bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinated, Differential Expression of Two Genes through Directed mRNA Cleavage and Stabilization by Secondary Structures
Authors: Smolke Christina D. , Carrier Trent A. , Keasling J. D.
Published: July 2002

Metabolic engineering and multisubunit protein production necessitate the expression of multiple genes at coordinated levels. In bacteria, genes for multisubunit proteins or metabolic pathways are often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to place multiple genes under the control of a single promoter and produce proteins encoded in that novel operon in different ratios over a range of inducer concentrations. RNase E sites identified in the
Rhodobacter capsulatus puf
operon and
Escherichia coli pap
operon were separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5′ and 3′ ends of the coding regions. The introduced RNase E site directed cleavage between the coding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease cleavage by engineered 3′ secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5′ end. The relative expression levels of two reporter genes could be varied up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the ribosome binding site, this technology should allow one to create new operons and coordinate, yet separately control, the expression levels of genes expressed in that operon.
The
Bradyrhizobium japonicum
Proline Biosynthesis Gene
proC
Is Essential for Symbiosis
Authors: King Natalie D. , Hojnacki David , O'Brian Mark R.
Published: July 2002

Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The
Bradyrhizobium japonicum proC
gene was isolated, and a
proC
mutant strain that behaved as a strict proline auxotroph in culture was constructed. The
proC
strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the
proC
gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.
A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk
Authors: Gunasekera Thusitha S. , Attfield Paul V. , Veal Duncan A.
Published: July 2002

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (≤1-h) FCM assay based on enzymatic clearing of milk to determine total bacteria in milk. When bacteria were added to ultra-heat-treated milk, a good correlation (
r
≥ 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (
P
< 0.01) and a good agreement (
r
= 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was ≤10
4
bacteria ml of milk
−1
. This limit is below the level of detection required to satisfy legislation in many countries and states.
Efficient Heterologous Expression in
Aspergillus oryzae
of a Unique Dye-Decolorizing Peroxidase, DyP, of
Geotrichum candidum
Dec 1
Authors: Sugano Yasushi , Nakano Ryosuke , Sasaki Katsuya , Shoda Makoto
Published: July 2002

Efficient expression of the dye-decolorizing peroxidase, DyP, from
Geotrichum candidum
Dec 1 in
Aspergillus oryzae
M-2-3 was achieved by fusing mature cDNA encoding
dyp
with the
A. oryzae
α-amylase promoter (
amyB
). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in
A. oryzae
. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by
A. oryzae
exhibits a function similar to that of native DyP.
Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity
Authors: Mason Maria G. , Ball Andrew S. , Reeder Brandon J. , Silkstone Gary , Nicholls Peter , Wilson Michael T.
Published: July 2002

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete
Thermomonospora fusca
BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete
Streptomyces viridosporus
T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.
Identification of Bacteria in Pasteurized Zucchini Purées Stored at Different Temperatures and Comparison with Those Found in Other Pasteurized Vegetable Purées
Authors: Guinebretiere Marie-Hélène , Berge Odile , Normand Philippe , Morris Cindy , Carlin Frédéric , Nguyen-The Christophe
Published: July 2002

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25°C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial
rrs
gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the
Bacillus circulans
(group 2) API taxon or in taxa with a low number of positive API characters such as
Brevibacillus brevis
. A phylogenetic analysis pointed to one new species of
Bacillus
and three new species of
Paenibacillus
among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as
B. circulans
(group 2) or
B. circulans/B. macerans/B.
polymyxa
were assigned to a new
Paenibacillus
taxon phylogenetically related to
P. azotofixans
. Storage conditions at 4°C favored the development of “
B. macroides/B. maroccanus
” and
Paenibacillus
spp. in zucchini purées and
Paenibacillus
spp. in other purées. Storage conditions at 20 to 25°C favored the development of
B. subtilis
group (
B. licheniformis
and
B. subtilis
) and
B. cereus
group strains. At 10°C,
Paenibacillus
spp. were always present at high frequencies, whereas the occurrence of
B. macroides/B. maroccanus
(in zucchini purées),
B. cereus
, and
B. pumilus
varied with the experiment.
Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria
Authors: Allen Andrew E. , Booth Melissa G. , Frischer Marc E. , Verity Peter G. , Zehr Jonathan P. , Zani Sabino
Published: July 2002

