bluuebunny/crossref_metadata_2025 huggingface dataset:
Intranasal Immunization with RecombinantAscaris suum14-Kilodalton Antigen Coupled with Cholera Toxin B Subunit Induces Protective Immunity toA. suumInfection in Mice
Authors: Tsuji Naotoshi , Suzuki Kayo , Kasuga-Aoki Harue , Matsumoto Yasunobu , Arakawa Takeshi , Ishiwata Kenji , Isobe Takashi
Published: July 2002

Animals can be rendered immune toAscarisparasites by immunization with infectious-stage larvae. The specific parasite gene products that mediate protective responses in ascariasis are unknown. We have identified a cDNA encodingAscaris suum14-kDa antigen (As14) and evaluated the vaccinal effect of theEscherichia coli-expressed recombinant protein (rAs14). GenBank analysis showed that As14 has low similarity at the amino acid level to aCaenorhabditis elegansgene product and to antigens of the filarial nematodes but not to other known proteins. In addition, As14 homologues were found to be expressed in human and dog roundworms. In mice that received intranasal administration of rAs14 coupled with cholera toxin B subunit (rAs14-CTB), there was a 64% reduction of recovery of larvae compared with that in the nontreated group. The vaccinated mice showed a significant increase in the total serum immunoglobulin G (IgG) levels and the mucosal IgA responses. Elevation of the rAs14-specific IgE response was also seen. Measurement of the IgG subclasses showed a higher level of IgG1 and a lower level of IgG2a antibody response in the sera of the immunized mice, suggesting that protection was associated with a type II immune response. As14 is the first protective antigen againstA. suuminfection to be identified. Our immunization trial results in laboratory animals suggest the possibility of developing a mucosal vaccine for parasitic diseases caused by ascarid nematodes.
Search for Correlates of Protective Immunity Conferred by Anthrax Vaccine
Authors: Reuveny Shaul , White Moshe D. , Adar Yaakov Y. , Kafri Yaron , Altboum Zeev , Gozes Yehusha , Kobiler David , Shafferman Avigdor , Velan Baruch
Published: July 2002

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (
r
2
= 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD
50
) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.
Multistage Multiantigen Heterologous Prime Boost Vaccine forPlasmodium knowlesiMalaria Provides Partial Protection in Rhesus Macaques
Authors: Rogers William O. , Baird J. Kevin , Kumar Anita , Tine John A. , Weiss Walter , Aguiar João C. , Gowda Kalpana , Gwadz Robert , Kumar Sanjai , Gold Mark , Hoffman Stephen L.
Published: July 2002

A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria speciesPlasmodium knowlesiincluding two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100P. knowlesisporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.
Identification and Characterization of
hsa
, the Gene Encoding the Sialic Acid-Binding Adhesin of
Streptococcus gordonii
DL1
Authors: Takahashi Yukihiro , Konishi Kiyoshi , Cisar John O. , Yoshikawa Masanosuke
Published: July 2002

Oral colonization by
Streptococcus gordonii
, an important cause of subacute bacterial endocarditis, involves bacterial recognition of sialic acid-containing host receptors. The sialic acid-binding activity of this microorganism was previously detected by bacterium-mediated hemagglutination and associated with a streptococcal surface component identified as the Hs antigen. The gene for this antigen (
hsa
) has now been cloned in
Escherichia coli
, and its expression has been detected by colony immunoblotting with anti-Hs serum. Mutants of
S. gordonii
containing
hsa
inactivated by the insertion of an erythromycin resistance gene or deletion from the chromosome were negative for Hs-immunoreactivity, bacterium-mediated hemagglutinating activity, and adhesion to α2-3-linked sialoglycoconjugates. The deletion in the latter mutants was complemented by plasmid-borne
hsa
, resulting in Hs antigen production and the restoration of cell surface sialic acid-binding activity. The
hsa
gene encodes a 203-kDa protein with two serine-rich repetitive regions in its 2,178-amino-acid sequence. The first serine-rich region occurs within the amino-terminal region of the molecule, between different nonrepetitive sequences that may be associated with sialic acid binding. The second serine-rich region, which is much longer than the first, is highly repetitive, containing 113 dodecapeptide repeats with a consensus sequence of SASTSASVSASE. This long repetitive region is followed by a typical gram-positive cell wall anchoring region at the carboxyl-terminal end. Thus, the predicted properties of Hsa, which suggest an amino-terminal receptor-binding domain attached to the cell surface by a molecular stalk, are consistent with the identification of this protein as the sialic acid-binding adhesin of
S. gordonii
DL1.
A Recombinant Carboxy-Terminal Domain of the Protective Antigen of
Bacillus anthracis
Protects Mice against Anthrax Infection
Authors: Flick-Smith Helen C. , Walker Nicola J. , Gibson Paula , Bullifent Helen , Hayward Sarah , Miller Julie , Titball Richard W. , Williamson E. Diane
Published: July 2002

