bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal Expression of the
Candida albicans
Genes
CHK1
and
CSSK1
, Adherence, and Morphogenesis in a Model of Reconstituted Human Esophageal Epithelial Candidiasis
Authors: Li Dongmei , Bernhardt Joern , Calderone Richard
Published: July 2002

We previously demonstrated that genes encoding a putative two-component histidine kinase (
CHK1
) or a response regulator (
CSSK1
) are each required for virulence in a murine model of hematogenously disseminated candidiasis and that strains with each gene deleted are also defective in morphogenesis under certain growth conditions. In the present study, the role of these two genes in the adherence to and colonization of reconstituted human esophageal tissue (RHE) is described. We compared strains of
Candida albicans
with deletions of
chk1
(strain CHK21) and
cssk1
(strain CSSK21) to wild-type cells (CAF2), as well as strains with
CHK1
and
CSSK1
reconstituted (strains CHK23 and CSSK23, respectively). Adherence and colonization of RHE were evaluated in periodic acid-Schiff-stained sections, as well as by SEM. We observed that both deletion-containing strains colonized the RHE to a lesser extent than did CAF2 and that the percent germination by both strains was reduced in comparison to that of control strains at 1 h postinfection. Expression of
CHK1
or
CSSK1
was quantitated by reverse transcription (RT)-PCR from RHE tissues infected with wild-type
C. albicans
yeast cells. Expression of both
CHK1
and
CSSK1
increased over the 48-h period following infection of the tissue, although expression of
CHK1
was greater than that of
CSSK1
. By RT-PCR, we have also shown that expression of
CHK1
and
CSSK1
in the strains with
cssk1
and
chk1
deleted, respectively, was similar to that of CAF2, indicating that
CHK1
and
CSSK1
do not regulate each other but probably encode signal proteins of different pathways. Our observations indicate that
CHK1
and
CSSK1
are each partially required for colonization and conversion to filamentous growth on RHE tissue.
Entamoeba histolytica
-Induced Dephosphorylation in Host Cells
Authors: Teixeira José E. , Mann Barbara J.
Published: July 2002

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting
Entamoeba histolytica
-mediated host cell killing, we investigated the involvement of PTPases during the attachment of
E. histolytica
to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to
E. histolytica
. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by
E. histolytica
. These results suggest that
E. histolytica
-mediated host cell death occurs by a mechanism that involves PTPase activation.
Clonal Polymorphism of
Borrelia burgdorferi
Strain B31 MI: Implications for Mutagenesis in an Infectious Strain Background
Authors: Elias Abdallah F. , Stewart Philip E. , Grimm Dorothee , Caimano Melissa J. , Eggers Christian H. , Tilly Kit , Bono James L. , Akins Darrin R. , Radolf Justin D. , Schwan Tom G. , Rosa Patricia
Published: July 2002

A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of
Borrelia burgdorferi
has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious
B. burgdorferi
strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious
B. burgdorferi
, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.
Pseudomonas aeruginosa
ExoT Acts In Vivo as a GTPase-Activating Protein for RhoA, Rac1, and Cdc42
Authors: Kazmierczak B. I. , Engel J. N.
Published: July 2002

The
Pseudomonas aeruginosa
protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases. Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells. We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases. GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other
P. aeruginosa
type III secreted proteins. We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.
Membrane Localization Contributes to the In Vivo ADP-Ribosylation of Ras by
Pseudomonas aeruginosa
ExoS
Authors: Riese Matthew J. , Barbieri Joseph T.
Published: July 2002

Type III-delivered exoenzyme S (ExoS) preferentially ADP-ribosylated membrane-associated His
6
HRas, relative to its cytosolic derivative His
6
HRasΔCAAX. This indicates that the subcellular protein distribution contributes to in vivo ADP-ribosylation by ExoS.
Transcription of Clumping Factor A in Attached and Unattached
Staphylococcus aureus
In Vitro and during Device-Related Infection
Authors: Wolz Christiane , Goerke Christiane , Landmann Regine , Zimmerli Werner , Fluckiger Ursula
Published: July 2002

