bluuebunny/crossref_metadata_2025 huggingface dataset:
Rhizobitoxine Production by
Bradyrhizobium elkanii
Enhances Nodulation and Competitiveness on
Macroptilium atropurpureum
Authors: Yuhashi Ken-Ichi , Ichikawa Norikazu , Ezura Hiroshi , Akao Shoichiro , Minakawa Yasuo , Nukui Noriyuki , Yasuta Tsuyoshi , Minamisawa Kiwamu
Published: July 2002

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of
Macroptilium atropurpureum
by
Bradyrhizobium elkanii. B. elkanii
produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in
B. elkanii
increased ethylene evolution and decreased nodulation and competitiveness on
M. atropurpureum
. These results suggest that rhizobitoxine enhances nodulation and competitiveness of
B. elkanii
on
M. atropurpureum
.
Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria
Authors: Ren Tie , Roy Réal , Knowles Roger
Published: July 2002

We studied nitrogen oxide production and consumption by methanotrophs
Methylobacter luteus
(group I),
Methylosinus trichosporium
OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as
Methylobacter
sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N
2
O). Only
Methylobacter
strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10
−17
mol of NO cell
−1
day
−1
, mostly after a culture became O
2
limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O
2
, and required CH
4
. Denitrification (methanol-supported N
2
O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore,

cd
1

and Cu nitrite reductases, NO reductase, and N
2
O reductase could not be detected by PCR amplification of the
nirS
,
nirK
,
norB
, and
nosZ
genes, respectively.
M. luteus
and
M. trichosporium
produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For
Methylobacter
strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as
Methylobacter
strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O
2
and nitrate availability occur.
Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of
Yersinia enterocolitica
Authors: Vishnubhatla A. , Fung D. Y. C. , Oberst R. D. , Hays M. P. , Nagaraja T. G. , Flood S. J. A.
Published: July 2002

We have developed a rapid procedure for the detection of virulent
Yersinia enterocolitica
in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal
yst
gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of
Y. enterocolitica
, other species of
Yersinia
(
Y. aldovae
,
Y. pseudotuberculosis
,
Y. mollaretti
,
Y. intermedia
,
Y. bercovieri
,
Y. ruckeri
,
Y. frederiksenii
, and
Y. kristensenii
), and other enteric bacteria (
Escherichia
,
Salmonella
,
Citrobacter
, and
Flavobacterium
). The assay was 100% specific in identifying the pathogenic strains of
Y. enterocolitica
. The sensitivity of the assay was found to be ≥10
2
CFU/ml in pure cultures and ≥10
3
CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with
Y. enterocolitica
serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent
Y. enterocolitica
with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.
Construction of a Multihybrid Display System: Flagellar Filaments Carrying Two Foreign Adhesive Peptides
Authors: Tanskanen Jarna , Korhonen Timo K. , Westerlund-Wikström Benita
Published: July 2002

A multivalent, bifunctional flagellum carrying two different adhesive peptides in separate flagellin subunits within a filament was constructed in
Escherichia coli
. The inserted peptides were the fibronectin-binding 115-mer D repeat region of
Staphylococcus aureus
and the 302-mer collagen-binding region of YadA of
Yersinia enterocolitica
. Western blotting, immunoelectron microscopy, and adhesion tests with hybrid flagella from an in
trans
-complemented Δ
fliC E. coli
strain showed that individual filaments consisted of both recombinant flagellins.
Light Conditions Affect the Measurement of Oceanic Bacterial Production via Leucine Uptake
Authors: Morán Xosé Anxelu G. , Massana Ramon , Gasol Josep M.
Published: July 2002