The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione
S
-transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA.
Passive Immunization with Bovine Milk Containing Antibodies to a Cell Surface Protein Antigen-Glucosyltransferase Fusion Protein Protects Rats against Dental Caries
Authors: Mitoma Morihide , Oho Takahiko , Michibata Naoko , Okano Kaoru , Nakano Yutaka , Fukuyama Masataka , Koga Toshihiko
Published: July 2002

Cell surface protein antigen (PAc) and glucosyltransferases (GTF) of
Streptococcus mutans
are major colonization factors of the organism. We prepared bovine milk containing antibodies against a fusion of the saliva-binding alanine-rich region of PAc with the glucan-binding domain of GTF-I. This study examined the effect of the immune milk on the cariogenicity of
S. mutans
in a rat model. Concentrated immune milk was fed to rats once a day for 55 days. The group that received immune milk had significantly less caries development than controls.
Regulation of the Overlapping
pic/set
Locus in
Shigella flexneri
and Enteroaggregative
Escherichia coli
Authors: Behrens Martin , Sheikh Jalaluddin , Nataro James P.
Published: July 2002

Most strains of
Shigella flexneri
2a and enteroaggregative
Escherichia coli
carry a highly conserved chromosomal locus which encodes a 109-kDa secreted mucinase (called Pic) and, on the opposite strand in overlapping fashion, an oligomeric enterotoxin called ShET1, encoded by the
setA
and
setB
genes. Here, we characterize the genetic regulation of these overlapping genes. Our data suggest that
pic
and the
setBA
loci are transcribed as complementary 4-kb mRNA species. The major
pic
promoter is maximally activated at 37°C in exponential growth phase. Our data suggest that the
setB
gene is transcribed from a promoter which lies more than 1.5 kb upstream of the
setB
structural gene;
setA
may be transcribed via readthrough of the
setB
transcript and possibly by its own promoter. The long leader of the
setB
gene provides a strong silencing effect on
setB
transcription. The signals which provide relief from
setB
silencing are not clear, but significant induction is observed in a continuous anaerobic culture of human fecal bacteria, suggesting that some complex characteristics of the human intestine act to lift repression of
setB
expression. Our studies provide the first insights into the mechanisms affecting expression of this unusual virulence locus.
Peptidoglycan from
Staphylococcus aureus
Induces Tissue Factor Expression and Procoagulant Activity in Human Monocytes
Authors: Mattsson Eva , Herwald Heiko , Björck Lars , Egesten Arne
Published: July 2002

Staphylococcus aureus
is one of the most significant pathogens in human sepsis and endocarditis.
S. aureus
can initiate blood coagulation, leading to the formation of microthrombi and multiorgan dysfunction in sepsis, whereas in endocarditis the bacterium induces fibrin clots on the inner surface of the heart, so-called endocardial vegetations. In the present study, we show that live and heat-killed
S. aureus
bacteria are potent inducers of procoagulant activity in human peripheral blood mononuclear cells. Furthermore, purified peptidoglycan, the main cell wall component of
S. aureus
, induced procoagulant activity in mononuclear cells in a concentration-dependent fashion. The procoagulant activity in these cells was dependent on expression of tissue factor, since antibodies to tissue factor inhibited the effect of peptidoglycan. In mononuclear cells stimulated with peptidoglycan, reverse transcription-PCR showed tissue factor gene expression, and the gene product was detected by enzyme-linked immunosorbent assay. Finally, flow cytometry identified tissue factor at the surface of CD14-positive monocytes. Peptidoglycan is known to induce proinflammatory cytokine production in monocytes. The present investigation shows that peptidoglycan also activates the extrinsic pathway of coagulation by inducing the expression of tissue factor in these cells. This mechanism helps to explain the procoagulant activity, which plays such an important role in the pathogenicity of severe
S. aureus
infections.
Identification of a
Neospora caninum
Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans
Authors: Keller Nadine , Naguleswaran Arunasalam , Cannas Angela , Vonlaufen Nathalie , Bienz Marianne , Björkman Camilla , Bohne Wolfgang , Hemphill Andrew
Published: July 2002