Staphylococcus aureus
is one of the pathogens most frequently isolated in device-related infections.
S. aureus
is equipped with surface-associated proteins promoting specific binding to matrix molecules. Clumping factor A (ClfA, encoded by
clfA
) mediates adhesion to fibrinogen. Whereas the contribution of ClfA to pathogenicity is well documented, the influence of different growth and host parameters on gene activity is unclear. To elucidate this question, we investigated
clfA
transcript levels in an animal model of device-related infection and in planktonic and sessile bacteria grown in vitro. Specific mRNA from the
S. aureus
strains Newman, Reynolds, and RN6390 was quantified by LightCycler reverse transcription-PCR. In vitro,
clfA
transcript levels were low in the early logarithmic growth phase, but a clear increase was observed after the late logarithmic phase. Quantities of
clfA
transcripts were four to six times higher in the planktonic than in the sessile bacterial subpopulations grown to the stationary phase. During infection, in strains Newman and Reynolds levels of
clfA
transcripts in exudates accumulating in the infected devices were lower than those in the bacteria grown in vitro to stationary phase.
clfA
mRNA levels in the exudates increased during the initial phase of infection and remained constant after 96 h postinoculation. In contrast to the in vitro results, quantities of
clfA
transcripts in the unattached bacteria of the exudates never exceeded the level of
clfA
transcripts in the sessile bacteria attached to glass beads. However, a clear increase in
clfA
quantities in the sessile bacteria was observed late in infection after 144 h. In conclusion, maximal
clfA
transcript levels are reached late during growth in vitro and in vivo.
Correlation of ESAT-6-Specific Gamma Interferon Production with Pathology in Cattle followingMycobacterium bovisBCG Vaccination against Experimental Bovine Tuberculosis
Authors: Vordermeier H. Martin , Chambers Mark A. , Cockle Paul J. , Whelan Adam O. , Simmons Jennifer , Hewinson R. Glyn
Published: July 2002

Vaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated withMycobacterium bovisbacillus Calmette-Guérin (BCG) and were then challenged with virulentM. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses followingM. bovischallenge demonstrated that proliferative T-cell and gamma interferon (IFN-γ) responses towards theM. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-γ measured by enzyme-linked immunosorbent assay afterM. bovischallenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-γ, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.
Differential Activation of the
tcpPH
Promoter by AphB Determines Biotype Specificity of Virulence Gene Expression in
Vibrio cholerae
Authors: Kovacikova Gabriela , Skorupski Karen
Published: July 2002

Vibrio cholerae
strains of the classical biotype express the genes encoding cholera toxin (CT) and toxin-coregulated pilus (TCP) under a variety of environmental conditions in vitro, whereas El Tor biotype strains express these genes only under specialized culture conditions. We show here that a single base-pair difference at positions −65 and −66 of the classical and El Tor
tcpPH
promoters, respectively, is responsible for the differential regulation of virulence gene expression in these two disease-causing biotypes. Analysis of
tcpP-lacZ
fusions in both
V. cholerae
and
Escherichia coli
indicated that transcriptional activation of the El Tor
tcpPH
promoter by the LysR regulator AphB was significantly reduced relative to that of the classical promoter. Reciprocal exchange of the
tcpPH
promoter between the two biotypes in
V. cholerae
showed that the ability to activate the transcription of
tcpPH
is not dependent on the biotype of the strain per se but on the
tcpPH
promoter itself. Classical and El Tor
tcpP-lacZ
promoter chimeras in
E. coli
localized the region responsible for the differential activation of
tcpPH
by AphB to within 75 bp of the transcriptional start site. Individual base-pair changes within this region showed that the presence of either an A or a G at position −65 or −66 conferred the classical or El Tor, respectively, pattern of
tcpPH
activation by AphB. Reciprocal exchange of these base pairs between biotypes in
V. cholerae
switched the biotype-specific pattern of expression of
tcpPH
as well as the production of CT and TCP in response to environmental stimuli.
Exopolysaccharide Production Is Required for Development of
Escherichia coli
K-12 Biofilm Architecture
Authors: Danese Paul N. , Pratt Leslie A. , Kolter Roberto
Published: July 2002

Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of
Escherichia coli
K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of
E. coli
biofilms.
Identification of
Escherichia coli ubiB
, a Gene Required for the First Monooxygenase Step in Ubiquinone Biosynthesis
Authors: Poon Wayne W. , Davis Diana E. , Ha Huan T. , Jonassen Tanya , Rather Philip N. , Clarke Catherine F.
Published: July 2002

It was recently discovered that the
aarF
gene in
Providencia stuartii
is required for coenzyme Q (CoQ) biosynthesis. Here we report that
yigR
, the
Escherichia coli
homologue of
aarF
, is
ubiB
, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the
P. stuartii aarF
and
E. coli ubiB
(
yigR
) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the
E. coli
strain AN59, which contains the
ubiB409
mutant allele. Analysis of the mutation in the
E. coli
strain AN59 reveals no mutations within the
ubiB
gene, but instead shows the presence of an IS
1
element at position +516 of the
ubiE
gene. The
ubiE
gene encodes a
C
-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5′ gene in an operon containing
ubiE
,
yigP
, and
ubiB
. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the
ubiE
::IS
1
mutation on the downstream
ubiB
gene. AN59 is complemented by a DNA segment containing the contiguous
ubiE
,
yigP
, and
ubiB
genes. Although transformation of AN59 with a DNA segment containing the
ubiB
coding region fails to restore CoQ biosynthesis, transformation with the
ubiE
coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the
fre
gene, previously considered to correspond to
ubiB
, was found not to be involved in CoQ biosynthesis. The
ubiB
gene is a member of a predicted protein kinase family of which the
Saccharomyces cerevisiae ABC1
gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.
Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase
Authors: Smith Kerry S. , Cosper Nathaniel J. , Stalhandske Christina , Scott Robert A. , Ferry James G.
Published: July 2002

The β-class carbonic anhydrase from the archaeon
Methanobacterium thermoautotrophicum
(Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the
Spinacia oleracea
(spinach) β-class carbonic anhydrase indicate that the secondary structure of the β-class enzymes is predominantly α-helical, unlike that of the α- or γ-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters
k
cat
and
k
cat
/

K
m

for CO
2
hydration are pH dependent. The steady-state parameter
k
cat
is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5,
k
cat
is 2.1-fold higher in H
2
O than in D
2
O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter
k
cat
/

K
m

is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic β-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic β-class enzymes and firmly establish that the α-, β-, and γ-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.
Identification of Genes in the RosR Regulon of
Rhizobium etli
Authors: Bittinger Mark A. , Handelsman Jo
Published: July 2002

RosR is a determinant of nodulation competitiveness and cell surface characteristics of
Rhizobium etli
and has sequence similarity to a family of transcriptional repressors. To understand how RosR affects these phenotypes, we mutagenized a
rosR
mutant derivative of
R. etli
strain CE3 with a mini-Tn
5
that contains a promoterless
gusA
gene at one end, which acts as a transcriptional reporter. Using a mass-mating technique, we introduced
rosR
into each mutant in
trans
and screened for mutants that expressed different levels of β-glucuronidase activity in the presence and absence of
rosR
. A screen of 18,000 mutants identified 52 insertions in genes negatively regulated by RosR and 1 insertion in a gene positively regulated by RosR. Nucleotide sequence analysis of the regions flanking the insertions suggests that RosR regulates genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism and those in a region containing sequence similarity to
virC1
and
virD3
from
Agrobacterium tumefaciens
. Two of the mutants produced colonies with altered morphology and were more competitive in nodulation than was CE3Δ
rosR
, the
rosR
parent. One mutant that contained an insertion in a gene with similarity to
exsH
of
Sinorhizobium meliloti
did not nodulate the plant host
Phaseolus vulgaris
without
rosR
. These results indicate that RosR directly or indirectly influences expression of diverse genes in
R. etli
, some of which affect the cell surface and nodulation competitiveness.
Pyruvate Kinase of the Hyperthermophilic Crenarchaeote
Thermoproteus tenax
: Physiological Role and Phylogenetic Aspects
Authors: Schramm Alexander , Siebers Bettina , Tjaden Britta , Brinkmann Henner , Hensel Reinhard
Published: July 2002

