bluuebunny/crossref_metadata_2025 huggingface dataset:
The HilA Box and Sequences outside It Determine the Magnitude of HilA-Dependent Activation of P

prgH

from
Salmonella
Pathogenicity Island 1
Authors: Lostroh C. Phoebe , Lee Catherine A.
Published: July 2002

Salmonella
requires genes on the
Salmonella
pathogenicity island 1 (SPI1) for the intestinal phase of infection in several models of pathogenesis. In
Salmonella enterica
serovar Typhimurium, most SPI1 genes are arranged in operons that are coordinately regulated by the SPI1-encoded protein HilA. In the past, it has been shown that HilA directly activates two promoters on SPI1, P

invF-1

and P

prgH

. P

invF-1

contains a HilA binding site, termed a HilA box, that is necessary and sufficient for activation by HilA. The HilA box is 17 nucleotides long and contains a direct repeat comprised of two hexamers separated by 5 nucleotides, centered at −45 relative to the start site of transcription. P

prgH

also contains a HilA box, and here we investigate its role at P

prgH

. We have found that the HilA box is necessary, but not sufficient, for HilA-dependent activation of P

prgH

. Instead, half-site-like hexamers outside the HilA box appear to be required for HilA-dependent activation of P

prgH

, even though HilA binds to the HilA box in the absence of these hexamers. Thus, although HilA-dependent activation of P

invF-1

and P

prgH

coordinates the expression of the structural genes for a type III secretion apparatus and the effectors secreted by that apparatus, it is also possible that mechanisms not apparent under in vitro inducing conditions could separate the expression of
invFGEABC-spaMNOPQRS-sicA-sipBCDA-iacP-sicP-sptP
and
prgHIJK-orgABC
.
Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Sperandio Vanessa , Torres Alfredo G. , Girón Jorge A. , Kaper James B.
Published: July 2002

Enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the
luxS
gene. In this study we hybridized an
E. coli
gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic
luxS
mutant. We observed that 404 genes were regulated by
luxS
at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the
luxS
mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the
luxS
mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::
lacZ
fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the
luxS
mutant. The average swimming speeds for the wild-type strain and the
luxS
mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of
E. coli
as well as for virulence factors.
Selection and Characterization of
Escherichia coli
Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue
Authors: Bacher Jamie M. , Ellington Andrew D.
Published: July 2002

Escherichia coli
isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (
aroP
) and tryptophanyl-tRNA synthetase (
trpS
) contained several amino acid substitutions, and the tyrosine repressor (
tyrR
) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.
Identification of thenikGene Cluster ofBrucella suis: Regulation and Contribution to Urease Activity
Authors: Jubier-Maurin Véronique , Rodrigue Agnès , Ouahrani-Bettache Safia , Layssac Marion , Mandrand-Berthelot Marie-Andrée , Köhler Stephan , Liautard Jean-Pierre
Published: July 2002

Analysis of aBrucella suis1330 gene fused to agfpreporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous tonikA, the first gene of theEscherichia colioperon encoding the specific transport system for nickel. DNA sequence analysis of the correspondingB. suis niklocus showed that it was highly similar to that ofE. coliexcept for localization of thenikRregulatory gene, which lies upstream from the structuralnikABCDEgenes and in the opposite orientation. Protein sequence comparisons suggested that the deducednikABCDEgene products belong to a periplasmic binding protein-dependent transport system. ThenikApromoter-gfpfusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl2excess. Insertional inactivation ofnikAstrongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, thenikAmutant ofB. suiswas functionally complemented with theE. coli nikgene cluster, leading to the recovery of urease activity. Reciprocally, anE. colistrain harboring a deletednikoperon recovered hydrogenase activity by heterologous complementation with theB. suis niklocus. Taking into account these results, we propose that theniklocus ofB. suisencodes a nickel transport system. The results further suggest that nickel could enterB. suisvia other transport systems. Intracellular growth rates of theB. suiswild-type andnikAmutant strains in human monocytes were similar, indicating thatnikAwas not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.
Growth Phase and Growth Rate Regulation of the
rapA
Gene, Encoding the RNA Polymerase-Associated Protein RapA in
Escherichia coli
Authors: Cabrera Julio E. , Jin Ding Jun
Published: July 2002