A PCR approach was used to construct a database of
nasA
genes (called
narB
genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A
nasA
-specific PCR primer set that could be used to selectively amplify the
nasA
gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced
nasA
genes. Our results indicate that several groups of heterotrophic bacterial
nasA
genes are common and widely distributed in oceanic environments.
Effects of the 20-Kilodalton Helper Protein on Cry1Ac Production and Spore Formation in
Bacillus thuringiensis
Authors: Shao Zongze , Liu Ziduo , Yu Ziniu
Published: July 2002

Bacillus thuringiensis
produces large amounts of various pesticidal proteins during the stationary phase. In order to achieve a high yield and form crystals, some pesticidal proteins require the presence of other proteins. Helper protein P20 is required for efficient production of both the Cyt1A and Cry11A crystal proteins in
B. thuringiensis
subsp.
israelensis
. Although full-length Cry1 protoxins are usually independent in terms of expression and crystallization in
B. thuringiensis
, in this study P20 significantly enhanced production of Cry1Ac protoxin (133 kDa) in an acrystalliferous and plasmid-negative strain. In the presence of P20, the yield of Cry1Ac protoxin increased 2.5-fold, and on average the resulting crystals were 1.85 μm long and 0.85 μm wide, three times the size of the crystals formed in the control lacking P20. Correspondingly, the recombinant strain that coexpressed P20 and Cry1Ac exhibited higher toxicity against
Heliothis armigera
larvae than the control. Furthermore, serious degradation of Cry1Ac in vivo was observed, which has seldom been reported previously. Actually, most protein was completely degraded during synthesis, and after synthesis about one-third of the expressed protoxins were degraded further before crystallization. In this process, P20 protected only nascent Cry1Ac from degradation, indicating that it acted as a molecular chaperon. In addition, spores were smaller and rounder and had a thinner exosporium layer when they were produced in the presence of P20. In summary, Cry1Ac was severely degraded during synthesis; this degradation was effectively relieved by P20, which resulted in enhanced production. Our results indicated that P20 is an effective tool for optimizing protein production in vivo.
Efficient Homolactic Fermentation by
Kluyveromyces lactis
Strains Defective in Pyruvate Utilization and Transformed with the Heterologous
LDH
Gene
Authors: Bianchi Michele M. , Brambilla Luca , Protani Francesca , Liu Chi-Li , Lievense Jefferson , Porro Danilo
Published: July 2002

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a
Kluyveromyces lactis
yeast strain lacking the single pyruvate decarboxylase gene (
KlPDC1
) and transformed with the heterologous lactate dehydrogenase gene (
LDH
). The
LDH
gene was placed under the control the
KlPDC1
promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g
−1
, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used
K. lactis LDH
transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g
−1
(maximum theoretical yield, 1 g g
−1
), and with high LDH activity.
Construction and Analysis of Photolyase Mutants of
Pseudomonas aeruginosa
and
Pseudomonas syringae
: Contribution of Photoreactivation, Nucleotide Excision Repair, and Mutagenic DNA Repair to Cell Survival and Mutability following Exposure to UV-B Radiation
Authors: Kim Jae J. , Sundin George W.
Published: July 2002

Based on nucleotide sequence homology with the
Escherichia coli
photolyase gene (
phr
), the
phr
sequence of
Pseudomonas aeruginosa
PAO1 was identified from the genome sequence, amplified by PCR, cloned, and shown to complement a known
phr
mutation following expression in
Escherichia coli
SY2. Stable, insertional
phr
mutants containing a tetracycline resistance gene cassette were constructed in
P. aeruginosa
PAO1 and
P. syringae
pv. syringae FF5 by homologous recombination and sucrose-mediated counterselection. These mutants showed a decrease in survival compared to the wild type of as much as 19-fold after irradiation at UV-B doses of 1,000 to 1,550 J m
−2
followed by a recovery period under photoreactivating conditions. A
phr uvrA
mutant of
P. aeruginosa
PAO1 was markedly sensitive to UV-B irradiation exhibiting a decrease in survival of 6 orders of magnitude following a UV-B dose of 250 J m
−2
. Complementation of the
phr
mutations in
P. aeruginosa
PAO1 and
P. syringae
pv. syringae FF5 using the cloned
phr
gene from strain PAO1 resulted in a restoration of survival following UV-B irradiation and recovery under photoreactivating conditions. The UV-B survival of the
phr
mutants could also be complemented by the
P. syringae
mutagenic DNA repair determinant
rulAB
. Assays for increases in the frequency of spontaneous rifampin-resistant mutants in UV-B-irradiated strains containing
rulAB
indicated that significant UV-B mutability (up to a 51-fold increase compared to a nonirradiated control strain) occurred even in the wild-type PAO1 background in which
rulAB
only enhanced the UV-B survival by 2-fold under photoreactivating conditions. The frequency of occurrence of spontaneous nalidixic acid-resistant mutants in the PAO1
uvrA
and
uvrA phr
backgrounds complemented with
rulAB
were 3.8 × 10
−5
and 2.1 × 10
−3
, respectively, following a UV-B dose of 1,550 J m
−2
. The construction and characterization of
phr
mutants in the present study will facilitate the determination of the roles of light and dark repair systems in organisms exposed to solar radiation in their natural habitats.
Proline-Rich Peptide from the Coral Pathogen
Vibrio shiloi
That Inhibits Photosynthesis of Zooxanthellae
Authors: Banin Ehud , Khare Sanjay K. , Naider Fred , Rosenberg Eugene
Published: July 2002