Pyruvate kinase (PK; EC
2.7.1.40
) of
Thermoproteus tenax
was purified to homogeneity, and its coding gene was cloned and expressed in
Escherichia coli
. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg
2+
and Mn
2+
. Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K
+
ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the
T. tenax
-specific variant of the Embden-Meyerhof-Parnas pathway.
T. tenax
PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in
Archaea
, show activity without effectors but are commonly regulated by the energy charge of the cell.
Role of Ger Proteins in Nutrient and Nonnutrient Triggering of Spore Germination in
Bacillus subtilis
Authors: Paidhungat Madan , Setlow Peter
Published: July 2002

Dormant
Bacillus subtilis
spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the
gerA
family of operons, which includes
gerA
,
gerB
, and
gerK
. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known
gerA
-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca
2+
and dipicolinic acid (DPA). These observations showed that proteins encoded by
gerA
family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca
2+
–DPA-induced germination showed that the effect of Ca
2+
–DPA on spore germination was saturated at 60 mM and had a

K
m

of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca
2+
–DPA but not in nutrient germinants, indicating that Ca
2+
–DPA and nutrient germinants probably act through parallel arms of the germination pathway.
Activation by Gene Amplification of
pitB
, Encoding a Third Phosphate Transporter of
Escherichia coli
K-12
Authors: Hoffer Sally M. , Schoondermark Paul , van Veen Hendrik W. , Tommassen Jan
Published: July 2002

Two systems for the uptake of inorganic phosphate (P
i
) in
Escherichia coli
, PitA and Pst, have been described. A revertant of a
pitA pstS
double mutant that could grow on P
i
was isolated. We demonstrate that the expression of a new P
i
transporter, PitB, is activated in this strain by a gene amplification event.
Dual Repression by Fe
2+
-Fur and Mn
2+
-MntR of the
mntH
Gene, Encoding an NRAMP-Like Mn
2+
Transporter in
Escherichia coli
Authors: Patzer Silke I. , Hantke Klaus
Published: July 2002

The uptake of Mn
2+
, a cofactor for several enzymes in
Escherichia coli
, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe
2+
with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe
2+
and Mn
2+
transporters. In
E. coli
strains with chromosomal
mntH-lacZ
fusions,
mntH
was partially repressed by both Mn
2+
and Fe
2+
. Inactivation of
fur
resulted in the loss of Fe
2+
-dependent repression of
mntH
transcription, demonstrating that Fe
2+
repression depends on the global iron regulator Fur. However, these
fur
mutants still showed Mn
2+
-dependent repression of
mntH
. The Mn
2+
-responsive transcriptional regulator of
mntH
was identified as the gene product of
o155
(renamed MntR).
mntR
mutants were impaired in Mn
2+
but not Fe
2+
repression of
mntH
transcription. Binding of purified MntR to the
mntH
operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the
mntH
operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.
pbpB
, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium
Anabaena
sp. Strain PCC7120
Authors: Lázaro Sara , Fernández-Piñas Francisca , Fernández-Valiente Eduardo , Blanco-Rivero Amaya , Leganés Francisco
Published: July 2002

Transposon mutagenesis of
Anabaena
sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced
Anabaena
sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the
Escherichia coli
nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as
pbpB
and its product as PBP2. The wild-type form of
pbpB
on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N
2
, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.
Hydrogen Peroxide Fluxes and Compartmentalization inside Growing
Escherichia coli
Authors: Seaver Lauren Costa , Imlay James A.
Published: July 2002