The
Escherichia coli rapA
gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied the
rapA
promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of
rapA
is growth phase dependent, peaking at the early log phase. The growth phase control of
rapA
is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most
E. coli
promoters. We also found that the
rapA
promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the −10 and +1 positions of the
rapA
promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.
DNA as a Nutrient: Novel Role for Bacterial Competence Gene Homologs
Authors: Finkel Steven E. , Kolter Roberto
Published: July 2002

The uptake and stable maintenance of extracellular DNA, genetic transformation, is universally recognized as a major force in microbial evolution. We show here that extracellular DNA, both homospecific and heterospecific, can also serve as the sole source of carbon and energy supporting microbial growth. Mutants unable to consume DNA suffer a significant loss of fitness during stationary-phase competition. In
Escherichia coli
, the use of DNA as a nutrient depends on homologs of proteins involved in natural genetic competence and transformation in
Haemophilus influenzae
and
Neisseria gonorrhoeae
. Homologs of these
E. coli
genes are present in many members of the γ subclass of
Proteobacteria
, suggesting that the mechanisms for consumption of DNA may have been widely conserved during evolution.
Precise Deletion of
tagD
and Controlled Depletion of Its Product, Glycerol 3-Phosphate Cytidylyltransferase, Leads to Irregular Morphology and Lysis of
Bacillus subtilis
Grown at Physiological Temperature
Authors: Bhavsar Amit P. , Beveridge Terry J. , Brown Eric D.
Published: July 2002

Using a previously reported conditional expression system for use in
Bacillus subtilis
(A. P. Bhavsar, X. Zhao, and E. D. Brown, Appl. Environ. Microbiol. 67:403–410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (
tag
) gene,
tagD,
in
B. subtilis
. This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background. This
tagD
mutant was subject to full phenotypic rescue upon expression of the complementing copy of
tagD.
Depletion of the
tagD
gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of
tagD
from the
amyE
locus revealed structural defects centered on shape, septation, and division. Thickening of the wall and ultimately lysis followed these events.
Genetic Evidence for Parallel Pathways of Chaperone Activity in the Periplasm of
Escherichia coli
Authors: Rizzitello Amy E. , Harper Jill R. , Silhavy Thomas J.
Published: July 2002

The periplasm of
Escherichia coli
contains many proteins proposed to have redundant functions in protein folding. Using depletion analysis, we directly demonstrated that null mutations in
skp
and
surA,
as well as in
degP
and
surA,
result in synthetic phenotypes, suggesting that Skp, SurA, and DegP are functionally redundant. The Δ
skp surA
::
kan
combination has a bacteriostatic effect and leads to filamentation, while the
degP
::Tn
10 surA
::
kan
combination is bactericidal. The steady-state levels of several envelope proteins are greatly reduced upon depletion of a wild-type copy of
surA
in both instances. We suggest that the functional redundancy of Skp, SurA, and DegP lies in the periplasmic chaperone activity. Taken together, our data support a model in which the periplasm of
E. coli
contains parallel pathways for chaperone activity. In particular, we propose that Skp and DegP are components of the same pathway and that SurA is a component of a separate pathway. The loss of either pathway has minimal effects on the cell, while the loss of both pathways results in the synthetic phenotypes observed.
Biochemical Analysis of Replication Factor C from the Hyperthermophilic ArchaeonPyrococcus furiosus
Authors: Cann Isaac K. O. , Ishino Sonoko , Yuasa Mihoko , Daiyasu Hiromi , Toh Hiroyuki , Ishino Yoshizumi
Published: July 2002

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domainEucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases δ and ɛ, onto primed DNA templates. RFC-like genes, arranged in tandem in thePyrococcus furiosusgenome, were cloned and expressed individually inEscherichia colicells to determine their roles in DNA synthesis. TheP. furiosusRFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) andP. furiosusPCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II fromP. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved inArchaeaandEucarya.
Isogenic Strain Construction and Gene Targeting in
Candida dubliniensis
Authors: Staib Peter , Moran Gary P. , Sullivan Derek J. , Coleman David C. , Morschhäuser Joachim
Published: July 2002