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in
Neospora caninum
tachyzoites which, due to its homology to MIC1 in
Toxoplasma gondii
(TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in
Escherichia coli
, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.
Identification of a Small RNA within the
pdh
Gene Cluster of
Mycoplasma pneumoniae
and
Mycoplasma genitalium
Authors: Göhlmann Hinrich W. H. , Weiner January , Schön Astrid , Herrmann Richard
Published: July 2002

A highly abundant and heterogenous small RNA about 205 to 210 bases long named MP200 RNA has been identified in
Mycoplasma pneumoniae
. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (
pdh
) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the
pdh
gene cluster of
Mycoplasma genitalium
that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in
Mycoplasma pneumoniae
. The RNAs from both
M. pneumoniae
and
M. genitalium
have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.
Inactivation of the Stress- and Starvation-Inducible
gls24
Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression in
Enterococcus faecalis
Authors: Giard Jean-Christophe , Rince Alain , Capiaux Herve , Auffray Yanick , Hartke Axel
Published: July 2002

Enterococcus faecalis
induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl
2
and bile salts stresses, one of these proteins (Gls24) was qualified as a “general stress protein” and was analyzed at the molecular level. Its corresponding gene,
gls24
, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding
gls24
(
orf4
) showed very strong identity with
gls24
. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from
Lactococcus lactis
and an alkaline stress protein from
Staphylococcus aureus
with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i)
gls24
possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the
gls24
gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in
E. faecalis
. Comparison of two-dimensional protein gels from wild-type cells with those from
gls24
mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for
l
-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.
Expression of
Mycobacterium smegmatis
Pyrazinamidase in
Mycobacterium tuberculosis
Confers Hypersensitivity to Pyrazinamide and Related Amides
Authors: Boshoff Helena I. M. , Mizrahi Valerie
Published: July 2002

A pyrazinamidase (PZase)-deficient
pncA
mutant of
Mycobacterium tuberculosis
, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role of
pncA
in the PZA susceptibility of
M. tuberculosis
(A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the
pzaA
gene encoding the major PZase/nicotinamidase from
Mycobacterium smegmatis
(H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the
M. tuberculosis pncA
gene into the
pncA
mutant complemented its PZase/nicotinamidase defect. In both
pzaA
- and
pncA
-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The
pzaA
-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type and
pncA
-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on
M. smegmatis
(MIC, 150 μg/ml in all cases) and rendered
Escherichia coli
hypersensitive for growth at low pH.
fleN
, a Gene That Regulates Flagellar Number in
Pseudomonas aeruginosa
Authors: Dasgupta Nandini , Arora Shiwani K. , Ramphal Reuben
Published: July 2002

The single polar flagellum of
Pseudomonas aeruginosa
plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the
Pseudomonas
genome database, an open reading frame (ORF), flanked by flagellar genes
flhF
and
fliA
, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in
P. aeruginosa
PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in
P. aeruginosa
. The ORF was named
fleN
. An intact copy of
fleN
on a plasmid complemented the mutant by restoring motility and monoflagellate status. The β-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and
fleN
mutant strains revealed a direct correlation between six promoters that were upregulated in the
fleN
mutant (
fliLMNOPQ
,
flgBCDE
,
fliEFG
,
fliDS orf126
,
fleSR
, and
fliC
) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in
P. aeruginosa
.
The LE1 Bacteriophage Replicates as a Plasmid within
Leptospira biflexa
: Construction of an
L. biflexa-Escherichia coli
Shuttle Vector
Authors: Girons Isabelle Saint , Bourhy Pascale , Ottone Catherine , Picardeau Mathieu , Yelton David , Hendrix Roger W. , Glaser Philippe , Charon Nyles
Published: July 2002