The coral-bleaching bacterium
Vibrio shiloi
biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH
4
Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH
4
Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH
4
Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH
3
into the cell. It is known that uptake of NH
3
into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by
V. shiloi
.
Heterogeneity of Shiga Toxin-Producing
Escherichia coli
Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France
Authors: Pradel Nathalie , Boukhors Karima , Bertin Yolande , Forestier Christiane , Martin Christine , Livrelli Valérie
Published: July 2002

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing
Escherichia coli
(STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the
stx
genes or the plasmids (
stx
2
-restriction fragment length polymorphism [RFLP],
stx
2
gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However,
stx
2
-RFLP and
stx
2
variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of
stx
2
-RFLP,
stx
2
variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.
Anaerobic Mineralization of Stable-Isotope-Labeled 2-Methylnaphthalene
Authors: Sullivan Elise R. , Zhang Xiaoming , Phelps Craig , Young L. Y.
Published: July 2002

An active sulfate-reducing consortium that degrades 2-methylnaphthalene (2-MNAP) at rates of up to 25 μM day
−1
was established. Degradation was inhibited in the presence of molybdate and ceased in the absence of sulfate. As much as 87% of 2-[
14
C]MNAP was mineralized to
14
CO
2
. 2-Naphthoic acid (2-NA) was detected as a metabolite, and incubation with either deuterated 2-MNAP or [
13
C]bicarbonate indicates that 2-NA is the result of oxidation of the methyl group. Also detected were carboxylated 2-MNAPs, suggesting the presence of an alternative pathway for 2-MNAP degradation.
Method for Spiking Soil Samples with Organic Compounds
Authors: Brinch Ulla C. , Ekelund Flemming , Jacobsen Carsten S.
Published: July 2002

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction ofPseudomonas fluorescensVKI171 SJ132 genetically tagged withluxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introducedP. fluorescensVKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.
Evaluation of the Abbott AxSYM Cytomegalovirus (CMV) Immunoglobulin M (IgM) Assay in Conjunction with Other CMV IgM Tests and a CMV IgG Avidity Assay
Authors: Lazzarotto T. , Galli C. , Pulvirenti R. , Rescaldani R. , Vezzo R. , La Gioia A. , Martinelli C. , La Rocca S. , Agresti G. , Grillner L. , Nordin M. , van Ranst M. , Combs B. , Maine G. T. , Landini M. P.
Published: July 2002

The measurement of the avidity of cytomegalovirus (CMV) immunoglobulin G (IgG) antibodies has been shown by several investigators to be useful in identifying and excluding primary CMV infections in pregnant women. In this work, we examined the diagnostic utility of reflex testing of CMV IgM-positive specimens from pregnant women by using a CMV IgG avidity assay. The utility of this approach was directly dependent on the sensitivity of the CMV IgM assay employed during the initial screen. The higher initial reactivity rate of the AxSYM CMV IgM assay was necessary in order to detect CMV IgM in specimens containing low-avidity CMV IgG antibodies, indicative of a primary CMV infection, which other CMV IgM assays (Behring, Vidas, Captia, and Eurogenetics) fail to detect in some cases. The use of the AxSYM CMV IgM assay, followed by an avidity test, should result in more accurate diagnosis of CMV infection in pregnant women.
Syphilis Fast Latex Agglutination Test, a Rapid Confirmatory Test
Authors: Fears Martha B. , Pope Victoria
Published: July 2002