The effect of irradiance in the range of 400 to 700 nm or photosynthetically active radiation (PAR) on bacterial heterotrophic production estimated by the incorporation of
3
H-leucine (referred to herein as Leu) was investigated in the northwestern Mediterranean Sea and in a coastal North Atlantic site, with Leu uptake rates ranging over 3 orders of magnitude. We performed in situ incubations under natural irradiance levels of Mediterranean samples taken from five depths around solar noon and compared them to incubations in the dark. In two of the three stations large differences were found between light and dark uptake rates for the surfacemost samples, with dark values being on average 133 and 109% higher than in situ ones. Data obtained in coastal North Atlantic waters confirmed that dark enclosure may increase Leu uptake rates more than threefold. To explain these differences, on-board experiments of Leu uptake versus irradiance were performed with Mediterranean samples from depths of 5 and 40 m. Incubations under a gradient of 12 to 1,731 μmol of photons m
−2
s
−1
evidenced a significant increase in incorporation rates with increasing PAR in most of the experiments, with dark-incubated samples departing from this pattern. These results were not attributed to inhibition of Leu uptake in the light but to enhanced bacterial response when transferred to dark conditions. The ratio of dark to light uptake rates increased as dissolved inorganic nitrogen concentrations decreased, suggesting that bacterial nutrient deficiency was overcome by some process occurring only in the dark bottles.
Flux Analysis of the Metabolism of
Clostridium cellulolyticum
Grown in Cellulose-Fed Continuous Culture on a Chemically Defined Medium under Ammonium-Limited Conditions
Authors: Desvaux Mickaël , Petitdemange Henri
Published: July 2002

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium
Clostridium cellulolyticum
was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (
D
), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As
D
rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing
D
, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high
D
values, while cellodextrin excretion occurred mainly at lower ones. With increasing
D
, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010–2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells
−1
h
−1
could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of
C. cellulolyticum
under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.
Enzymatic Synthesis of Aminoglycoside Antibiotics: Novel Adenosylmethionine:2-Deoxystreptamine
N
-Methyltransferase Activities in Hygromycin B- and Spectinomycin-Producing
Streptomyces
spp. and Uses of the Methylated Products
Authors: Walker James B.
Published: July 2002

Aminocyclitols structurally related to streptamine, a 1,3-diaminocyclitol, are common components of the RNA-binding aminoglycoside antibiotics. The respective aminocyclitol cores of hygromycin B and spectinomycin are
N
3
-methyl-2-deoxy-
d
-streptamine and
N
1
,
N
3
-dimethyl-2-
epi
-streptamine. Adenosyl[
methyl
-
14
C]methionine:2-deoxystreptamine
N
-methyltransferase activities were detected in extracts of early-stationary-phase mycelia of the hygromycin B producer
Streptomyces hygroscopicus
subsp.
hygroscopicus
ATCC 27438 and the spectinomycin producer
Streptomyces flavopersicus
ATCC 19756. Extracts of both strains methylated the N
1
- and N
3
-amino groups of 2-deoxystreptamine, streptamine, and 2-
epi
-streptamine; the N
1
-amino group of
N
3
-methyl-2-deoxy-
d
-streptamine, and the N
3
-amino group of
N
1
-ethyl-2-deoxy-
d
-streptamine, the semisynthetic aminocyclitol of netilmicin. The mono[
14
C]methyl derivatives of 2-deoxystreptamine, streptamine, and 2-
epi
-streptamine were excellent substrates for
l
-glutamine:aminocyclitol aminotransferase and thereby provided a sensitive assay for derepression of this key enzyme, a generic biosynthetic marker that we have shown to be the only enzyme common to the biosyntheses of all major aminoglycoside antibiotics. Other prospective uses for these methyl-labeled 2-deoxystreptamine analogs are also described.
Genetic Characterization of
Cylindrospermopsis raciborskii
(Cyanobacteria) Isolates from Diverse Geographic Origins Based on
nifH
and
cpcBA
-IGS Nucleotide Sequence Analysis
Authors: Dyble Julianne , Paerl Hans W. , Neilan Brett A.
Published: July 2002

Isolates of the toxic, N
2
-fixing species
Cylindrospermopsis raciborskii
from various geographic locations were analyzed with respect to their genetic diversity based on the
nifH
and
cpcBA
-IGS genes. Gene sequences clustered according to their geographic origin, with the
nifH
sequences separating into European, Australian, and American groups and the
cpcBA
-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from
Cylindrospermopsis
species, and an additional primer set for
cpcBA
-IGS was designed to specifically amplify the American
C. raciborskii
strains.
Cocaine Causes Increased Type I Interferon Secretion by both L929 Cells and Murine Macrophages
Authors: Grattendick K. , Jansen D. B. , Lefkowitz D. L. , Lefkowitz S. S.
Published: July 2002