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the
Leptospira biflexa
saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131–1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in
Leptospira
. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the
bla
gene but carrying a kanamycin resistance marker from the gram-positive bacterium
Enterococcus
(
Streptococcus
)
faecalis
. These constructs were transformed into
L. biflexa
strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in
L. biflexa
. However, a larger region including an intact
parA
gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of
Leptospira
species.
Gene Cloning and Molecular Characterization of Lysine Decarboxylase from
Selenomonas ruminantium
Delineate Its Evolutionary Relationship to Ornithine Decarboxylases from Eukaryotes
Authors: Takatsuka Yumiko , Yamaguchi Yoshihiro , Ono Minenobu , Kamio Yoshiyuki
Published: July 2002

Lysine decarboxylase (LDC; EC
4.1.1.18
) from
Selenomonas ruminantium
comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both
l
-lysine and
l
-ornithine with similar

K
m

and

V
max

values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (
ldc
) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of
S. ruminantium
LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC
4.1.1.17
), including the mouse,
Saccharomyces cerevisiae
,
Neurospora crassa
,
Trypanosoma brucei
, and
Caenorhabditis elegans
enzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved in
S. ruminantium
LDC. Computer analysis of the putative secondary structure of
S. ruminantium
LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward both
l
-lysine and
l
-ornithine with a substrate specificity ratio of 0.83 (defined as the
k
cat
/

K
m

ratio obtained with
l
-ornithine relative to that obtained with
l
-lysine). We have succeeded in converting
S. ruminantium
LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in
S. ruminantium
LDC.
A Rac Homolog Is Required for Induction of Hyphal Growth in the Dimorphic Yeast
Yarrowia lipolytica
Authors: Hurtado Cleofe A. R. , Beckerich Jean-Marie , Gaillardin Claude , Rachubinski Richard A.
Published: July 2002

Dimorphism in fungi is believed to constitute a mechanism of response to adverse conditions and represents an important attribute for the development of virulence by a number of pathogenic fungal species. We have isolated
YlRAC1
, a gene encoding a 192-amino-acid protein that is essential for hyphal growth in the dimorphic yeast
Yarrowia lipolytica
and which represents the first Rac homolog described for fungi.
YlRAC1
is not an essential gene, and its deletion does not affect the ability to mate or impair actin polarization in
Y. lipolytica
. However, strains lacking functional
YlRAC1
show alterations in cell morphology, suggesting that the function of
YlRAC1
may be related to some aspect of the polarization of cell growth. Northern blot analysis showed that transcription of
YlRAC1
increases steadily during the yeast-to-hypha transition, while Southern blot analysis of genomic DNA suggested the presence of several
RAC
family members in
Y. lipolytica
. Interestingly, strains lacking functional
YlRAC1
are still able to grow as the pseudohyphal form and to invade agar, thus pointing to a function for
YlRAC1
downstream of
MHY1
, a previously isolated gene encoding a C
2
H
2
-type zinc finger protein with the ability to bind putative stress response elements and whose activity is essential for both hyphal and pseudohyphal growth in
Y. lipolytica.
Isolation of
PDX2
, a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria
Authors: Ehrenshaft Marilyn , Daub Margaret E.
Published: July 2002

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in
Escherichia coli
, recent characterization of the
Cercospora nicotianae
biosynthesis gene
PDX1
led to the discovery that most organisms contain a pyridoxine synthesis gene not found in
E. coli. PDX2
was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to
PDX1
-containing organisms. The role of
PDX2
in pyridoxine biosynthesis was confirmed by complementation of two
C. nicotianae
pyridoxine auxotrophs not mutant in
PDX1
. Also, targeted gene replacement of
PDX2
in
C. nicotianae
results in pyridoxine auxotrophy. Comparable to
PDX1, PDX2
homologues are not found in any of the organisms with homologues to the
E. coli
pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain
PDX1
homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.
SsrA-Mediated Tagging in
Bacillus subtilis
Authors: Wiegert Thomas , Schumann Wolfgang
Published: July 2002

A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the
ssrA
gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this
ssrA
-mediated mechanism in
Bacillus subtilis
, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the
bgaB
reporter gene coding for a heat-stable β-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter β-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the
hrcA
stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by
trans
translational addition of the proteolytic tag. Inactivating the
B. subtilis ssrA
or
smpB
(
yvaI
) gene prevented the
trans
translational tagging reaction. Various protease-deficient strains of
B. subtilis
were tested for proteolysis of tagged HrcA. HrcA remained stable only in
clpX
or
clpP
knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in
B. subtilis
.
ATPase-Defective Derivatives of
Escherichia coli
DnaK That Behave Differently with Respect to ATP-Induced Conformational Change and Peptide Release
Authors: Barthel Thomas K. , Zhang Jundong , Walker Graham C.
Published: July 2002

