bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium
Anabaena variabilis
ATCC 29413
Authors: Happe Thomas , Schütz Kathrin , Böhme Herbert
Published: July 2002

A 10-kb DNA region of the cyanobacterium
Anabaena variabilis
ATCC 29413 containing the structural genes of the uptake hydrogenase (
hupSL
) was cloned and sequenced. In contrast to the
hupL
gene of
Anabaena
sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the
hupL
gene during the heterocyst differentiation of
A. variabilis
. The
hupSL
genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The
hupSL
operon in
A. variabilis
was interrupted by an interposon deletion (mutant strain AVM13). Under N
2
-fixing conditions, the mutant strain exhibited significantly increased rates in H
2
accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H
2
developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.
DnaA, the Initiator of
Escherichia coli
Chromosomal Replication, Is Located at the Cell Membrane
Authors: Newman Gillian , Crooke Elliott
Published: July 2002

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in
Escherichia coli
, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in
E. coli
is a membrane-affiliated event.
Vibrio fischeriGeneshvnAandhvnBEncode Secreted NAD+-Glycohydrolases
Authors: Stabb Eric V. , Reich Karl A. , Ruby Edward G.
Published: July 2002

HvnA and HvnB are proteins secreted byVibrio fischeriES114, an extracellular light organ symbiont of the squidEuprymna scolopes, that catalyze the transfer of ADP-ribose from NAD+to polyarginine. Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases (ARTases), and it was hypothesized that they mediate bacterium-host signaling. We have clonedhvnAandhvnBfrom strain ES114.hvnAappears to be expressed as part of a four-gene operon, whereashvnBis monocistronic. The predicted HvnA and HvnB amino acid sequences are 46% identical to one another and share 44% and 34% identity, respectively, with an open reading frame present in thePseudomonas aeruginosagenome. Four lines of evidence indicate that HvnA and HvnB mediate polyarginine ADP-ribosylation not by ARTase activity, but indirectly through an NAD+-glycohydrolase (NADase) activity that releases free, reactive, ADP-ribose: (i) like other NADases, and in contrast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of not only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1,N6-etheno-NAD+and NAD+; (iii) incubation of HvnA and HvnB with [32P]NAD+resulted in the production of ADP-ribose; and (iv) purified HvnA displayed an NADaseVmaxof 400 mol min−1mol−1, which is within the range reported for other NADases and 102- to 104-fold higher than the minor NADase activity reported in bacterial ARTase toxins. Construction and analysis of anhvnA hvnBmutant revealed no other NADase activity in culture supernatants ofV. fischeri, and this mutant initiated the light organ symbiosis and triggered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.
Promoters of the CATG-Specific Methyltransferase Gene
hpyIM
Differ between
iceA1
and
iceA2 Helicobacter pylori
Strains
Authors: Xu Qing , Blaser Martin J.
Published: July 2002

Helicobacter pylori
strains can be divided into two groups, based on the presence of two unrelated genes,
iceA1
and
iceA2,
that occupy the same genomic locus.
hpyIM
, located immediately downstream of either gene, encodes a functional CATG-specific methyltransferase. Despite the strong conservation of the
hpyIM
open reading frame (ORF) among all
H. pylori
strains, the sequences upstream of the ORF in
iceA1
and
iceA2
strains are substantially different. To explore the roles of these upstream sequences in
hpyIM
regulation, promoter analysis of
hpyIM
was performed. Both deletion mutation and primer extension analyses demonstrate that the
hpyIM
promoters differ between
H. pylori
strains 60190 (
iceA1
) and J188 (
iceA2
). In strain 60190,
hpyIM
has two promoters, P
a
or P
I
, which may function independently, whereas only one
hpyIM
promoter, P
c
, was found in strain J188. The XylE assay showed that the
hpyIM
transcription level was much higher in strain 60190 than in strain J188, indicating that regulation of
hpyIM
transcription differs between the
H. pylori iceA1
strain (60190) and
iceA2
strains (J188). Since the
iceA1
and
iceA2
sequences are highly conserved within
iceA1
or
iceA2
strains, we conclude that promoters of the CATG-specific methylase gene
hpyIM
differ between
iceA1
and
iceA2
strains, which leads to differences in regulation of
hpyIM
transcription.
Novel Macrolide-Specific ABC-Type Efflux Transporter in
Escherichia coli
Authors: Kobayashi Nobuyoshi , Nishino Kunihiko , Yamaguchi Akihito
Published: July 2002