Candida dubliniensis
is a recently described opportunistic fungal pathogen that is closely related to
Candida albicans
but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of
C. albicans
and
C. dubliniensis
at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to
C. albicans
, no auxotrophic
C. dubliniensis
strains are available for genetic manipulations. Therefore, we constructed homozygous
ura3
mutants from a
C. dubliniensis
wild-type isolate by targeted gene deletion. The two
URA3
alleles were sequentially inactivated using the

MPA
R

-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The
URA3
gene from
C. albicans
(
CaURA3
) was then used as a selection marker for targeted integration of a fusion between the
C. dubliniensis MDR1
(
CdMDR1
) promoter and a
C. albicans
-adapted
GFP
reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent
ura3
-negative parent strains had correctly integrated the reporter gene fusion into the
CdMDR1
locus, demonstrating that the
CaURA3
gene can be used for efficient and specific targeting of recombinant DNA into the
C. dubliniensis
genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that
CdMDR1
is not significantly expressed under these conditions. Fluconazole had no effect on
MDR1
expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the
CdMDR1
gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.
ATP-Binding Cassette Transport System Involved in Regulation of Morphological Differentiation in Response to Glucose in
Streptomyces griseus
Authors: Seo Jeong-Woo , Ohnishi Yasuo , Hirata Aiko , Horinouchi Sueharu
Published: July 2002

Streptomyces griseus
NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from “submerged spores” in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (
dasR
) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (
dasA
) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named
dasB
and
dasC
, encoding a membrane-spanning protein were present downstream of
dasA
, which suggested that
dasRABC
comprises a gene cluster for an ABC transporter, probably for sugar import.
dasR
was transcribed actively during vegetative growth, and
dasA
was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of
dasRA
in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of
dasA
into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal
dasA
or
dasR
in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of
dasA
were independent of a microbial hormone, A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.
The Molybdate-Responsive
Escherichia coli
ModE Transcriptional Regulator Coordinates Periplasmic Nitrate Reductase
(napFDAGHBC)
Operon Expression with Nitrate and Molybdate Availability
Authors: McNicholas Paul M. , Gunsalus Robert P.
Published: July 2002

Expression of the
Escherichia coli napFDAGHBC
operon (also known as
aeg46.5
), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a
napF-lacZ
operon fusion, we demonstrate that
napF
gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the
napF
promoter with an apparent

K
d

of about 35 nM at a position centered at −133.5 relative to the start of
napF
transcription. Although the ModE binding site sequence is similar to other
E. coli
ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a
modE
phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered
napF
expression independent of
modE.
These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent
napF
expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of
napF
gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.
Multiple Pathways of Spx (YjbD) Proteolysis inBacillus subtilis
Authors: Nakano Shunji , Zheng Guolu , Nakano Michiko M. , Zuber Peter
Published: July 2002

ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways. Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development ofBacillus subtilis. Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation. Thespx(formallyyjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred byclpPandclpX. The level of Spx in wild-type cells grown in competence medium is low, and that inclpPmutants is high. This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from theclpPmutation. In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx. Western blot analysis showed that Spx accumulated inclpXmutant to the same level as that observed in theclpPmutant. In contrast, a very low concentration of Spx was detected in aclpCmutant. An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH. However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx. Transcription ofspx, as measured by expression ofspx-lacZ, was slightly increased by theclpXmutation. To exclude a possible effect ofclpXandclpPonspxtranscription, thespxgene was placed under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible Pspacpromoter. In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx. Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP. The putative proteolysis by ClpXP might require another adapter protein. Spx probably is degraded by ClpCP under as yet unidentified conditions. This study suggests that the level of Spx is tightly controlled by two different ClpP proteases.
Genetic Variability of
Yersinia pestis
Isolates as Predicted by PCR-Based IS
100
Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (
glpD
)
Authors: Motin Vladimir L. , Georgescu Anca M. , Elliott Jeffrey M. , Hu Ping , Worsham Patricia L. , Ott Linda L. , Slezak Tomas R. , Sokhansanj Bahrad A. , Regala Warren M. , Brubaker Robert R. , Garcia Emilio
Published: July 2002

