bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA
Authors: Jasalavich Claudia A. , Ostrofsky Andrea , Jellison Jody
Published: July 2002

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.
Screening of Genes Involved in Isooctane Tolerance in
Saccharomyces cerevisiae
by Using mRNA Differential Display
Authors: Miura Shigenori , Zou Wen , Ueda Mitsuyoshi , Tanaka Atsuo
Published: July 2002

A
Saccharomyces cerevisiae
strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including
MUQ1
,
PRY2
,
HAC1
,
AGT1
,
GAC1
, and
ICT1
(
YLR099c
) was induced, while the expression of the remaining 16 genes, including
JEN1
,
PRY1
,
PRY3
, and
KRE1
, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by
HAC1
,
PRY1
, and
ICT1
have been reported to be associated with cell stress, and
AGT1
and
GAC1
have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively.
MUQ1
and
KRE1
, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.
Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of
Salmonella enterica
Serovar Enteritidis
Authors: Garaizar Javier , López-Molina Nuria , Laconcha Idoia , Lau Baggesen Dorte , Rementeria Aitor , Vivanco Ana , Audicana Ana , Perales Ildefonso
Published: July 2002

Strains of
Salmonella enterica
(
n
= 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (
D
= 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes
Xba
I,
Bln
I, and
Spe
I for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of
Salmonella
serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.
A Simple, Rapid Method for Demonstrating Bacterial Flagella
Authors: Grossart Hans-Peter , Steward Grieg F. , Martinez Josefina , Azam Farooq
Published: July 2002

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.
An Activator of Transcription Regulates Phage TP901-1 Late Gene Expression
Authors: Brøndsted Lone , Pedersen Margit , Hammer Karin
Published: July 2002

A promoter active in the late phase of the lytic cycle of lactococcal bacteriophage TP901-1 has been identified. The promoter is tightly regulated and requires the product of the phage TP901-1
orf29
for activity. A deletion analysis of the late promoter region showed that a fragment as small as 99 bp contains both the promoter and the region necessary for activation by ORF29. The transcriptional start site of the promoter was identified by primer extension to position 13073 on the TP901-1 genome, thus located 87 bp downstream of
orf29
in a 580-bp intergenic region between
orf29
and
orf30.
Furthermore, the region located −85 to −61 bp upstream of the start site was shown to be necessary for promoter activity. During infection, the transcript arising from the late promoter is fully induced at 40 min postinfection, and our results suggest that a certain level of ORF29 must be reached in order to activate transcription of the promoter. Several lactococcal bacteriophages encode ORF29 homologous proteins, indicating that late transcription may be controlled by a similar mechanism in these phages. With the identification of this novel regulator, our results suggest that within the P335 group of lactococcal phages at least two regulatory systems controlling transcription in the late stage of infection exist.
Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to
Chloroflexus
spp
Authors: Nübel Ulrich , Bateson Mary M. , Madigan Michael T. , Kühl Michael , Ward David M.
Published: July 2002

Filamentous bacteria containing bacteriochlorophylls
c
and
a
were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to
Chloroflexus
spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to
Chloroflexus
which exhibit different distributions along a gradient of salinity from 5.5 to 16%.
Immunological Diagnosis of Human Hydatid Cyst Relapse: Utility of the Enzyme-Linked Immunoelectrotransfer Blot and Discriminant Analysis
Authors: Gadea I. , Ayala G. , Diago M. T. , Cuñat A. , Garcia de Lomas J.
Published: July 2002