Using 255 serum samples with various reactivities, we evaluated the Syphilis Fast latex agglutination test (Syphilis Fast) against the
Treponema pallidum
particle agglutination test (TP-PA) for confirming a diagnosis of syphilis. We found 98.8% agreement between the Syphilis Fast and the TP-PA. The Syphilis Fast, however, had a couple of advantages over the TP-PA: the test takes only 8 min to perform and produces results that are easy to read. It appears to be a good confirmatory test for syphilis, especially for point-of-care clinics such as prenatal or sexually transmitted disease clinics.
Modulation of Release of Proinflammatory Bacterial Compounds by Antibacterials: Potential Impact on Course of Inflammation and Outcome in Sepsis and Meningitis
Authors: Nau Roland , Eiffert Helmut
Published: July 2002

Several bacterial components (endotoxin, teichoic and lipoteichoic acids, peptidoglycan, DNA, and others) can induce or enhance inflammation and may be directly toxic for eukaryotic cells. Bactericidal antibiotics which inhibit bacterial protein synthesis release smaller quantities of proinflammatory/toxic bacterial compounds than Β-lactams and other cell wall-active drugs. Among the Β-lactams, compounds binding to penicillin-binding protein 2 (PBP-2) release smaller amounts of bacterial substances than antibacterials inhibiting PBP-3. Generally, high antibiotic concentrations (more than 10 times the MIC) induce the release of fewer bacterial proinflammatory/toxic compounds than concentrations close to the MIC. In several in vitro and in vivo systems, bacteria treated with protein synthesis inhibitors or Β-lactams inhibiting PBP-2 induce less inflammation than bacteria treated with PBP-3-active Β-lactams. In mouse models of Escherichia coli peritonitis sepsis and of Streptococcus pneumoniae meningitis, lower release of proinflammatory bacterial compounds was associated with reduced mortality. In conclusion, sufficient evidence for the validity of the concept of modulating the release of proinflammatory bacterial compounds by antibacterials has been accumulated in vitro and in animal experiments to justify clinical trials in sepsis and meningitis. A properly conducted study addressing the potential benefit of bacterial protein synthesis inhibitors versus Β-lactam antibiotics will require both strict selection and inclusion of a large number of patients. The benefit of this approach should be greatest in patients with a high bacterial load.
A Postgenomic Approach to Identification ofMycobacterium leprae-Specific Peptides as T-Cell Reagents
Authors: Dockrell Hazel M. , Brahmbhatt Shweta , Robertson Brian D. , Britton Sven , Fruth Uli , Gebre Negussie , Hunegnaw Mesfin , Hussain Rabia , Manandhar Rakesh , Murillo Luis , Pessolani Maria Cristina V. , Roche Paul , Salgado Jorge L. , Sampaio Elizabeth , Shahid Firdaus , Thole Jelle E. R. , Young Douglas B.
Published: July 2002

To identifyMycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure toM. lepraefrom exposure toMycobacterium tuberculosisor to environmental mycobacteria or from immune responses followingMycobacterium bovisBCG vaccination, 15-mer synthetic peptides were synthesized based on data from theM. lepraegenome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n= 59) and healthy leprosy contacts (n= 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-γ) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences inM. lepraeandM. tuberculosisand would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had ≥5 of 15 amino acid mismatches between the equivalentM. lepraeandM. tuberculosissequences; of these, eight gave specificities of ≥90% (percentage of United Kingdom donors who were nonresponders for IFN-γ secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.
Mutation and Recombination in the Upstream Homology Box-Flanked
ospE
-Related Genes of the Lyme Disease Spirochetes Result in the Development of New Antigenic Variants during Infection
Authors: Sung Shian Ying , McDowell John V. , Carlyon Jason A. , Marconi Richard T.
Published: July 2002

The
ospE
gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The
ospE
genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that
ospE
-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to
ospE
diversity and thus ultimately to antigenic variation. Using an isogeneic clone of
Borrelia burgdorferi
B31G (designated B31Gc1), we demonstrate that the
ospE
-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.
Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of NontypeableHaemophilus influenzaein Middle Ear Mucosa by Intranasal Immunization
Authors: Kodama Satoru , Suenaga Satoshi , Hirano Takashi , Suzuki Masashi , Mogi Goro
Published: July 2002