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of
Escherichia coli
DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The
dnaK
alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a Δ
dnaK
mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.
Carboxy-Terminal Region Involved in Activity of
Escherichia coli
TolC
Authors: Yamanaka Hiroyasu , Izawa Hiroshi , Okamoto Keinosuke
Published: July 2002

The
Escherichia coli
TolC acts as a channel tunnel in the transport of various molecules across the outer membrane. Partial-deletion studies of
tolC
revealed that the region extending from the 50th to the 60th amino acid residue from the carboxy terminus plays an important role in this transport activity of TolC.
MxiM and MxiJ, Base Elements of the Mxi-Spa Type III Secretion System of
Shigella
, Interact with and Stabilize the MxiD Secretin in the Cell Envelope
Authors: Schuch Raymond , Maurelli Anthony T.
Published: July 2002

The type III secretion pathway is broadly distributed across many parasitic bacterial genera and serves as a mechanism for delivering effector proteins to eukaryotic cell surface and cytosolic targets. While the effectors, as well as the host responses elicited, differ among type III systems, they all utilize a conserved set of 9 to 11 proteins that together form a bacterial envelope-associated secretory organelle or needle complex. The general structure of the needle complex consists of a transenvelope base containing at least three ring-forming proteins (MxiD, MxiJ, and MxiG in
Shigella
) that is connected to a hollow needle-like extension that projects away from the cell surface. Several studies have shown that the initial steps in needle complex assembly require interactions among the base proteins, although specific details of this process remain unknown. Here we identify a role for another base element in
Shigella
, MxiM, in interactions with the major outer-membrane-associated ring-forming protein, MxiD. MxiM affects several features of MxiD, including its stability, envelope association, and assembly into homomultimeric structures. Interestingly, many of the effects were also elicited by the inner-membrane-associated base element, MxiJ. We confirmed that MxiM-MxiD and MxiJ-MxiD interactions occur in vivo in the cell envelope, and we present evidence that together these base elements can form a transmembrane structure which is likely an important intermediary in the process of needle complex assembly.
Evidence of Multiple Regulatory Functions for the PtsN (IIA
Ntr
) Protein of
Pseudomonas putida
Authors: Cases Ildefonso , Lopez Juan-Antonio , Albar Juan-Pablo , De Lorenzo Vı́ctor
Published: July 2002

The
ptsN
gene of
Pseudomonas putida
encodes IIA
Ntr
, a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the ς
54
-dependent promoter
Pu
of the TOL (toluate degradation) plasmid pWW0. Using two-dimensional gel electrophoresis, we have examined the effect of
ptsN
disruption on the general expression pattern of
P. putida
. To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the
P. putida
proteome was used as a reference for comparisons. Among all gene products whose expression was lowered by this carbon source (247 spots [about 22%]), only 6 behaved as
Pu
(i.e., were depressed in the
ptsN
background). This evidenced only a minor role for IIA
Ntr
in the extensive inhibition of gene expression in
P. putida
caused by glucose. However, the same experiments revealed a large incidence of glucose-independent effects brought about by the
ptsN
mutation. As many as 108 spots (ca. 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIA
Ntr
. By matching this pattern with that of an
rpoN
::ΩKm strain of
P. putida
, which lacks the ς
54
protein, we judge that most proteins whose expression was affected by
ptsN
were unrelated to the alternative sigma factor. These data suggest a role of IIA
Ntr
as a general regulator, independent of the presence of repressive carbon sources and not limited to ς
54
-dependent genes.
Suppression of Hypersensitivity of
Escherichia coli acrB
Mutant to Organic Solvents by Integrational Activation of the
acrEF
Operon with the IS
1
or IS
2
Element
Authors: Kobayashi Kei , Tsukagoshi Norihiko , Aono Rikizo
Published: July 2002