Escherichia coli
generates about 14 μM hydrogen peroxide (H
2
O
2
) per s when it grows exponentially in glucose medium. The steady-state intracellular concentration of H
2
O
2
depends on the rates at which this H
2
O
2
is dissipated by scavenging enzymes and by efflux from the cell. The rates of H
2
O
2
degradation by the two major scavenging enzymes, alkyl hydroperoxide reductase and catalase, were quantified. In order to estimate the rate of efflux, the permeability coefficient of membranes for H
2
O
2
was determined. The coefficient is 1.6 × 10
−3
cm/s, indicating that permeability is substantial but not unlimited. These data allowed internal H
2
O
2
fluxes and concentrations to be calculated. Under these growth conditions, Ahp scavenges the majority of the endogenous H
2
O
2
, with a small fraction degraded by catalase and virtually none persisting long enough to penetrate the membrane and exit the cell. The robust scavenging activity maintains the H
2
O
2
concentration inside glucose-grown cells at <10
−7
M, substantially below the level (10
−6
M) at which toxicity is evident. When extracellular H
2
O
2
is present, its flux into the cell can be rapid, but the internal concentration may still be an order of magnitude lower than that outside. The presence of such gradients was confirmed in experiments that revealed different degrees of oxidative stress in cocultured scavenger-deficient mutants. The limited permeability of membranes to H
2
O
2
rationalizes the compartmentalization of scavenging systems and predicts that bacteria that excrete redox-cycling drugs do not experience the same H
2
O
2
dose that they impose on their competitors.
PatS and Products of Nitrogen Fixation Control Heterocyst Pattern
Authors: Yoon Ho-Sung , Golden James W.
Published: July 2002

The filamentous cyanobacterium
Anabaena
sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The
patS
gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of
patS
expression and heterocyst pattern formation. A
patS
-
gfp
reporter strain revealed clusters of
patS
-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of
patS
during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5′ end of a transcript for
patS
was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5′ end at −39 bases replaced the vegetative cell transcript. A
patS
mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The
patS
mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.
An Iron-Binding Protein, Dpr, fromStreptococcus mutansPrevents Iron-Dependent Hydroxyl Radical Formation In Vitro
Authors: Yamamoto Yuji , Poole Leslie B. , Hantgan Roy R. , Kamio Yoshiyuki
Published: July 2002

Thedprgene is an antioxidant gene which was isolated from theStreptococcus mutanschromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant ofEscherichia coli, and it was proven to play an indispensable role in oxygen tolerance inS. mutans. Here, we purified the 20-kDadprgene product, Dpr, from a crude extract ofS. mutansas an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. UnlikeE. coliDps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 μM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein inS. mutansand may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.
Biosynthesis Pathway of ADP-
l
-
glycero
-β-
d
-
manno
-Heptose in
Escherichia coli
Authors: Kneidinger Bernd , Marolda Cristina , Graninger Michael , Zamyatina Alla , McArthur Fiona , Kosma Paul , Valvano Miguel A. , Messner Paul
Published: July 2002

The steps involved in the biosynthesis of the ADP-
l
-
glycero
-β-
d
-
manno-
heptose (ADP-
l
-β-
d
-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-
d
-β-
d
-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene,
gmhB
(formerly
yaeD
), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-
d
-β-
d
-heptose. Our results demonstrate that the synthesis of ADP-
d
-β-
d
-heptose in
Escherichia coli
requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional
d
-β-
d
-heptose 7-phosphate kinase/
d
-β-
d
-heptose 1-phosphate adenylyltransferase), and GmhB (
d
,
d
-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-
l
-β-
d
-heptose precursor utilized in the assembly of inner core LPS.
Characterization of an ADP-Ribosyltransferase Toxin (AexT) from
Aeromonas salmonicida
subsp.
salmonicida
Authors: Braun Martin , Stuber Katja , Schlatter Yvonne , Wahli Thomas , Kuhnert Peter , Frey Joachim
Published: July 2002

An ADP-ribosylating toxin named
Aeromonas salmonicida
exoenzyme T (AexT) in
A. salmonicida
subsp.
salmonicida
, the etiological agent of furunculosis in fish, was characterized. Gene
aexT
, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of
Pseudomonas aeruginosa
and to the YopE cytotoxin of different
Yersinia
species. The
aexT
gene was detected in all of the 12
A. salmonicida
subsp.
salmonicida
strains tested but was absent from all other
Aeromonas
species. Recombinant AexT produced in
Escherichia coli
possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by
A. salmonicida
subsp.
salmonicida
and cross-reacted with ExoS and ExoT of
P. aeruginosa
. AexT toxin could be detected in a wild type (wt) strain of
A. salmonicida
subsp.
salmonicida
freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when
A. salmonicida
subsp.
salmonicida
was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt
A. salmonicida
subsp.
salmonicida
, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An
aexT
mutant which was constructed from the wt strain with an insertionally inactivated
aexT
gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of
A. salmonicida
subsp.
salmonicida
for RTG-2 fish cells.
Immunodiagnosis of
Ehrlichia canis
Infection with Recombinant Proteins
Authors: McBride Jere W. , Corstvet Richard E. , Breitschwerdt Edward B. , Walker David H.
Published: July 2002