Cocaine has been demonstrated to have a number of different effects on immune cell functions. We have reported alterations of cellular functions by macrophages (Mφ) exposed to cocaine in vitro, including the inhibition of mouse hepatitis virus replication. Here, we present evidence that cocaine stimulates the secretion of an antiviral product that is neutralized by anti-interferon (anti-IFN). A dose-dependent increase in the secretion of IFN by both Mφ and L929 cells incubated with cocaine, with a concomitant decrease in virus replication, is also reported. The increase in IFN secretion was most pronounced when cells were cultured in the presence of the IFN inducer poly(I·C). The effect of cocaine on IFN production was found to be primarily at the transcript level in both Mφ and L929 cells. These findings further support our previous research demonstrating an antiviral activity of cocaine in vitro. The relevance of this activity to viral infections in general remains to be determined.
Prevalence and Phylogenetic Analysis of TT Virus DNA in Western India
Authors: Arankalle V. A. , Gandhe S. S. , Deshmukh T. M. , Chadha M. S. , Walimbe A. M.
Published: July 2002

In western India, TT virus (TTV) DNA positivity varied from 6.7% (5 of 75) in chronic hepatitis patients to 24.4% (10 of 41) in hemophiliacs; 7.4% (4 of 54) of voluntary blood donors had circulating TTV DNA. Phylogenetic analysis revealed a predominance of genotype 1a. In India, TTV is transmitted mainly by nonparenteral routes and is not an important cause of chronic liver diseases.
Comparison of a Chemiluminescent Immunoassay with Two Microparticle Enzyme Immunoassays for Detection of Hepatitis B Virus Surface Antigen
Authors: Diepersloot Robert J. A. , van Zantvliet-van Oostrom Yvonne , Gleaves Curt A.
Published: July 2002

A comparative evaluation of the following commercial immunoassays for the detection of hepatitis B virus surface antigen (HBsAg) was performed: the Abbott AxSYM, Abbott IMx, and DPC IMMULITE assays. The specificity was 100% for all assays. Twelve samples were identified and were confirmed to be positive for HBsAg by all three methods. One additional sample was identified as reactive and was confirmed to be positive by the Abbott AxSYM assay only. Prior to confirmation testing the DPC IMMULITE assay produced significantly fewer false-positive results than the Abbott AxSYM assay (
P
< 0.05).
Seroepidemiology of Human Group C Rotavirus in Japan Based on a Blocking Enzyme-Linked Immunosorbent Assay
Authors: Kuzuya Mitsutaka , Fujii Ritsushi , Hamano Masako , Ohata Ritsuko , Ogura Hajime , Yamada Masao
Published: July 2002

A novel blocking enzyme-linked immunosorbent assay (BL-ELISA) was developed for detection of antibodies to human group C rotavirus (CHRV). The specificity of the BL-ELISA was confirmed by using animal sera hyperimmunized to group A and group C rotaviruses and paired sera from five patients with acute CHRV gastroenteritis. Furthermore, there was concordance between the BL-ELISA and a neutralization assay for CHRV in 226 (95%) of 238 samples. By using the BL-ELISA, we determined the seroprevalence of CHRV in 704 serum samples obtained from nine different age groups of inhabitants of Okayama Prefecture, Japan, in 1992, 1994, and 1996. As a result, 211 sera (30%) were found to be positive for CHRV antibodies. The seroprevalence gradually increased with age and reached 52.7% in the oldest individuals. A further analysis of the youngest age group suggested that CHRVs predominantly prevail in persons older than 3 years of age in Japan. When comparing the three sampling years, a larger percentage of antibody-positive sera was detected in 1994 than in either 1992 or 1996 in individuals between 6 and 15 years of age, reflecting the occurrence of a CHRV outbreak among children during the winter of 1992 to 1993 that was previously documented. These results indicate that CHRV infections may occur more frequently in spite of the relatively low detection rate of the virus.
Analysis of
Chlamydia pneumoniae
Growth in Cells by Reverse Transcription-PCR Targeted to Bacterial Gene Transcripts
Authors: Haranaga Shusaku , Ikejima Hideaki , Yamaguchi Hiroyuki , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