In the
Escherichia coli
genome, five putative open reading frame (ORF) clusters,
mdlAB
,
ybjYZ
,
yddA
,
yojHI
, and
yhiH
, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573–592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes
acrAB
. Among them, only
ybjYZ
gave significant erythromycin resistance and significantly decreased the accumulation of [
14
C]erythromycin. Therefore,
ybjYZ
was renamed
macAB
(macrolide-specific ABC-type efflux carrier). Plasmids carrying both the
macA
and -
B
genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in
E. coli
. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.
Identification of the Structural Gene for the TDP-Fuc4NAc:Lipid II Fuc4NAc Transferase Involved in Synthesis of Enterobacterial Common Antigen in
Escherichia coli
K-12
Authors: Rahman Arifur , Barr Kathleen , Rick Paul D.
Published: July 2002

The polysaccharide chains of enterobacterial common antigen (ECA) are comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy
-d-
galactose, ManNAcA is
N
-acetyl
-d-
mannosaminuronic acid, and GlcNAc is
N
-acetyl
-d-
glucosamine. Individual trisaccharide repeat units are assembled as undecaprenyl-linked intermediates in a sequence of reactions that culminate in the transfer of Fuc4NAc from TDP-Fuc4NAc to ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) to yield Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III), the donor of trisaccharide repeat units for ECA polysaccharide chain elongation. Most of the genes known to be involved in ECA assembly are located in the
wec
gene cluster located at ca. 85.4 min on the
Escherichia coli
chromosome. The available data suggest that the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase also resides in the
wec
gene cluster; however, the location of this gene has not been unequivocally defined. Previous characterization of the nucleotide sequence of the
wec
gene cluster in the region between
o416
and
wecG
revealed that it contained three open reading frames:
o74
,
o204
, and
o450
. In contrast, the results of experiments described in the current investigation revealed that it contains only two open reading frames,
o359
and
o450
. Mutants of
E. coli
possessing null mutations in
o359
were unable to synthesize ECA, and they accumulated lipid II. In addition, the in vitro incorporation of [
3
H]FucNAc from TDP-[
3
H]Fuc4NAc into lipid II was not observed in reaction mixtures using cell extracts obtained from these mutants as a source of enzyme. The ECA-negative phenotype of these mutants was complemented by plasmid constructs containing the wild-type
o359
allele, and Fuc4NAc transferase activity was demonstrated by using cell extracts obtained from the complemented mutants. Furthermore, partially purified
o359
gene product, expressed as recombinant C-terminal His-tagged protein, was able to catalyze the in vitro transfer of [
3
H]Fuc4NAc from TDP-[
3
H]Fuc4NAc to lipid II. Our data support the conclusion that
o359
of the
wec
gene cluster of
E. coli
is the structural gene for the TDP-Fuc4NAc:lipid II Fuc4NAc transferase involved in the synthesis ECA trisaccharide repeat units.
Identification and Inactivation of Three Group 2 Sigma Factor Genes in
Anabaena
sp. Strain PCC 7120
Authors: Khudyakov Ivan Y. , Golden James W.
Published: July 2002

Three new
Anabaena
sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of
sigD
,
sigE
, and
sigF
genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of
sigD
and
sigE
mutant strains to establish diazotrophic growth. A
sigD sigE
double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of
sigD
or
sigE
, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the
sigE
gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially
sigE sigF
, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes.
luxAB
reporter fusions with
sigD
,
sigE
, and
sigF
all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains,
Anabaena
sp. strain PCC 7120,
Synechococcus
sp. strain PCC 7002, and
Synechocystis
sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains,
Anabaena
sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.
ADP-Dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea
Authors: Verhees Corné H. , Tuininga Judith E. , Kengen Servé W. M. , Stams Alfons J. M. , van der Oost John , de Vos Willem M.
Published: July 2002

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PP
i
-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order
Thermococcales
, including
Pyrococcus
and
Thermococcus
spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders
Methanococcales
and
Methanosarcinales
, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from
Methanococcus jannaschii
was functionally expressed in
Escherichia coli
, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order
Thermococcales
, the
M. jannaschii
ADP-PFK has an extremely low

K
m

for fructose 6-phosphate (9.6 μM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.
RNA Arbitrarily Primed PCR Survey of Genes Regulated by ToxR in the Deep-Sea Bacterium
Photobacterium profundum
Strain SS9
Authors: Bidle Kelly A. , Bartlett Douglas H.
Published: July 2002