Ehrlichia canis
causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive
E. canis
proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive
E. canis
surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in
E. chaffeensis
DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with
E. chaffeensis
as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for
E. canis
, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for
E. canis
infections.
Genotyping
Encephalitozoon cuniculi
by Multilocus Analyses of Genes with Repetitive Sequences
Authors: Xiao Lihua , Li Lixia , Visvesvara Govinda S. , Moura Hercules , Didier Elizabeth S. , Lal Altaf A.
Published: July 2002

Encephalitozoon cuniculi
infects a wide range of mammalian hosts. Three genotypes based on the number of GTTT repeats in the internal transcribed spacer (ITS) of the rRNA have been described, of which genotypes I and III have been identified in humans. In this study, the genetic diversity of
E. cuniculi
was examined at the polar tube protein (PTP) and spore wall protein I (SWP-1) loci. Nucleotide sequence analysis of the PTP gene divided 11
E. cuniculi
isolates into three genotypes in congruence with the result of analysis of the ITS of the rRNA gene. The three PTP genotypes differed from one another by the copy number of the 78-bp central repeat as well as point mutations. These
E. cuniculi
isolates also differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some
E. cuniculi
isolates had heterogeneous copies of the SWP-1 gene with various numbers of repeats. Intragenotypic variation was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate
E. cuniculi
in clinical samples.
Change of Major Genotype of Enterovirus 71 in Outbreaks of Hand-Foot-and-Mouth Disease in Taiwan between 1998 and 2000
Authors: Wang Jen-Ren , Tuan Yen-Chang , Tsai Huey-Pin , Yan Jing-Jou , Liu Ching-Chuan , Su Ih-Jen
Published: July 2002

Two outbreaks of hand-foot-and-mouth disease (HFMD) occurred in Taiwan between 1998 and 2000. Enteroviruses were isolated from a total of 1,892 patients in this laboratory during this period. Of the virus isolates, enterovirus 71 (EV71) was diagnosed in 44.4% of the patients (132 of 297) in 1998, 2% (13 of 646) in 1999, and 20.5% (195 of 949) in 2000. Genetic analyses of the 5′-untranslated and VP1 regions of EV71 isolates by reverse transcription-PCR and sequencing were performed to understand the diversity of EV71 in these outbreaks of HFMD. Most EV71 isolates from the 1998 epidemic belonged to genotype C, while only one-tenth of the isolates were genotype B. Interestingly, all EV71 isolates tested from 1999 to 2000 belonged to genotype B. This study indicated that two genogroups of EV71 capable of inducing severe clinical illness have been circulating in Taiwan. Furthermore, the predominant EV71 genotypes responsible for each of the two major HFMD outbreaks within the 3-year period in Taiwan were different.
Carriage Frequency, Intensity of Carriage, and Strains of Oral Yeast Species Vary in the Progression to Oral Candidiasis in Human Immunodeficiency Virus-Positive Individuals
Authors: Vargas Kaaren G. , Joly Sophie
Published: July 2002

Candidasamples were taken over a period of 2 years from 54 human immunodeficiency virus (HIV)-positive asymptomatic subjects to evaluate changes in yeast carriage, intensity of carriage, and genotype over time. Overall, we found that HIV-positive patients with CD4+-cell counts of between 200 and 400/μl had significantly more yeast colonization than healthy control subjects. Of the 54 patients, 11 developed thrush. We found that intensity of carriage in these 11 patients increased significantly in the progression from asymptomatic yeast carrier to an episode of oral thrush. Also, the most common yeast species isolated wasCandidaalbicans; however, we did see a number of patients harboring multiple species at the same time. Using theC.albicans-specific probe Ca3, we found that 54% (n= 6) of the 11 patients who developed thrush maintained genetically similar strains throughout the study period, with minor genetic variations in all patients except one. Forty-six percent of these patients had either multiple strains throughout the study period (n= 2), strain replacement (n= 1), or species replacement (n= 2). Of the patients who had multiple strains, one (I4) was infected by two different strains ofCandidadubliniensisdistinguished by a recently developed species-specific probe. These results suggest that commensal strains colonizing HIV-positive individuals can undergo alterations prior to producing an episode of thrush.
Environmental Occurrence ofMadurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan
Authors: Ahmed Abdalla , Adelmann Daniel , Fahal Ahmed , Verbrugh Henri , van Belkum Alex , de Hoog Sybren
Published: July 2002