A discriminant technique was applied to the different serological patterns obtained by enzyme-linked immunoelectrotransfer blotting (EITB) and by conventional immunological tests, in order to differentiate the residual antibody patterns present in healed hydatidosis from the ones present in patients with active hydatidosis. For this purpose, specific antibodies against
Echinococcus granulosus
were detected by indirect hemagglutination, agglutination of latex particles, basophil degranulation, and EITB for 23 patients with active hydatidosis and 45 patients with surgically cured hydatidosis. Discriminant analysis of the different serological patterns obtained by EITB and conventional serology correctly classified 92.54% of patients (93.3% if the patients are differentiated according to the time elapsed since surgery). This method detected the presence of active hydatidosis in 95.6% of patients for whom abdominal ultrasonography had confirmed the presence of active hydatid cysts. The global specificity was 88.9%. The specificity was 97.1% for patients who had been operated on 3 years ago or more and 63.6% for patients with less time since surgery.
N-Formyl-Methionyl-Leucyl-Phenylalanine Inhibits both Gamma Interferon- and Interleukin-10-Induced Expression of FcγRI on Human Monocytes
Authors: Beigier-Bompadre Macarena , Barrionuevo Paula , Alves-Rosa Fernanda , Rubel Carolina J. , Palermo Marina S. , Isturiz Martı́n A.
Published: July 2002

Three different classes of receptors for the Fc portion of immunoglobulin G (FcγRs), FcγRI, FcγRII, and FcγRIII, have been identified on human leukocytes. One of them, FcγRI, is a high-affinity receptor capable of induction of functions that include phagocytosis, respiratory burst, antibody-dependent cell-mediated cytotoxicity (ADCC), and secretion of cytokines. This receptor is expressed on mononuclear phagocytes, and this expression is regulated by cytokines and hormones such as gamma interferon (IFN-γ), IFN-β, interleukin-10 (IL-10), and glucocorticoids. We have recently demonstrated that the chemotactic peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP) is capable of inducing a time-dependent downregulation of both FcγRIIIB and FcγRII in human neutrophils, altering FcγR-dependent functions. Considering the biological relevance of the regulation of FcγRI, we investigated the effect of FMLP on the overexpression of FcγRI induced by both IFN-γ and IL-10 on human monocytes. We demonstrate that FMLP significantly abrogated IFN-γ- and IL-10-induced FcγRI expression, although its basal level of expression was not altered. However, other IFN-γ-mediated effects such as the overexpression of the major histocompatibility complex class II antigens and the enhancement of lipopolysaccharide-induced secretion of tumor necrosis factor alpha were not affected by FMLP treatment. The formyl peptide completely inhibited the IFN-γ- and IL-10-induced enhancement of ADCC and phagocytosis carried out by adherent cells. The inhibitory effect of FMLP on FcγRI upregulation could exert an important regulatory effect during the evolution of bacterial infections.
Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with
Lactococcus lactis
Producing Bovine Beta-Lactoglobulin
Authors: Chatel Jean-Marc , Langella Philippe , Adel-Patient Karine , Commissaire Jacqueline , Wal Jean-Michel , Corthier Gérard
Published: July 2002

The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism
Lactococcus lactis,
which has GRAS (“generally regarded as safe”) status, as a delivery vehicle. The cDNA of the
blg
gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in
L. lactis
. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 μg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.
Sensitive Microplate Assay for Detection of Bactericidal Antibodies to
Vibrio cholerae
O139
Authors: Attridge Stephen R. , Johansson Camilla , Trach Dang D. , Qadri Firdausi , Svennerholm Ann-Mari
Published: July 2002

A microplate assay for the detection of bactericidal antibodies to
Vibrio cholerae
O139 is described. The assay is sensitive, highly reproducible, specific, and convenient to perform. It has been used to demonstrate the induction of serum bactericidal antibodies in Vietnamese recipients of an oral, inactivated, bivalent O1/O139 vaccine, as well as in Bangladeshi patients with O139 disease. In both study groups there was a significant inverse correlation between the preexposure level of antibodies in serum and the magnitude of the subsequent bactericidal response. Although infection generated stronger responses than vaccination, the proportion of responders was similar among individuals with low background titers.
Bordetella pertussisTonB, a Bvg-Independent Virulence Determinant
Authors: Pradel Elizabeth , Guiso Nicole , Menozzi Franco D. , Locht Camille
Published: July 2002

In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, theBordetella pertussis tonB exbB exbDlocus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream fromtonBwas detected in a Fur titration assay, indicating that thetonB exbB exbDoperon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the β-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream fromtonBand downstream fromexbD, respectively. AB. pertussis ΔtonB exbB::Kmrmutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB+/TonB−pair. The ΔtonB exbBmutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.
Epitope Mapping of the Immunodominant Invariable Region ofBorrelia burgdorferiVlsE in Three Host Species
Authors: Liang Fang Ting , Philipp Mario T.
Published: July 2002