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in
Escherichia coli. E. coli
OST5500 is hypersensitive to solvents due to inactivation of the
acrB
gene by insertion of IS
30
. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS
1
or IS
2
) was found integrated upstream of the
acrEF
operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the
acrEF
operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the
acrEF
operon with IS
1
or IS
2
integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated
acrEF
, resulting in functional complementation of the
acrB
mutation. The
acrB
mutation was also complemented by a plasmid containing
acrF
or
acrEF
under the control of P
lac
. The wild-type
tolC
gene was found to be essential for complementation of the
acrB
mutation by
acrEF
. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.
Bacteriophage SP6 Is Closely Related to Phages K1-5, K5, and K1E but Encodes a Tail Protein Very Similar to That of the Distantly Related P22
Authors: Scholl Dean , Adhya Sankar , Merril Carl R.
Published: July 2002

The lytic salmonella phage SP6 encodes a tail protein with a high degree of sequence similarity to the tail protein of the biologically unrelated lysogenic salmonella phage P22. The SP6 tail gene is flanked by an upstream region that contains a promoter and a downstream region that contains a putative Rho-independent transcription terminator, giving it a cassette or modular structure almost identical to the structure of the tail genes of coliphages K1E, K5, and K1-5. It now appears that SP6, K1-5, K5, and K1E are very closely related but have different tail fiber proteins, giving them different host specificities.
Identification by Flagellum Display of an Epithelial Cell- and Fibronectin-Binding Function in the SlpA Surface Protein of
Lactobacillus brevis
Authors: Hynönen Ulla , Westerlund-Wikström Benita , Palva Airi , Korhonen Timo K.
Published: July 2002

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of
Lactobacillus brevis
ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the
slpA
gene were expressed as internal in-frame fusions in the variable region of the
fliC
H7
gene of
Escherichia coli
. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of
L. brevis
ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.
Osmoregulation of Dimer Resolution at the Plasmid pJHCMW1
mwr
Locus by
Escherichia coli
XerCD Recombination
Authors: Pham Huong , Dery Ken J. , Sherratt David J. , Tolmasky Marcelo E.
Published: July 2002

Xer-mediated dimer resolution at the
mwr
site of plasmid pJHCMW1 is osmoregulated in
Escherichia coli
. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the
E. coli
ARG box consensus. The central region of the
mwr
core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.
Bursitis Due to
Mycobacterium goodii
, a Recently Described, Rapidly Growing Mycobacterium
Authors: Friedman N. Deborah , Sexton Daniel J.
Published: July 2002

We report a case of olecranon bursitis due to
Mycobacterium goodii
in a 60-year-old man. Prior to recognition of his infection, he received intrabursal steroids and underwent olecranon bursectomy. His infection was cured with antimicrobial therapy consisting of doxycycline and ciprofloxacin. This case illustrates that previously unrecognized members of the
Mycobacterium smegmatis
group of mycobacteria have pathogenic potential.
Quantification of
Candida albicans
Actin mRNA by the LightCycler System as a Means of Assessing Viability in a Model of Cutaneous Candidiasis
Authors: Okeke Charles N. , Tsuboi Ryoji , Ogawa Hideoki
Published: July 2002

The LightCycler system (two-step reverse transcription-PCR–fluorescent hybridization [LC RT-PCR–FH]) was used to quantify
Candida albicans
actin mRNA as a means of assessing its viability in a reconstituted skin model of cutaneous candidiasis following the application of an antimycotic. A 192-bp
ACT
exon fragment was ligated into the pCR2.1 plasmid vector, and dilutions of the cloned insert (pACT; 4.092 kb) were used as the standard reference template. The LC RT-PCR–FH system could detect 1 fg of pACT, equivalent to 2.2 copies of the plasmid. The
ACT
exon-based PCR primers and FH probes were
C. albicans
specific, and electrophoretic analysis of the LC RT-PCR–FH assay product showed a 174-bp band in agarose gel. The number of copies of
C. albicans ACT
mRNA per milligram of tissue decreased with increasing amounts of amorolfine applied to a
C. albicans
-infected skin model, showing a reduction in viability. Detection and quantification of
ACT
mRNA in tissue by the LC RT-PCR–FH assay corresponded with cultural isolation of
C. albicans
from samples. The
ACT
mRNA-targeted LC RT-PCR–FH assay represents a sensitive, specific, rapid, and quantitative means of assessing the viability of
C. albicans
in infected tissue. This method may also be useful in evaluating the therapeutic efficacies of antifungal drugs in the treatment of various forms of candidiasis and other fungal diseases.
Detection of Antibodies to a Disease-Associated Herpesvirus of the Green Turtle,Chelonia mydas
Authors: Coberley Sadie S. , Herbst Lawrence H. , Brown Daniel R. , Ehrhart Llewellyn M. , Bagley Dean A. , Schaf Susan A. , Moretti Ritchie H. , Jacobson Elliott R. , Klein Paul A.
Published: July 2002