Madurella mycetomatisis the main causative agent of human eumycetoma, a severe debilitating disease endemic in Sudan. It has been suggested that eumycetoma has a soil-borne or thorn prick-mediated origin. For this reason, efforts were undertaken to cultureM. mycetomatisfrom soil samples (n= 43) and thorn collections (n= 35) derived from areas in which it is endemic. However, ribosomal sequencing data revealed that the black fungi obtained all belonged to other fungal species. In addition, we performed PCR-mediated detection followed by restriction fragment length polymorphism (RFLP) analysis for the identification ofM. mycetomatisDNA from the environmental samples as well as biopsies from patients with mycetoma. In the case of the Sudanese soil samples, 17 out of 74 (23%) samples were positive forM. mycetomatisDNA. Among the thorn collections, 1 out of 22 (5%) was positive in the PCR. All PCR RFLP patterns clearly indicated the presence ofM. mycetomatis. In contrast, 15 Dutch and English control soil samples were all negative. Clinically and environmentally obtained fungal PCR products share the same PCR RFLP patterns, suggesting identity, at least at the species level. These observations support the hypothesis that eumycetoma is primarily environmentally acquired and suggest thatM. mycetomatisneeds special conditions for growth, as direct isolation from the environment seems to be impossible.
Fatal Case of
Campylobacter lari
Prosthetic Joint Infection and Bacteremia in an Immunocompetent Patient
Authors: Werno Anja M. , Klena John D. , Shaw Geoffrey M. , Murdoch David R.
Published: July 2002

Campylobacter lari
is an infrequent cause of intestinal and extraintestinal infection in humans. We report a case of
C. lari
prosthetic joint infection and bacteremia in an 81-year-old immunocompetent man. The infection was associated with septic shock and fatal outcome.
C. lari
may cause severe disease, even in an immunocompetent host.
Relatively Alcohol-Resistant Mycobacteria Are Emerging Pathogens in Patients Receiving Acupuncture Treatment
Authors: Woo Patrick C. Y. , Leung Kit-Wah , Wong Samson S. Y. , Chong Ken T. K. , Cheung Elim Y. L. , Yuen Kwok-Yung
Published: July 2002

Acupuncture has been gaining popularity as a form of alternative medicine. In the past, only blood-borne viruses and anecdotal reports of bacterial infections have been associated with acupuncture. We report on four patients with mycobacterial infections complicating acupuncture who were encountered in a 2-year period. All had clinical and/or radiological lesions at acupuncture point- and meridian-specific locations. There was no other history of trauma or other clinical foci of infections, and the chest radiographs were normal. Histological studies of biopsy specimens of all four patients showed changes compatible with chronic inflammation, with granulomatous inflammation present in three patients and acid-fast bacilli present in two. Conventional biochemical tests and whole-cell fatty acid analysis for identification were inconclusive for all four nonpigmented mycobacteria recovered from tissue biopsies. 16S rRNA gene sequencing showed that the strains from two patients were
Mycobacterium chelonae
and that those from the other two were
Mycobacterium nonchromogenicum
. Alcohol resistance assay using the quantitative suspension test revealed that all four strains showed prolonged survival in 75% alcohol compared to other skin flora. Mycobacterial infections transmitted by acupuncture are an emerging problem. A high index of suspicion is essential to recognize this clinical syndrome, and strict implementation of proper infection control guidelines for acupuncture is mandatory.
Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat
A/D
Junction Element
Authors: Haberman Rebecca P. , McCown Thomas J. , Samulski Richard Jude
Published: July 2002

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the “off” state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other
cis
-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the
A/D
elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.