VlsE, the variable surface antigen ofBorrelia burgdorferi, contains a 26-amino-acid-long immunodominant invariable region, IR6. In the present study, three overlapping 14-mer peptides reproducing the sequence of IR6were used as peptide-based enzyme-linked immunosorbent assay antigens to map this invariable region in infected monkeys, mice, and human Lyme disease patients. Antibodies of the two primate species appeared to recognize IR6as a single antigenic determinant, while mouse antibodies recognized multiple epitopes within this region.
Expression of
Staphylococcus aureus
Clumping Factor A in
Lactococcus lactis
subsp.
cremoris
Using a New Shuttle Vector
Authors: Que Yok-Ai , Haefliger Jacques-Antoine , Francioli Patrick , Moreillon Philippe
Published: July 2002

Staphylococcus aureus
harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in
Lactococcus lactis
subsp.
cremoris
1363. This bacterium is devoid of virulence factors and has a known genetic background. A new
Escherichia coli-L. lactis
shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the
oriCol
E1 origin, generating pOri253 that could replicate in
E. coli
. Second, the lactococcal promoters
P23
or
P59
were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying
P23
(named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the
P59
-containing plasmid. Transcription was absent in
E. coli
. The staphylococcal clumping factor A (
clfA
) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-
clfA
produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of
S. aureus
Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.
Use of RNA Arbitrarily Primed-PCR Fingerprinting To Identify
Vibrio cholerae
Genes Differentially Expressed in the Host following Infection
Authors: Chakrabortty Amit , Das Soumita , Majumdar Sabita , Mukhopadhyay Kanchan , Roychoudhury Susanta , Chaudhuri Keya
Published: July 2002

Evidence suggests that a repertoire of
Vibrio cholerae
genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo- and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The
V. cholerae murE
gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the
murE
gene of
V. cholerae
was transformed into
V. cholerae
569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When
V. cholerae
569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions.
Expression Analysis of the Yersiniabactin Receptor Gene
fyuA
and the Heme Receptor
hemR
of
Yersinia enterocolitica
In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase
Authors: Jacobi Christoph A. , Gregor Sebastian , Rakin Alexander , Heesemann Jürgen
Published: July 2002

The enteropathogenic
Yersinia enterocolitica
strains have several systems for scavenging iron from their environment. We have studied the expression of the
fyuA
gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the
hemR
gene, which encodes the receptor for heme, using the reporter genes
gfp
(encoding green fluorescent protein) and
luc
(encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of
fyuA
and
hemR
or expression of one gene by a
gfp
-
luc
tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that
fyuA
and
hemR
are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27°C. In the in vivo BALB/C mouse infection model, tissue-specific expression of
fyuA
and
hemR
reporter fusions was observed. Surprisingly,
fyuA
and
hemR
reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying
fyuA
or
hemR
reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of
fyuA
and
hemR
under peritoneal growth conditions. Differential expression of the
fyuA
and
hemR
reporter fusions could not be observed, suggesting similar regulation of
fyuA
and
hemR
in the mouse infection model.
Vacuolating Cytotoxin of
Helicobacter pylori
Plays a Role during Colonization in a Mouse Model of Infection
Authors: Salama Nina R. , Otto Glen , Tompkins Lucy , Falkow Stanley
Published: July 2002

Helicobacter pylori
, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells.
H. pylori
strains possessing different alleles of
vacA
differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. However, the specific role(s) played by VacA during the course of infection and disease is not clear. We have used a mouse model of
H. pylori
infection to begin to address this role. A null mutation of
vacA
compromises
H. pylori
in its ability to initially establish infection. If an infection by a
vacA
mutant is established, the bacterial load and degree of inflammation are similar to those associated with an isogenic wild-type strain. Thus, in this infection model,
vacA
plays a role in the initial colonization of the host, suggesting that strains of
H. pylori
expressing active alleles of
vacA
may be better adapted for host-to-host transmission.
Fate ofMycobacterium tuberculosiswithin Murine Dendritic Cells
Authors: Bodnar Kendra A. , Serbina Natalya V. , Flynn JoAnne L.
Published: July 2002