NontypeableHaemophilus influenzae(NTHI) is a major pathogen of otitis media. One of the outer membrane proteins of NTHI, P6, is an antigen common to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against nontypeableHaemophilus influenzae(NTHI) and to investigate mucosal immune responses in the middle ear, mice were immunized intranasally with the P6 outer membrane protein of NTHI, and P6-specific immune responses in the middle ear mucosa were examined. Mice were given with P6 and cholera toxin intranasally as an adjuvant on days 0, 7, and 14 and were killed on day 21. The P6-specific immunoglobulin A (IgA) antibody titer in ear wash was significantly elevated. Mononuclear cells were isolated from middle ear mucosa, and an increase in P6-specific IgA-producing cells was shown with an enzyme-linked immunospot assay. In addition, an increase in memory T cells in middle ear mucosa was detected with flow cytometric analysis after intranasal immunization. Moreover, in vitro stimulation with P6 resulted in proliferation of purified CD4+T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. These results indicate that P6-specific IgA–B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion.
Cryptosporidium parvum
Induces Host Cell Actin Accumulation at the Host-Parasite Interface
Authors: Elliott David A. , Clark Douglas P.
Published: July 2002

Cryptosporidium parvum
is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that
Cryptosporidium
resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which
Cryptosporidium
invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein α-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including α-catenin, β-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus,
Cryptosporidium
induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and α-actinin into a host-parasite junctional complex.
UV-B Irradiation Increases Susceptibility of Mice to Malarial Infection
Authors: Yamamoto Kiichi , Ito Reiko , Koura Minako , Kamiyama Tsuneo
Published: July 2002

We here examined whether exposure of mice to UV-B affected their susceptibility to the murine malaria parasitePlasmodium chabaudi. When BALB/c mice with depilated skin were irradiated with UV-B and subsequently infected with the parasite, 80 to 100% of the UV-B-irradiated mice died within 12 days of infection with a sublethal dose. In addition, UV-B irradiation of C57BL/10 (B-10) mice, which are otherwise naturally resistant to the parasites, rendered them susceptible, and 100% of irradiated B-10 mice died within 11 days postinfection. The level of plasma gamma interferon (IFN-γ) in unirradiated B-10 mice at 5 days after infection increased to 566 pg/ml, whereas the UV-B exposure of mice impaired the production of IFN-γ, which showed a maximum level of 65 pg/ml in response to the parasite infection. The maximum level of plasma interleukin-10 in UV-B-irradiated mice in response to the parasite infection was ∼1,100 pg/ml, which was approximately fourfold higher than the maximum level in unirradiated control mice. When UV-B-irradiated B-10 mice were administered murine recombinant IFN-γ after infection, the mice regained parasite resistance. These results demonstrated that the UV-B exposure of mice enhances the susceptibility to the malaria parasites and suggested that the enhanced susceptibility following UV-B exposure was mediated by impairment of IFN-γ production in response to the parasite infection.
In Vivo-Induced Genes in
Pseudomonas aeruginosa
Authors: Handfield Martin , Lehoux Dario E. , Sanschagrin François , Mahan Michael J. , Woods Donald E. , Levesque Roger C.
Published: July 2002

In vivo expression technology was used for testing
Pseudomonas aeruginosa
in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight
ivi
proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called
pvdI
) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).
The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in
Yersinia pestis
Authors: Oyston Petra C. F. , Dorrell Nick , Williams Kerstin , Li Shu-Rui , Green Michael , Titball Richard W. , Wren Brendan W.
Published: July 2002

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in
Salmonella
species. We have identified, cloned, and sequenced PhoP orthologues from
Yersinia pestis
,
Yersinia pseudotuberculosis
, and
Yersinia enterocolitica
. To investigate the role of PhoP in the pathogenicity of
Y. pestis
, an isogenic
phoP
mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and
phoP
mutant strains, grown at either 28 or 37°C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in
Y. pestis
. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the
phoP
mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of
Y. pestis
.
An OmpA-Like Protein fromAcinetobacterspp. Stimulates Gastrin and Interleukin-8 Promoters
Authors: Ofori-Darko Ernest , Zavros Yana , Rieder Gabriele , Tarlé Susan A. , Van Antwerp Mary , Merchant Juanita L.
Published: July 2002

Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 108organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified anAcinetobactersp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of theAcinetobactersp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.
Regulation of
Brucella abortus
Catalase
Authors: Kim Jeong-a , Sha Zengyu , Mayfield John E.
Published: July 2002