Chlamydia pneumoniae
is an obligate intracellular bacterium and has a unique development cycle consisting of an elementary body (EB) and reticular body (RB). EBs survive in extracellular environments as well as infect susceptible host cells. However, EBs display no measurable metabolic activity. In contrast, RBs are metabolically active and can replicate in a host cell but are noninfectious. Therefore, analysis of
C. pneumoniae
growth in infected cells by conventional bacterial culture may not permit sufficient information about growth of the bacteria in cells. In this study, therefore, we examined the usefulness of the reverse transcription (RT)-PCR method for analysis of bacterial transcripts to evaluate
C. pneumoniae
growth in HEp-2 cells because the levels of bacterial gene transcripts are known to show the metabolic activity of bacteria. The transcripts for the
C. pneumoniae hsp60
gene and 16S rRNA in the cells were easily detected just after infection, followed by a marked increase. In contrast,
pyk
and
omcB
transcripts slowly increased after a latent period. The hydrocortisone treatment of
C. pneumoniae
-infected cells induced an increase of all bacterial transcripts tested compared with the control group. The treatment of the infected cells with the antibiotic minocycline showed a selective inhibition of bacterial gene transcripts, even though the complete inhibition of EB production determined by the bacterial culture assay was evident. These results indicate that the determination of bacterial gene transcripts by RT-PCR might be a powerful method to analyze in detail growth of
C. pneumoniae
in host cells, particularly altered bacterial growth caused by agents such as antimicrobials.
Immunocytochemical Method for Early Laboratory Diagnosis of Tuberculous Meningitis
Authors: Sumi M. G. , Mathai A. , Reuben S. , Sarada C. , Radhakrishnan V. V.
Published: July 2002

A simple immunocytochemical method was standardized for the direct demonstration of mycobacterial antigen in cerebrospinal fluid (CSF) specimens of patients with tuberculous meningitis (TBM). CSF-cytospin smears were prepared from 22 patients with a clinical diagnosis of TBM and also from an equal number of patients with nontuberculous neurological diseases (disease control). Immunocytological demonstration of mycobacterial antigens in the cytoplasm of monocytoid cells was attempted, by using rabbit immunoglobulin G to
Mycobacterium tuberculosis
as the primary antibody. Of the 22 CSF-cytospin smears from TBM patients, 16 showed positive immunostaining, while all of the CSF-cytospin smears from the disease control showed negative immunostaining for mycobacterial antigen. The technical aspects of this immunocytological method for the demonstration of mycobacterial antigens are simple, rapid, and reproducible, as well as specific, and therefore can be applied for the early diagnosis of TBM, particularly in patients in whom bacteriological methods did not demonstrate the presence of
M. tuberculosis
in the CSF.
Generation of Female Genital Tract Antibody Responses by Local or Central (Common) Mucosal Immunization
Authors: Wu Hong-Yin , Abdu Samira , Stinson Dana , Russell Michael W.
Published: July 2002

Genital antibody responses were compared in female mice immunized intravaginally (i.vag.) or intranasally (i.n.) with a bacterial protein antigen (AgI/II ofStreptococcus mutans) coupled to the B subunit of cholera toxin. Serum and salivary antibodies were also evaluated as measures of disseminated mucosal and systemic responses. Although i.vag. immunization induced local vaginal immunoglobulin A (IgA) and IgG antibody responses, these were not disseminated to a remote secretion, the saliva, and only modest levels of serum antibodies were generated. In contrast, i.n. immunization was substantially more effective at inducing IgA and IgG antibody responses in the genital tract and in the circulation, as well as at inducing IgA antibodies in the saliva. Moreover, mucosal and systemic antibodies induced by i.n. immunization persisted for at least 12 months. Analysis of the molecular form of genital IgA indicated that the majority of both total IgA and specific IgA antibody was polymeric, and likely derived from the common mucosal immune system.
The
Legionella pneumophila iraAB
Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence
Authors: Viswanathan V. K. , Edelstein Paul H. , Pope C. Dumais , Cianciotto Nicholas P.
Published: July 2002

Legionella pneumophila
, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn
10
mutagenesis, we previously isolated a
L. pneumophila
mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (
iraA
) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (
iraB
) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the
iraAB
locus was largely limited to strains of
L. pneumophila
, the most pathogenic of the
Legionella
species. A newly derived mutant containing a targeted disruption of
iraB
showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that
iraA
is critical for virulence of
L. pneumophila
while
iraB
is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.
Dominant Nonresponsiveness toHelicobacter pyloriInfection Is Associated with Production of Interleukin 10 but Not Gamma Interferon
Authors: Sutton Philip , Kolesnikow Tassia , Danon Stephen , Wilson John , Lee Adrian
Published: July 2002