The interaction of microbes with dendritic cells (DCs) is likely to have an enormous impact on the initiation of the immune response against a pathogen. In this study, we compared the interaction ofMycobacterium tuberculosiswith murine bone marrow-derived DCs and macrophages (Mφ) in vitro.M. tuberculosisgrew equally well within nonactivated DCs and Mφ. Activation of DCs and Mφ with gamma interferon and lipopolysaccharide inhibited the growth of the intracellular bacteria in a nitric oxide synthase-dependent fashion. However, while this activation enabled Mφ to kill the intracellular bacteria, theM. tuberculosisbacilli within activated DCs were not killed. Thus, DCs could restrict the growth of the intracellular mycobacteria but were less efficient than Mφ at eliminating the infection. These results may have implications for priming immune responses toM. tuberculosis. In addition, they suggest that DCs may serve as a reservoir forM. tuberculosisin tissues, including the lymph nodes and lungs.
Differential Regulation of Bvg-Activated Virulence Factors Plays a Role in
Bordetella pertussis
Pathogenicity
Authors: Kinnear Susan M. , Marques Ryan R. , Carbonetti Nicholas H.
Published: July 2002

Bordetella pertussis
, the causative agent of whooping cough, regulates expression of many virulence factors via a two-component signal transduction system encoded by the
bvgAS
regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated.
fha
is transcribed within 10 min following a
bvg
AS-inducing signal, while
prn
is transcribed after 1 h and
ptx
is not transcribed until 2 to 4 h after induction. These genes therefore represent early, intermediate, and late classes of
bvg
-activated promoters, respectively. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in
B. pertussis
pathogenicity has not been characterized. We provide evidence that alterations to the promoter regions of
bvg
-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition,
B. pertussis
strains containing these promoter alterations that express either
ptx
at an early time or
fha
at a late time demonstrate a significant reduction in their ability to colonize respiratory tracts in an intranasal mouse model of infection. These data suggest a role for differential regulation of
bvg
-activated genes, and therefore for the BvgAS regulatory system, in the pathogenicity of
B. pertussis
.
LYT1 Protein Is Required for Efficient In Vitro Infection by
Trypanosoma cruzi
Authors: Manning-Cela Rebeca , Cortés Arantxa , González-Rey Elena , Van Voorhis Wesley C. , Swindle John , González Antonio
Published: July 2002

Trypanosoma cruzi
invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of the
LYT1
gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of
LYT1
resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of
LYT1
restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments with
LYT1
-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.
Candidate Vaccine against Botulinum Neurotoxin Serotype A Derived from a Venezuelan Equine Encephalitis Virus Vector System
Authors: Lee John S. , Pushko Peter , Parker Michael D. , Dertzbaugh Mark T. , Smith Leonard A. , Smith Jonathan F.
Published: July 2002

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and
cis
-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H
C
) of the BoNT/A heavy chain was cloned into the replicon vector (H
C
-replicon). Cotransfection of BHK cells in vitro with the H
C
-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H
C
VEE replicon particles (H
C
-VRP). Cells infected with H
C
-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H
C
-VRP, mice were vaccinated with various doses of H
C
-VRP at different intervals. Mice inoculated subcutaneously with H
C
-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.
Identification of Novel
Plasmodium gallinaceum
Zygote- and Ookinete-Expressed Proteins as Targets for Blocking Malaria Transmission
Authors: Langer Rebecca C. , Li Fengwu , Vinetz Joseph M.
Published: July 2002