A PCR-based genotyping system that detects divergence of IS
100
locations within the
Yersinia pestis
genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS
100
and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in
Y. pestis
suggested that a 93-bp deletion within the
glpD
gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The
glpD
gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the
glpD
version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different
Yersinia
strains in nature. The relevance of this observation on the emergence of epidemic
Y. pestis
strains is discussed.
RecA Protein from the Extremely Radioresistant Bacterium
Deinococcus radiodurans
: Expression, Purification, and Characterization
Authors: Kim Jong-Il , Sharma Ajay K. , Abbott Stephen N. , Wood Elizabeth A. , Dwyer David W. , Jambura Aaron , Minton Kenneth W. , Inman Ross B. , Daly Michael J. , Cox Michael M.
Published: July 2002

The RecA protein of
Deinococcus radiodurans
(RecA
Dr
) is essential for the extreme radiation resistance of this organism. The RecA
Dr
protein has been cloned and expressed in
Escherichia coli
and purified from this host. In some respects, the RecA
Dr
protein and the
E. coli
RecA (RecA
Ec
) proteins are close functional homologues. RecA
Dr
forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA
Ec
. The RecA
Dr
protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the
E. coli
SSB protein. As is the case with the
E. coli
RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA
Dr
protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA
Dr
protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA
Dr
protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA
Dr
protein binds much better to duplex DNA than the RecA
Ec
protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in
Deinococcus
.
Protective Role of
tolC
in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate
Authors: Shyu J. Bruce H. , Lies Douglas P. , Newman Dianne K.
Published: July 2002

Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer. Here we provide genetic evidence that AQDS enters
Shewanella oneidensis
strain MR-1 and causes cell death if it accumulates past a critical concentration. A
tolC
homolog protects the cell from toxicity by mediating the efflux of AQDS. Electron transfer to AQDS appears to be independent of the
tolC
pathway, however, and requires the outer membrane protein encoded by
mtrB
. We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics.
Genetic Diversity of
Mycobacterium africanum
Clinical Isolates Based on IS
6110
-Restriction Fragment Length Polymorphism Analysis, Spoligotyping, and Variable Number of Tandem DNA Repeats
Authors: Viana-Niero Cristina , Gutierrez Cristina , Sola Christophe , Filliol Ingrid , Boulahbal Fadila , Vincent Véronique , Rastogi Nalin
Published: July 2002

A collection of 105 clinical isolates originally identified as
Mycobacterium africanum
were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the
M. tuberculosis
complex, whereas genotypic characterization was based on IS
6110
-restriction fragment length polymorphism (IS
6110
-RFLP) analysis, IS
1081
-RFLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyping), variable number of tandem DNA repeats (VNTR), and the polymorphism of the
oxyR
,
pncA
, and
mtp
40 loci. The results obtained showed that a majority of
M. africanum
isolates were characterized by a specific spoligotyping pattern that was intermediate between those of
M. tuberculosis
and
M. bovis
, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respectively. A tentative
M. africanum
-specific spoligotyping signature appeared to be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as the polymorphism of
oxyR
and
pncA
, a total of 24 isolates were excluded from the final study (19 were identified as
M. tuberculosis
, 2 were identified as
M. canetti
, and 3 were identified as
M. bovis
). The remaining 81
M. africanum
isolates were efficiently subtyped in three distinct subtypes (A1 to A3) by IS
6110
-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of the A2 subtype but showed significant variations for subtypes A1 and A3. A phylogenetic tree based on a selection of isolates representing the three subtypes using VNTR and spoligotyping alone or in combination confirmed the subtypes described as well as the heterogeneity of subtype A3.
Identification of Aichi Virus Infection by Measurement of Immunoglobulin Responses in an Enzyme-Linked Immunosorbent Assay
Authors: Yamashita Teruo , Ito Miyabi , Tsuzuki Hideaki , Sakae Kenji
Published: July 2002