Helicobacter pylori-induced gastritis is an essential precursor lesion for the development of peptic ulcers or gastric adenocarcinoma. We demonstrate that nonresponsiveness toH. pyloriSS1 infection is dominantly inherited in mice. F1hybrid crosses between a nonresponder mouse and three responder strains all possessed the nonresponder phenotype. Secretion of interleukin-10 but not gamma interferon was associated with nonresponsiveness to infection.
Diminished Diarrheal Response to
Vibrio cholerae
Strains Carrying the Replicative Form of the CTXΦ Genome instead of CTXΦ Lysogens in Adult Rabbits
Authors: Faruque Shah M. , Rahman M. Mostafizur , Hasan A. K. M. Mahbub , Nair G. Balakrish , Mekalanos John J. , Sack David A.
Published: July 2002

Toxigenic
Vibrio cholerae
strains are lysogens of CTXΦ, a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient
V. cholerae
cells by CTXΦ, the phage genome either integrates into the host chromosome at a specific attachment site (
att
RS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring
att
RS-negative nontoxigenic
V. cholerae
or attenuated (CTX
−
att
RS negative) derivatives of wild-type toxigenic strains with CTXΦ and examined the diarrheagenic potential of the strains carrying the RF of the CTXΦ genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTXΦ produced more CT than corresponding lysogens as assayed by a G
M1
-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the
att
RS-negative strains (which carried the CTXΦ genome as the RF) were either negative or produced mild diarrhea, whereas the
att
RS-positive strains with integrated CTXΦ produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the
att
RS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTXΦ are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTXΦ genome. These results may have implications for the development of live
V. cholerae
vaccine candidates impaired in chromosomal integration of CTXΦ. These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic
V. cholerae
strains.
DNA Immunization byPlasmodium falciparumLiver-Stage Antigen 3 Induces Protection againstPlasmodium yoeliiSporozoite Challenge
Authors: Sauzet Jean-Pierre , Perlaza Blanca-Liliana , Brahimi Karima , Daubersies Pierre , Druilhe Pierre
Published: July 2002

DNA-based immunization of mice byPlasmodium falciparumliver-stage antigen 3 (PfLSA3), a novel highly conservedP. falciparumpreerythrocytic antigen, was evaluated. Animals developed a dominant Th1 immune response (high gamma interferon T-cell responses and predominance of immunoglobulin G2a) to each of three recombinant proteins spanning the molecule. We have exploited the immunological cross-reactivity of PfLSA3 with its putative homologue on sporozoites of the rodent parasitePlasmodium yoelii, and we show for the first time that responses induced by PfLSA3 in mice significantly protect against a heterologous challenge byP. yoeliisporozoites. These results support a significant effect of DNA-induced immune responses on preerythrocytic stages.
Conservation of Outer Membrane Protein E among Strains of
Moraxella catarrhalis
Authors: Murphy Timothy F. , Brauer Aimee L. , Yuskiw Norine , McNamara Erin R. , Kirkham Charmaine
Published: July 2002

Outer membrane protein E (OMP E) is a 50-kDa protein of
Moraxella catarrhalis
which has several features that suggest that the protein may be an effective vaccine antigen. To assess the conservation of OMP E among strains of
M. catarrhalis,
22 isolates were studied with eight monoclonal antibodies which recognize epitopes on different regions of the protein. Eighteen of 22 strains were reactive with all eight antibodies. The sequences of
ompE
from 16 strains of
M. catarrhalis
were determined, including the 4 strains which were nonreactive with selected monoclonal antibodies. Analysis of sequences indicate a high degree of conservation among strains, with sequence differences clustered in limited regions of the gene. To assess the stability of
ompE
during colonization of the human respiratory tract, the sequences of
ompE
of isolates collected from patients colonized with the same strain for 3 to 9 months were determined. The sequences remained unchanged. These results indicate that OMP E is highly conserved among strains of
M. catarrhalis,
and preliminary studies indicate that the gene which encodes OMP E remains stable during colonization of the human respiratory tract.
Intrachromosomal Recombination within the
vsp
Locus of
Mycoplasma bovis
Generates a Chimeric Variable Surface Lipoprotein Antigen
Authors: Lysnyansky Inessa , Ron Yael , Sachse Konrad , Yogev David
Published: July 2002