The development of transmission-blocking vaccines is one approach to malaria control. To identify novel
Plasmodium
zygote- and ookinete-secreted proteins as targets of blocking malaria transmission, monoclonal antibodies (MAbs) were produced against parasite-secreted proteins found in
Plasmodium gallinaceum
ookinete culture supernatants. Four MAbs—1A6, 2A5, 2B5, and 4B6—were identified that bound to
P. gallinaceum
zygotes and ookinetes in diverse patterns in terms of spatial localization on parasites, time course of antigen expression, and Western immunoblot patterns. MAbs 2A5 and 4B6 recognized more than one protein band as detected by Western immunoblot of
P. gallinaceum
ookinete supernatants. Beginning at 0 h postfertilization, MAb 2A5 recognized a diverse set of antigens; at 10 h postfertilization, MAb 4B6 recognized several antigens as well. MAb 1A6 recognized a single ∼17-kDa protein, and 2B5 recognized a single ∼32-kDa protein at 15 h postfertilization. In membrane feeding assays to assess the effect of these MAbs on
P. gallinaceum
infectivity for
Aedes aegypti
mosquitoes, the addition of MAbs 1A6 and 2B5 to infectious blood meals significantly inhibited oocyst development in the mosquito midgut. In contrast, MAb 2A5 seemed to enhance infectivity. These results demonstrate that
Plasmodium
ookinetes secrete proteins (in addition to previously characterized chitinases) that may be targets for blocking malaria transmission. Future investigation of ookinete-secreted neutralization-sensitive molecules should provide valuable insight into mechanisms by which ookinetes exit the blood meal, penetrate and transverse the peritrophic matrix, and invade the mosquito midgut epithelium.
Characterization and Transcriptional Analysis of Gene Clusters for a Type IV Secretion Machinery in Human Granulocytic and Monocytic Ehrlichiosis Agents
Authors: Ohashi Norio , Zhi Ning , Lin Quan , Rikihisa Yasuko
Published: July 2002

Anaplasma
(
Ehrlichia
)
phagocytophila
and
Ehrlichia chaffeensis
, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight
virB
and
virD
genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight
virB
and
virD
genes were clustered at two separate loci in each genome. Five of the genes (
virB8
,
virB9
,
virB10
,
virB11
, and
virD4
) were located downstream from a
ribA
gene. These five genes in both
A. phagocytophila
and
E. chaffeensis
were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the
virB8
gene in human leukemia cell lines. The
virB9
gene of
A. phagocytophila
was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (
virB3
,
virB4
, and
virB6
) of both
A. phagocytophila
and
E. chaffeensis
were arranged downstream from a
sodB
gene and cotranscribed with the
sodB
gene through one or more
sodB
promoters in human leukocytes. This suggests that transcription of the three
virB
genes in these two
Anaplasma
and
Ehrlichia
spp. is regulated by factors that influence the
sodB
gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of
Anaplasma
and
Ehrlichia
spp. in granulocytes or monocytes.
A Regulatory RNA (PrrB RNA) Modulates Expression of Secondary Metabolite Genes in
Pseudomonas fluorescens
F113
Authors: Aarons Simon , Abbas Abdelhamid , Adams Claire , Fenton Anne , O'Gara Fergal
Published: July 2002

The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in
Pseudomonas
spp. Screening of a
Pseudomonas fluorescens
F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both
gacS
and
gacA
mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious
Pseudomonas
protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement
gacS
and
gacA
mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a ς
70
promoter sequence from positions −10 to −35. Inactivation of the
prrB
gene in F113 resulted in a significant reduction of 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when
prrB
was overexpressed. The
prrB
gene sequence contains a number of imperfect repeats of the consensus sequence 5′-AGGA-3′, and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in
Escherichia coli
and
Erwinia carotovora
, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacS-mediated regulation of Phl and HCN production in
P. fluorescens
F113.
Analysis of the Role of
recA
in Phenotypic Switching of
Pseudomonas tolaasii
Authors: Sinha Himanshu , Pain Arnab , Johnstone Keith
Published: July 2002