Using inhibitory enzyme-linked immunosorbent assay, seroconversions to Aichi virus were detected in 24 (42.9%) of 56 patients with gastroenteritis in six outbreaks. Virus-specific immunoglobulin M (IgM) was detected in convalescent-phase sera from 7 of 24 patients. Of the other 17 patients, 12 developed a significant increase in both IgA and IgG levels and 5 developed a significant increase in IgG alone.
Molecular Characterization of New Clinical Isolates of
Candida albicans
and
C. dubliniensis
in Japan: Analysis Reveals a New Genotype of
C. albicans
with Group I Intron
Authors: Tamura Miki , Watanabe Kayo , Mikami Yuzuru , Yazawa Katsukiyo , Nishimura Kazuko
Published: July 2002

The genetic diversity of recent clinical isolates of
Candida albicans
in Japan was studied on the basis of amplified DNA band lengths determined with a specific PCR primer reported to have been designed to span a transposable intron region in the 25S rRNA gene. Our analyses of 301 clinical isolates of
C. albicans
showed that they could be classified into five genotypes: genotype A (172 isolates), genotype B (66 isolates), genotype C (56 isolates), genotype D (
C. dubliniensis
; 5 isolates), and a new genotype (designated genotype E; 2 isolates). The new genotype E was characterized to have a group I intron-like sequence, which is longer than hitherto reported ones and which has a nucleotide sequence length of 962 bp. Our analysis of the 962-bp sequence indicated that it is composed of an intron similar to that of
C. dubliniensis
of 621 bp with a 341-bp insertion. Analysis of the sequence of the internal transcribed spacer (ITS) region of the genotype E strain showed that its sequence is identical to those of strains of other genotypes, with only a few base substitution differences. Throughout the study, the possible horizontal transfer of the group I intron between
C. dubliniensis
and
C. albicans
was suggested. A high degree of correlation between the presence of a group I intron in
C. albicans
genotype E and susceptibility to the antifungal agent flucytosine was observed. The five isolates of
C. dubliniensis
examined in the present study showed genetic diversity when they were compared by randomly amplified polymorphic DNA fingerprinting pattern analysis, and this diversity was also confirmed by the analysis of ITS region sequences.
Persistent ICT Malaria P.f/P.v Panmalarial and HRP2 Antigen Reactivity after Treatment of
Plasmodium falciparum
Malaria Is Associated with Gametocytemia and Results in False-Positive Diagnoses of
Plasmodium vivax
in Convalescence
Authors: Tjitra Emiliana , Suprianto Sri , McBroom James , Currie Bart J. , Anstey Nicholas M.
Published: July 2002

A problem with rapid
Plasmodium falciparum
-specific antigen histidine-rich protein 2 (HRP2) detection tests for malaria is the persistence of antigen in blood after the disappearance of asexual-stage parasitemia and clinical symptoms, resulting in false-positive (FP) test results following treatment. The ICT P.f/P.v immunochromatographic test detects both HRP2 and a panmalarial antigen (PMA) found in both
P. falciparum
and
Plasmodium vivax
. To examine posttreatment antigen persistence with this test and whether persistent sexual-stage forms (gametocytes) are a cause of FP tests after treatment, we compared serial antigen test results with microscopy results from patients symptomatic with
P. falciparum
malaria in Indonesia for 28 days following treatment with chloroquine (CQ;
n
= 66), sulfadoxine-pyrimethamine (SP;
n
= 36), and artesunate plus sulfadoxine-pyrimethamine (ART + SP;
n
= 15). Persistent FP antigenemia following SP treatment occurred in 29% (HRP2) and 42% (PMA) of the patients on day 7 and in 10% (HRP2) and 23% (PMA) on day 14. The high rates of persistent HRP2 and PMA antigenemia following CQ and SP treatment were strongly associated with the presence of gametocytemia, with the proportion with gametocytes on day 7 posttreatment being significantly greater in those with FP results than in those with true-negative PMA and HRP2 results. Gametocyte frequency on day 14 post-SP treatment was also greater in those with FP PMA results. Following SP treatment, PMA persisted longer than HRP2, giving an FP diagnosis of
P. vivax
in up to 16% of patients on day 14, with all FP
P. vivax
diagnoses having gametocytemia. In contrast, PMA was rapidly cleared following ART + SP treatment in association with rapid clearance of gametocytemia. Gametocytes appear to be an important cause of persistent posttreatment panmalarial antigenemia in areas of endemicity and may also contribute in part to persistent HRP2 antigenemia following treatment.
Novel Penicillin-, Cephalosporin-, and Macrolide-Resistant Clones of
Streptococcus pneumoniae
Serotypes 23F and 19F in Taiwan Which Differ from International Epidemic Clones
Authors: Chiou Christine C. , McEllistrem Mary Catherine
Published: July 2002

