of
Photorhabdus
as a model system for bacterial infection.
Genetic Analysis of the
Sinorhizobium meliloti
BacA Protein: Differential Effects of Mutations on Phenotypes
Authors: LeVier Kristin , Walker Graham C.
Published: July 2002

Sinorhizobium meliloti
strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a
bacA-
null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in
bacA
were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.
H2O2-Forming NADH Oxidase with Diaphorase (Cytochrome) Activity fromArchaeoglobus fulgidus
Authors: Reed David W. , Millstein Jack , Hartzell Patricia L.
Published: July 2002

An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobeArchaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in theA. fulgidusgenome. The gene AF0395 was cloned and its product was purified fromEscherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochromec. During purification NoxA2 remains associated with the enzyme responsible ford-lactate oxidation, thed-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.
Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins
Authors: Thomas Nikhil A. , Jarrell Ken F.
Published: July 2002

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each ofMethanococcus voltae,Methanococcus maripaludis,Methanococcus thermolithotrophicus, andMethanococcus jannaschiihas been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes,flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes ofM. voltaewere successfully overexpressed as His-tagged proteins inEscherichia coli.These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionatedM. voltaecells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly,flaDwas found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes ofM. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.
Role for Vitamin B
12
in Light Induction of Gene Expression in the Bacterium
Myxococcus xanthus
Authors: Cervantes María , Murillo Francisco J.
Published: July 2002

A light-inducible promoter (P
B
) drives the
carB
operon (carotenoid genes) of the bacterium
Myxococcus xanthus
. A gene encoding a regulator of carotenoid biosynthesis was identified by studying mutant strains carrying a transcriptional fusion to P
B
and deletions in three candidate genes. Our results prove that the identified gene, named
carA
, codes for a repressor of the P
B
promoter in the dark. They also show that the
carA
gene product does not participate in the light activation of two other promoters connected with carotenoid synthesis or its regulation in
M. xanthus
. CarA is a novel protein consisting of a DNA-binding domain of the family of MerR helix-turn-helix transcriptional regulators, directly joined to a cobalamin-binding domain. In support of this, we report here that the presence of vitamin B
12
or some other cobalamin derivatives is absolutely required for activation of the P
B
promoter by light.
Overexpression of the
Escherichia coli sugE
Gene Confers Resistance to a Narrow Range of Quaternary Ammonium Compounds
Authors: Chung Yong Joon , Saier Milton H.
Published: July 2002

SugE of
Escherichia coli
, first identified as a suppressor of
groEL
mutations but a member of the small multidrug resistance family, has not previously been shown to confer a drug resistance phenotype. We show that high-level expression of
sugE
leads to resistance to a subset of toxic quaternary ammonium compounds.
Phylogenetic Analysis of
Lacazia loboi
Places This Previously Uncharacterized Pathogen within the Dimorphic Onygenales
Authors: Herr Roger A. , Tarcha Eric J. , Taborda Paulo R. , Taylor John W. , Ajello Libero , Mendoza Leonel
Published: July 2002

Lacazia loboi
is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics. To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens
Pneumocystis carinii
and
Rhinosporidium seeberi
, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene. Our phylogenetic analysis indicated that
L. loboi
is the sister taxon of the human dimorphic fungal pathogen
Paracoccidioides brasiliensis
and that both species belong with the other dimorphic fungal pathogens in the order Onygenales. The low nucleotide variation among three
P. brasiliensis
18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of
L. loboi
used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding.
Molecular Characterization of
Cryptosporidium
Isolates Obtained from Humans in France
Authors: Guyot K. , Follet-Dumoulin A. , Lelièvre E. , Sarfati C. , Rabodonirina M. , Nevez G. , Cailliez J. C. , Camus D. , Dei-Cas E.
Published: July 2002

Cryptosporidium parvum
is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of
Cryptosporidium
species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom
Cryptosporidium
oocysts were recognized by clinical laboratories. A fragment of the
Cryptosporidium
18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of
Cryptosporidium parvum
. However, a number of immunocompromised patients were infected with
C. meleagridis
(3 of 57),
C. felis
(6 of 57), or a new genotype of
C. muris
(1 of 57). This is the first report of the last three species of
Cryptosporidium
in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of
Cryptosporidium
species and genotypes.
Rapid Detection of Herpes Simplex Virus DNA in Genital Ulcers by Real-Time PCR Using SYBR Green I Dye as the Detection Signal
Authors: Aldea Carmen , Alvarez Carmen P. , Folgueira Lola , Delgado Rafael , Otero Joaquín R.
Published: July 2002

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.
Household Transmission of
Streptococcus pneumoniae
among Siblings with Acute Otitis Media
Authors: Shimada Jun , Yamanaka Noboru , Hotomi Muneki , Suzumoto Masaki , Sakai Akihiro , Ubukata Kimiko , Mitsuda Toshihiro , Yokota Shumpei , Faden Howard
Published: July 2002

Nasopharyngeal transmission of
Streptococcus pneumoniae
was evaluated among 23 siblings with acute otitis media (AOM). Restriction fragment length polymorphism revealed that the nasopharyngeal strains were identical between siblings in 12 of 13 clusters of AOM experienced in 11 families. This study demonstrated person-to-person transmission of
S. pneumoniae
, especially drug-resistant strains, among siblings with AOM.