All aerobic organisms have mechanisms that protect against oxidative compounds. Catalase, peroxidase, superoxide dismutase, glutathione, and thioredoxin are widely distributed in many taxa and constitute elements of a nearly ubiquitous antioxidant metabolic strategy. Interestingly, the regulatory mechanisms that control these elements are rather different depending on the nature of the oxidative stress and the organism. Catalase is well documented to play an important role in protecting cells from oxidative stress. In particular, pathogenic bacteria seem to use this enzyme as a defensive tool against attack by the host. To investigate the significance of catalase in hostile environments, we made catalase deletion mutations in two different
B. abortus
strains and used two-dimensional gel analysis, survival tests, and adaptation experiments to explore the behavior and role of catalase under several oxidative stress conditions. These studies show that
B. abortus
strains that do not express catalase activity exhibit increased sensitivity to hydrogen peroxide. We also demonstrate that catalase expression is regulated in this species, and that preexposure to a sublethal concentration of hydrogen peroxide allows
B. abortus
to adapt so as to survive subsequent exposure to higher concentrations of hydrogen peroxide.
Secreted Aspartic Proteinase Family of
Candida tropicalis
Authors: Zaugg Christophe , Borg-von Zepelin Margarete , Reichard Utz , Sanglard Dominique , Monod Michel
Published: July 2002

Medically important yeasts of the genus
Candida
secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like
Candida albicans
,
Candida tropicalis
secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene
SAPT1
as a probe and under low-stringency hybridization conditions, three new closely related gene sequences,
SAPT2
to
SAPT4
, encoding secreted proteinases were cloned from a
C. tropicalis
λEMBL3 genomic library. All bands identified by Southern blotting of
Eco
RI-digested
C. tropicalis
genomic DNA with
SAPT1
could be assigned to a specific
SAP
gene. Therefore, the
SAPT
gene family of
C. tropicalis
is likely to contain only four members. Interestingly, the
SAPT2
and
SAPT3
gene products, Sapt2p and Sapt3p, which have not yet been detected in
C. tropicalis
cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast
Pichia pastoris
as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong
SAPT1
signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other
SAPT
genes under several conditions tested, showing that these
SAPT
genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.
Ferrochelatase Is Present in
Brucella
abortus
and Is Critical for Its Intracellular Survival and Virulence
Authors: Almirón Marta , Martı́nez Marcela , Sanjuan Norberto , Ugalde Rodolfo A.
Published: July 2002

Brucella
spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established,
Brucella
spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of
Brucella
abortus
. During a
B
.
abortus
genomic sequencing project, a clone coding a putative gene homologous to
hemH
was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type
hemH
. These data demonstrate that
B
.
abortus
synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of
B
.
abortus
and thus for the establishment of chronic disease as well.
Regulation of the Cytotoxic Enterotoxin Gene in
Aeromonas hydrophila
: Characterization of an Iron Uptake Regulator
Authors: Sha Jian , Lu Muping , Chopra Ashok K.
Published: July 2002

The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of
Aeromonas hydrophila
is aerolysin related and crucial to the pathogenesis of
Aeromonas
infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (
act
), a portion of the
act
gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (
phoA
) of
Escherichia coli
. The
act
::
phoA
reporter gene was then introduced into the chromosome of
A. hydrophila
by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the
act
promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The
act
promoter exhibited optimal activity at pH 7.0 and at 37°C, and maximal PhoA activity was noted when the culture was aerated. Using a
Vibrio cholerae
iron uptake regulator gene (
fur
) as a probe, a 2.6-kb
Sal
I/
Hin
dIII DNA fragment from an
A. hydrophila
chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the
fur
gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of
V. cholerae
. Complementation experiments demonstrated that the
A. hydrophila fur
gene could restore iron regulation in an
E. coli fur
-minus mutant. Using the suicide vector pDMS197, we generated a
fur
isogenic mutant of wild-type
A. hydrophila
SSU. Northern blot analysis data indicated that the repression in the transcription of the
act
gene by iron was relieved in the
fur
isogenic mutant. Further, iron regulation in the
fur
isogenic mutant of
A. hydrophila
could be restored by complementation. These results are important in understanding the regulation of the
act
gene under in vivo conditions.
Potential Role for Extracellular Glutathione-Dependent Ferric Reductase in Utilization of Environmental and Host Ferric Compounds byHistoplasma capsulatum
Authors: Timmerman Michelle M. , Woods Jon P.
Published: July 2002

The mammalian host specifically limits iron duringHistoplasma capsulatuminfection, and fungal acquisition of iron is essential for productive infection.H. capsulatumexpresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. TheH. capsulatumsiderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).