A cluster (14 of 18) of
Streptococcus pneumoniae
serotype 23F isolates that were resistant to penicillin (PEN), cephalosporin, and macrolide was found in one day care center in Kaohsiung, Taiwan. We analyzed the 18 isolates by pulsed field gel electrophoresis (PFGE). All but one serotype 23F isolate demonstrated identical PFGE patterns, which were different from the established pattern of the internationally spread Spanish 23F clone. The three strains of serotype 19F also showed a uniform pattern. These data strongly suggest that two novel clones of PEN-, cephalosporin-, and macrolide-resistant
S. pneumoniae
serotypes 23F and 19F are present in Taiwan.
Development of a Species-Specific PCR Assay for Detection of
Leishmania donovani
in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis
Authors: Salotra Poonam , Sreenivas G. , Pogue Gregory P. , Lee Nancy , Nakhasi Hira L. , Ramesh V. , Negi N. S.
Published: July 2002

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of
Leishmania donovani
in a species-specific manner among Old World leishmanias. With Indian strains and isolates of
L. donovani
the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz.,
Mycobacterium tuberculosis, Mycobacterium leprae
, and
Plasmodium
spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.
Novel Approach To Reduce the Hepatitis C Virus (HCV) Window Period: Clinical Evaluation of a New Enzyme-Linked Immunosorbent Assay for HCV Core Antigen
Authors: Icardi Giancarlo , Ansaldi Filippo , Bruzzone Bianca Marisa , Durando Paolo , Lee Stephen , de Luigi Carla , Crovari Pietro
Published: July 2002

The window period in hepatitis C virus (HCV) infection is still a major problem in ensuring blood safety. HCV RNA detection by nucleic acid amplification technology-based tests has contributed to reduce the infectivity of blood products, but it is expensive, time-consuming and affected by a high prevalence of false-positive results. The aim of this study was to assess the performance of a newly developed enzyme immunoassay for the detection of HCV core antigen and its suitability for use in the screening of blood units in order to identify infecting samples that do not contain specific antibodies. For evaluation of laboratory performance, different samples were selected: to evaluate specificity, we tested 2,586 sera from blood donors, 500 general population samples, and 58 “difficult sera”. All samples were tested by two screening assays, and results were negative. To estimate clinical sensitivity, 103 HCV RNA-positive, anti-HCV-negative samples, 6 natural seroconversion panels, and 9 commercial seroconversion panels were tested. Intra- and interassay precision were determined on two HCV-RNA-positive, anti-HCV-negative sera. Seventeen (0.66%) blood donor samples, 2 (0.4%) general population samples, and 2 (3.44%) difficult sera were initially reactive; 3 sera were positive on repetition. These 21 samples tested by reverse transcription-PCR were negative. The clinical sensitivity calculated with seroconversion panels and seroconverted patient samples was very similar to PCR sensitivity: 95% of PCR-positive, antibody-negative samples contained detectable HCV antigen. Data on intra- and interassay precision showed dispersion indices with values of less than 10%. In conclusion, the HCV antigen assay showed high sensitivity and specificity and could become a useful means of improving the safety of blood and blood products.
Molecular Epidemiology of Astrovirus Infection in Barcelona, Spain
Authors: Guix Susana , Caballero Santiago , Villena Cristina , Bartolomé Rosa , Latorre Cristina , Rabella Nuria , Simó Maria , Bosch Albert , Pintó Rosa M.
Published: July 2002