A family of 13 related but divergent
vsp
genes was recently found in the chromosome of the bovine pathogen
Mycoplasma bovis
. The
vsp
genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the
vsp
gene repertoire is subject to changes. Genetic analysis of
M. bovis
clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the
vsp
gene family, the formerly expressed
vspA
gene and the
vspO
gene, led to the formation of a new chimeric and functional
vsp
gene,
vspC
. The 5′ end of the recombination event was identified within the highly conserved
vsp
-upstream region, while the 3′ end was localized within the first repetitive domain (R
A
1) present in both
vspA
and
vspO
structural genes. As a result, the
vspC
gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved
vsp
-upstream region provided by the
vspO
gene; and a C-terminus-encoding region and the more distal and divergent
vsp
-upstream region acquired from the
vspA
gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.
Haemophilus ducreyi
Lipooligosaccharide Mutant Defective in Expression of β-1,4-Glucosyltransferase Is Virulent in Humans
Authors: Young Royden S. , Filiatrault Melanie J. , Fortney Kate R. , Hood Antoinette F. , Katz Barry P. , Munson Robert S. , Campagnari Anthony A. , Spinola Stanley M.
Published: July 2002

The lipooligosaccharide (LOS) of
Haemophilus ducreyi
contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of
H. ducreyi
from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by
H. ducreyi
in humans.
Extracellular Arginine Aminopeptidase from
Streptococcus gordonii
FSS2
Authors: Goldstein J. M. , Nelson D. , Kordula T. , Mayo J. A. , Travis J.
Published: July 2002

Streptococcus gordonii
is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), producing thrombus formation and tissue damage on the surfaces of heart valves. This is ironic, considering its normal role as a benign inhabitant of the oral microflora. However, strain FSS2 of
S. gordonii
has been found to produce several extracellular aminopeptidase- and fibrinogen-degrading activities during growth in a pH-controlled batch culture. In this report, we describe the purification, characterization, and partial cloning of a predicted serine class arginine aminopeptidase (RAP) with some cysteine class characteristics. Isolation of this enzyme by anion-exchange, gel filtration, and isoelectric focusing chromatography yielded a protein monomer of approximately 70 kDa, as shown by matrix-assisted laser desorption ionization, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Nested-PCR cloning enabled the isolation of a 324-bp-long DNA fragment encoding the 108-amino-acid N terminus of RAP. Culture activity profiles and N-terminal sequence analysis indicated the export of this protein from the cell surface. Homology was found with a putative dipeptidase from
Streptococcus pyogenes
and nonspecific dipeptidases from
Lactobacillus helveticus
and
Lactococcus lactis
. We believe that RAP may serve as a critical factor for arginine acquisition during nutrient stress in vivo and also in the proteolysis of host proteins and peptides during SBE pathology.
A Superoxide Dismutase C Mutant of
Haemophilus ducreyi
Is Virulent in Human Volunteers
Authors: Bong Cliffton T. H. , Fortney Kate R. , Katz Barry P. , Hood Antoinette F. , Mateo Lani R. San , Kawula Thomas H. , Spinola Stanley M.
Published: July 2002

Haemophilus ducreyi
produces a periplasmic copper-zinc superoxide dismutase (Cu-Zn SOD), which is thought to protect the organism from exogenous reactive oxygen species generated by neutrophils during an inflammatory response. We had previously identified the gene,
sodC
, responsible for the production and secretion of Cu-Zn SOD and constructed an isogenic
H. ducreyi
strain with a mutation in the
sodC
gene (35000HP-
sodC-cat
). Compared to the parent, the mutant does not survive in the presence of exogenous superoxide (L. R. San Mateo, M. Hobbs, and T. H. Kawula, Mol. Microbiol. 27:391-404, 1998) and is impaired in the swine model of
H. ducreyi
infection (L. R. San Mateo, K. L. Toffer, P. E. Orndorff, and T. H. Kawula, Infect. Immun. 67:5345-5351, 1999). To test whether Cu-Zn SOD is important for bacterial survival in vivo, six human volunteers were experimentally infected with 35000HP and 35000HP-
sodC-cat
and observed for papule and pustule formation. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule formation rates were 75% (95% confidence intervals [CI], 43 to 95%) at 12 parent-inoculated sites and 67% (95% CI, 41 to 88%) at 18 mutant-inoculated sites (
P
= 0.47). There was no significant difference in levels of
H. ducreyi
recovery from mutant- and parent-inoculated biopsy sites. These results suggest that expression of Cu-Zn SOD does not play a major role in the survival of this pathogen in the initial stages of experimental infection of humans.
Antibodies against a Synthetic Peptide of SagA Neutralize the Cytolytic Activity of Streptolysin S from Group A Streptococci
Authors: Dale James B. , Chiang Edna Y. , Hasty David L. , Courtney Harry S.
Published: July 2002

Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the
sagA
gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections.
A Promoter Region Binding Protein and DNA Gyrase Regulate Anaerobic Transcription of
nifLA
in
Enterobacter cloacae
Authors: Hu Biao , Zhu Jiabi , Shen San Chiun , Yu Guan-qiao
Published: July 2002

Our work provides evidence that a sequence characteristic of FNR binding sites, when interacted with by a
trans
-acting factor, activates anaerobic transcription of the
nifLA
operon in
Enterobacter cloacae
. DNA gyrase activity has been found to be important for the anaerobic transcription of the
nifLA
promoter. Our results suggest that anaerobic regulation of the
nifLA
operon is mediated through the control of the promoter region-binding
trans
-acting factor at the transcriptional level, while DNA supercoiling functions in providing a topological requirement for the activation of transcription.
Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental
Brevibacterium
Isolate Using mRNA Differential Display
Authors: Brzostowicz Patricia C. , Gibson Katharine L. , Thomas Stuart M. , Blasko Mary Sue , Rouvière Pierre E.
Published: July 2002

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant
Brevibacterium
environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in
Escherichia coli
and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.
Analysis of
tnrA
Alleles Which Result in a Glucose-Resistant Sporulation Phenotype in
Bacillus subtilis
Authors: Shin Byung-Sik , Choi Soo-Keun , Smith Issar , Park Seung-Hwan
Published: July 2002

Bacillus subtilis
cells cannot sporulate in the presence of catabolites such as glucose. During the analysis of Tn
10
-generated mutants, we found that deletion of the C-terminal region of the
tnrA
gene, which encodes a global regulator that positively regulates a number of genes in response to nitrogen limitation, results in a catabolite-resistant sporulation phenotype. Analyses of
nrg-lacZ
and
nasB-lacZ
, which are activated by TnrA under nitrogen limitation, showed that C-terminally truncated TnrA activates nitrogen-regulated genes constitutively. The relief of catabolite repression of sporulation may result from the uncontrolled expression of the TnrA-regulated genes.
Catabolite Repression and Induction of the Mg
2+
-Citrate Transporter CitM of
Bacillus subtilis
Authors: Warner Jessica B. , Krom Bastiaan P. , Magni Christian , Konings Wil N. , Lolkema Juke S.
Published: July 2002

In
Bacillus subtilis
the
citM
gene encodes the Mg
2+
-citrate transporter. A target site for carbon catabolite repression (
cre
site) is located upstream of
citM
. Fusions of the
citM
promoter region, including the
cre
sequence, to the β-galactosidase reporter gene were constructed and integrated into the
amyE
site of
B. subtilis
to study catabolic effects on
citM
expression. In parallel with β-galactosidase activity, the uptake of Ni
2+
-citrate in whole cells was measured to correlate
citM
promoter activity with the enzymatic activity of the CitM protein. In minimal media, CitM was only expressed when citrate was present. The presence of glucose in the medium completely repressed
citM
expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate. Studies with
B. subtilis
mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system. During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of
citM
expression by glucose. A reasonable correlation between
citM
promoter activity and CitM transport activity was observed in this study, indicating that the Mg
2+
-citrate uptake activity of
B. subtilis
is mainly regulated at the transcriptional level.
Involvement of ς
S
in Starvation-Induced Transposition of
Pseudomonas putida
Transposon Tn
4652
Authors: Ilves Heili , Hõrak Rita , Kivisaar Maia
Published: July 2002

Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn
4652
is elevated in stationary-phase
Pseudomonas putida
and suppressed in an isogenic ς
S
-defective strain. We demonstrate that transcription from the Tn
4652
transposase promoter is controlled by the stationary-phase-specific sigma factor ς
S
. To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase. Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.
Ancestral Divergence, Genome Diversification, and Phylogeographic Variation in Subpopulations of Sorbitol-Negative, β-Glucuronidase-Negative Enterohemorrhagic
Escherichia coli
O157
Authors: Kim Jaehyoung , Nietfeldt Joseph , Ju Jingliang , Wise John , Fegan Narelle , Desmarchelier Patricia , Benson Andrew K.
Published: July 2002