Lung-eye-trachea disease-associated herpesvirus (LETV) is linked with morbidity and mortality in mariculture-reared green turtles, but its prevalence among and impact on wild marine turtle populations is unknown. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of anti-LETV antibodies and could distinguish LETV-exposed green turtles from those with antibodies to fibropapillomatosis-associated herpesvirus (FPHV). Plasma from two captive-reared green turtles immunized with inactivated LETV served as positive controls. Plasma from 42 healthy captive-reared green turtles and plasma from 30 captive-reared green turtles with experimentally induced fibropapillomatosis (FP) and anti-FPHV antibodies had low ELISA values on LETV antigen. A survey of 19 wild green turtles with and 27 without FP (with and without anti-FPHV antibodies, respectively) identified individuals with antibodies to LETV regardless of their FP status. The seroprevalence of LETV infection was 13%. The presence of antibodies to LETV in plasma samples was confirmed by Western blot and immunohistochemical analyses. These results are the first to suggest that wild Florida green turtles are exposed to LETV or to an antigenically closely related herpesvirus(es) other than FPHV and that FPHV and LETV infections are most likely independent events. This is the first ELISA developed to detect antibodies for a specific herpesvirus infection of marine turtles. The specificity of this ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting exposure to this specific herpesvirus and enhances our ability to conduct seroepidemiological studies of these disease-associated agents in marine turtles.
Ehrlichia ruminantium
Major Antigenic Protein Gene (
map1
) Variants Are Not Geographically Constrained and Show No Evidence of Having Evolved under Positive Selection Pressure
Authors: Allsopp M. T. E. P. , Dorfling C. M. , Maillard J. C. , Bensaid A. , Haydon D. T. , van Heerden H. , Allsopp B. A.
Published: July 2002

In a search for tools to distinguish antigenic variants of
Ehrlichia ruminantium
, we sequenced the major antigenic protein genes (
map1
genes) of 21 different isolates and found that the sequence polymorphisms were too great to permit the design of probes which could be used as markers for immunogenicity. Phylogenetic comparison of the 21 deduced MAP1 sequences plus another 9 sequences which had been previously published did not reveal any geographic clustering among the isolates. Maximum likelihood analysis of codon and amino acid changes over the phylogeny provided no statistical evidence that the gene is under positive selection pressure, suggesting that it may not be important for the evasion of host immune responses.
Colonic Infection by
Bilophila wadsworthia
in Pigs
Authors: McOrist Alexandra L. , Warhurst Michelle , McOrist Steven , Bird Anthony R.
Published: July 2002

Bilophila wadsworthia
is a common inhabitant of the human colon and has been associated with appendicitis and other local sites of inflammation in humans. Challenge-exposure or prevalence studies in laboratory and other animals have not been reported.
B. wadsworthia
is closely related phylogenetically to
Desulfovibrio
sp. and
Lawsonia intracellularis
, which are considered colon pathogens. We developed a PCR specific for
B. wadsworthia
DNA. Samples of bacterial DNA extracted from the feces of pigs on six farms in Australia and four farms in Venezuela were examined. Specific DNA of
B. wadsworthia
was detected in the feces of 58 of 161 Australian and 2 of 45 Venezuelan pigs, results comprising 100% of the neonatal pigs, 15% of the weaned grower pigs, and 27% of the adult sows tested. Single-stranded conformational polymorphism analysis of PCR product DNA derived from pigs or from known human strains showed an identical pattern. Histologic examination of the intestines of weaned
B. wadsworthia
-positive pigs found no or minor specific lesions in the small and large intestines, respectively.
B. wadsworthia
is apparently a common infection in neonatal pigs, but its prevalence decreases after weaning. The possible role of
B. wadsworthia
as an infection in animals and in human colons requires further study.