Switching between the pathogenic smooth (1116S) and nonpathogenic rough (1116R) forms of
Pseudomonas tolaasii
occurs due to the reversible duplication of a 661-bp element within the
pheN
locus. Disruption of the chromosomal
recA
locus of 1116S and 1116R produced strains 1116S
recA
and 1116R
recA
, respectively, which showed typical loss of UV resistance. Switching from the smooth to the rough form was virtually eliminated in the 1116S
recA
strain, whereas the extent of switching from the rough to the smooth form was almost identical in 1116R and 1116R
recA
. It is concluded that phenotypic switching from 1116S to 1116R is
recA
dependent whereas that from 1116R to 1116S is
recA
independent.
Functionality of Purified ς
N
(ς
54
) and a NifA-Like Protein from the Hyperthermophile
Aquifex aeolicus
Authors: Studholme David J. , Wigneshwereraraj Siva R. , Gallegos María-Trinidad , Buck Martin
Published: July 2002

The genome sequence of the extremely thermophilic bacterium
Aquifex aeolicus
encodes alternative sigma factor ς
N
(ς
54
, RpoN) and five potential ς
N
-dependent transcriptional activators. Although
A. aeolicus
possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative ς
N
-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in
Escherichia coli
), and purified
A. aeolicus
ς
N
and the NifA homologue, AQ_218. Purified
A. aeolicus
ς
N
bound to
E. coli
core RNA polymerase and bound specifically to a DNA fragment containing
E. coli
promoter
glnHp2
and to several
A. aeolicus
DNA fragments containing putative ς
N
-dependent promoters. When combined with
E. coli
core RNA polymerase,
A. aeolicus
ς
N
supported
A. aeolicus
NifA-dependent transcription from the
glnHp2
promoter. The
E. coli
activator PspFΔHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the
A. aeolicus glnBA
operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the ς
N
enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and ς
N
have coevolved.
Effect on Heterocyst Differentiation of Nitrogen Fixation in Vegetative Cells of the Cyanobacterium
Anabaena variabilis
ATCC 29413
Authors: Thiel Teresa , Pratte Brenda
Published: July 2002

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions.
Anabaena variabilis
ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a
nif1
mutant; however, that expression did not prevent differentiation of heterocysts and expression of the
nif1
nitrogenase in either the
nif1
mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.
phzO
, a Gene for Biosynthesis of 2-Hydroxylated Phenazine Compounds in
Pseudomonas aureofaciens
30-84
Authors: Delaney Shannon M. , Mavrodi Dmitri V. , Bonsall Robert F. , Thomashow Linda S.
Published: July 2002

Certain strains of root-colonizing fluorescent
Pseudomonas
spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent
Pseudomonas
spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of
Pseudomonas aureofaciens
30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA).
P. aureofaciens
30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.
Aspartic Peptide Hydrolases in
Salmonella enterica
Serovar Typhimurium
Authors: Larsen Rachel A. , Knox Tina M. , Miller Charles G.
Published: July 2002

Two well-characterized enzymes in
Salmonella enterica
serovar Typhimurium and
Escherichia coli
are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of
pepB pepE
mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from
E. coli
, the product of the
iadA
gene. The other is the product of the serovar Typhimurium homolog of
E. coli ybiK
, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated
iaaA
(isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.
Identification and Characterization of a Second Superoxide Dismutase Gene (
sodM
) from
Staphylococcus aureus
Authors: Valderas Michelle Wright , Hart Mark E.
Published: July 2002

A gene encoding superoxide dismutase (SOD),
sodM
, from
S. aureus
was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the
S. aureus
SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The
sodM
gene expressed from a plasmid rescued an
Escherichia coli
double mutant (
sodA sodB
) under conditions that are otherwise lethal. SOD activity gels of
S. aureus
RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a
sodM
mutant, while the two lower bands were absent in a
sodA
mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the
sodA
and
sodM sodA
mutants but not the
sodM
mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the
sodM sodA
mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in
S. aureus
throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an
S. aureus sodA
mutant are maintained.
Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of
Streptomyces venezuelae
Authors: Wilson Daniel J. , Xue Yongquan , Reynolds Kevin A. , Sherman David H.
Published: July 2002

The
Streptomyces venezuelae pikD
gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (
pikD
) from the chromosome of
S. venezuelae
, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying
pikD
restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the
pikD
mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from
pik
cluster loci were linked to the
xylE
reporter and placed in
S. venezuelae
wild-type and
pikD
mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of
pikRII
,
pikAI
, and
desI
but not those controlling
pikRI
or
pikC
.