The O157:H7 lineage of enterohemorrhagic
Escherichia coli
is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and β-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and β-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, β-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United Stares by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, β-glucuronidase negative O157:H7 and O157:H− strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.
Type 1 Capsule Genes of
Staphylococcus aureus
Are Carried in a Staphylococcal Cassette Chromosome Genetic Element
Authors: Luong Thanh T. , Ouyang Shu , Bush Kelly , Lee Chia Y.
Published: July 2002

The
cap1
genes are required for the synthesis of type 1 capsular polysaccharide (CP1) in
Staphylococcus aureus
. We previously showed that the
cap1
locus was associated with a discrete genetic element in
S. aureus
M. In this report, we defined the boundaries of the
cap1
element by comparing its restriction pattern to that of a corresponding region from the CP1-negative strain Becker. The element was located in the
Sma
I-G chromosomal fragment of the standard mapping strain NCTC8325. The sequences of the entire
cap1
element and the flanking regions were determined. We found that there were two additional
cap1
genes not previously identified. The
cap1
operon was located in a staphylococcal cassette chromosome (SCC) element similar to the resistance island SCC
mec
recently described for methicillin resistance in
S. aureus
. Notably, the SCC
cap1
element was located at the same insertion site as all the SCC
mec
elements in the staphylococcal chromosome. The excision of SCC
cap1
could be demonstrated only in the presence of the recombinase genes from an SCC
mec
element, verifying that SCC
cap1
is a genuine SCC element but defective in mobilization. A novel enterotoxin gene, whose transcript was detected by Northern blotting, was found next to the SCC
cap1
locus. We propose that the enterotoxin gene and SCC
cap1
were inserted into this locus at the juxtaposition by independent events. Sequence comparison revealed numerous DNA rearrangements and mutations in SCC
cap1
and the left flanking region, suggesting that the SCC
cap1
had been inserted at the SCC
attC
site a long time ago. In addition, most genes in this region were incomplete, with the exception of the 15
cap1
genes, implying that the
cap1
genes confer a survival advantage on strain M.
Control of Anthrax Toxin Gene Expression by the Transition State Regulator
abrB
Authors: Saile Elke , Koehler Theresa M.
Published: July 2002

Bacillus anthracis
produces the anthrax toxin proteins protective antigen (PA), lethal factor (LF), and edema factor (EF) in a growth phase-dependent manner when cultured in liquid medium. Expression of the toxin genes
pagA
,
lef
, and
cya
peaks in late log phase, and steady-state levels of the toxin proteins are highest during the transition into stationary phase. Here we show that an apparent transition state regulator negatively regulates toxin gene expression. We identified two orthologues of the
B. subtilis
transition state regulator
abrB
in the
B. anthracis
genome: one on the chromosome and one on the 182-kb virulence plasmid pXO1. The orthologue located on the chromosome is predicted to encode a 94-amino-acid protein that is 85% identical to
B. subtilis
AbrB. The hypothetical protein encoded on pXO1 is 41% identical to
B. subtilis
AbrB but missing 27 amino acid residues from the amino terminus compared to the
B. subtilis
protein. Deletion of the pXO1-encoded
abrB
orthologue did not affect toxin gene expression under the conditions tested. However, a
B. anthracis
mutant in which the chromosomal
abrB
gene was deleted expressed
pagA
earlier and at a higher level than the parent strain. Expression of a transcriptional
pagA-lacZ
fusion in the
abrB
mutant was increased up to 20-fold during early exponential growth compared to the parent strain and peaked in mid-exponential rather than late exponential phase. In contrast to the strong effect of
abrB
on
pagA
expression,
lef-lacZ
and
cya-lacZ
expression during early-log-phase growth was increased only two- to threefold in the
abrB
null mutant. Western hybridization analysis showed increased PA, LF, and EF synthesis by the mutant. As is true in
B. subtilis
, the
B. anthracis abrB
gene is negatively regulated by
spo0A
. Our findings tie anthrax toxin gene expression to the complex network of postexponential phase adaptive responses that have been well studied in
B. subtilis
.