A 3-year study involving 2,347 gastroenteritis samples was conducted to determine the prevalence, time distribution, and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus-positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8, and HAstV-2. HAstV-5, HAstV-6, and HAstV-7 were not detected during these 3 years. From our serotype data for each age group, we observed that HAstV-1, HAstV-2, and HAstV-3 affected mostly children younger than 3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.
Variable-Number Tandem Repeat Typing ofMycobacterium tuberculosisIsolates with Low Copy Numbers of IS6110by Using Mycobacterial Interspersed Repetitive Units
Authors: Cowan Lauren Steinlein , Mosher Laura , Diem Lois , Massey Jeffrey P. , Crawford Jack T.
Published: July 2002

A study set of 180Mycobacterium tuberculosisandMycobacterium bovisisolates having low copy numbers of IS6110were genotyped using the recently introduced method based on the variable-number tandem repeats ofmycobacterialinterspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.
18S Ribosomal DNA Typing and Tracking of
Acanthamoeba
Species Isolates from Corneal Scrape Specimens, Contact Lenses, Lens Cases, and Home Water Supplies of
Acanthamoeba
Keratitis Patients in Hong Kong
Authors: Booton G. C. , Kelly D. J. , Chu Y.-W. , Seal D. V. , Houang E. , Lam D. S. C. , Byers T. J. , Fuerst P. A.
Published: July 2002

We examined partial 18S ribosomal DNA (
Rns
) sequences of
Acanthamoeba
isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with
Acanthamoeba
. We also looked for evidence of mixed infections. Identification of
Acanthamoeba Rns
genotypes was based on sequences of ∼113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were
Rns
genotype T4 and the remaining one was
Rns
genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two
Acanthamoeba
keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this
Rns
region for tracking
Acanthamoeba
keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of
Rns
genotype T4 strains in
Acanthamoeba
keratitis infections.
Prevalence of Bacterial Vaginosis and Vaginal Flora Changes in Peri- and Postmenopausal Women
Authors: Cauci Sabina , Driussi Silvia , De Santo Davide , Penacchioni Paola , Iannicelli Teresa , Lanzafame Paolo , De Seta Francesco , Quadrifoglio Franco , de Aloysio Domenico , Guaschino Secondo
Published: July 2002

Our aim was to evaluate the prevalence of bacterial vaginosis and decrease in lactobacillus colonization in women 40 years old or older in relation to menopausal status by evaluation of Gram-stained smears. A total of 1,486 smears from Italian Caucasian women aged 40 to 79 years were examined. Women were classified as follows: fertile (regular cycles) (
n
= 328), perimenopausal (irregular cycles) (
n
= 237), and postmenopausal (
n
= 921), including 331 women on estroprogestinic hormone replacement therapy (HRT). The prevalences of bacterial vaginosis (assessed as a Nugent score of ≥7) in fertile (9.8%) and perimenopausal (11.0%) women were not statistically different, whereas the prevalence was significantly lower overall in postmenopausal women (6.0%) (
P
= 0.02). Specifically, 6.3% of postmenopausal women without HRT and 5.4% of postmenopausal women with HRT were positive for bacterial vaginosis. The Nugent score system was not adequate for evaluating the normal and intermediate vaginal flora in women over the age of 40 years. High numbers of peri- and postmenopausal women had no lactobacilli and no bacterial-vaginosis-associated microorganisms. This nonpathological absence of lactobacilli in women with a Nugent score of 4 was scored as 4∗, and this group was considered separately from the intermediate flora group. A score of 4∗ was obtained for 2.1% of fertile women, 11.4% of perimenopausal women, 44.1% of postmenopausal women without HRT, and 6.9% of postmenopausal women with HRT. The physiological reduction in lactobacillus colonization of the vagina in postmenopausal women does not cause an increase in bacterial-vaginosis prevalence. Reversion of lactobacillus flora to premenopausal levels due to HRT does not increase the prevalence of bacterial vaginosis in postmenopausal women.
Lysosomotropic Agents and Cysteine Protease Inhibitors Inhibit Scrapie-Associated Prion Protein Accumulation
Authors: Doh-ura Katsumi , Iwaki Toru , Caughey Byron
Published: July 2002

We report that lysosomotropic agents and cysteine protease inhibitors inhibited protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells. The inhibition occurred without either apparent effects on normal prion protein biosynthesis or turnover or direct interactions with prion protein molecules. The findings introduce two new classes of inhibitors of the formation of protease-resistant prion protein.