We are currently investigating the role of ToxR-mediated gene regulation in
Photobacterium profundum
strain SS9. SS9 is a moderately piezophilic (“pressure loving”) psychrotolerant marine bacterium belonging to the family
Vibrionaceae
. In
Vibrio cholerae
, ToxR is a transmembrane DNA binding protein involved in mediating virulence gene expression in response to various environmental signals. A homolog to
V. cholerae
ToxR that is necessary for pressure-responsive gene expression of two outer membrane protein-encoding genes was previously found in SS9. To search for additional genes regulated by ToxR in SS9, we have used RNA arbitrarily primed PCR (RAP-PCR) with wild-type and
toxR
mutant strains of SS9. Seven ToxR-activated transcripts and one ToxR-repressed transcript were identified in this analysis. The cDNAs corresponding to these partial transcripts were cloned and sequenced, and ToxR regulation of their genes was verified. The products of these genes are all predicted to fall into one or both of two functional categories, those whose products alter membrane structure and/or those that are part of a starvation response. The transcript levels of all eight newly identified genes were also characterized as a function of hydrostatic pressure. Various patterns of pressure regulation were observed, indicating that ToxR activation or repression cannot be used to predict the influence of pressure on gene expression in SS9. These results provide further information on the nature of the ToxR regulon in SS9 and indicate that RAP-PCR is a useful approach for the discovery of new genes under the control of global regulatory transcription factors.
Novel Lysophospholipase A Secreted by
Legionella pneumophila
Authors: Flieger Antje , Gong Shimei , Faigle Marion , Stevanovic Stefan , Cianciotto Nicholas P. , Neumeister Birgid
Published: July 2002

We show that
Legionella pneumophila
possesses lysophospholipase A activity, which releases fatty acids from lysophosphatidylcholine. The NH
2
-terminal sequence of the enzyme contained FGDSLS, corresponding to a catalytic domain in a recently described group of lipolytic enzymes. Culture supernatants of a
L. pneumophila pilD
mutant lost the ability to cleave lysophosphatidylcholine.
Pseudomonas stutzeri
Has Two Closely Related
pilA
Genes (Type IV Pilus Structural Protein) with Opposite Influences on Natural Genetic Transformation
Authors: Graupner Stefan , Wackernagel Wilfried
Published: July 2002

Pseudomonas stutzeri
has type IV pili for which the
pilA
gene (here termed
pilAI
) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of
pilAI
we identified a gene, termed
pilAII
, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to
lacZ
revealed that
pilAII
is expressed only about 10% compared to
pilAI,
although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation of
pilAII
produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in
pilAI pilAII
double mutants expressing heterologous
pilA
genes of nontransformable bacteria, like
Pseudomonas aeruginosa
or
Dichelobacter nodosus
. The overexpression of
pilAII
decreased transformation up to 5,000-fold compared to that of the
pilAII
mutant. However, neither inactivation of
pilAII
nor its overexpression affected the amounts of [
3
H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In the
pilAII
mutant, the transformation by purified single-stranded DNA (which depends on
comA
and
exbB,
as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.
Reciprocal Regulation of Anaerobic and Aerobic Cell Wall Mannoprotein Gene Expression in
Saccharomyces cerevisiae
Authors: Abramova Natalia , Sertil Odeniel , Mehta Sapna , Lowry Charles V.
Published: July 2002

The
DAN/TIR
genes encode nine cell wall mannoproteins in
Saccharomyces cerevisiae
which are expressed during anaerobiosis (
DAN1
,
DAN2
,
DAN3
,
DAN4
,
TIR1
,
TIR2
,
TIR3
,
TIR4
, and
TIP1
). Most are expressed within an hour of an anaerobic shift, but
DAN2
and
DAN3
are expressed after about 3 h. At the same time,
CWP1
and
CWP2
, the genes encoding the major mannoproteins, are down-regulated, suggesting that there is a programmed remodeling of the cell wall in which Cwp1 and Cwp2 are replaced by nine anaerobic counterparts.
TIP1
,
TIR1
,
TIR2
, and
TIR4
are also induced during cold shock. Correspondingly,
CWP1
is down-regulated during cold shock. As reported elsewhere, Mox4 is a heme-inhibited activator, and Mot3 is a heme-induced repressor of the
DAN/TIR
genes (but not of
TIP1
). We show that
CWP2
(but not
CWP1
) is controlled by the same factors, but in reverse fashion—primarily by Mot3 (which can function as either an activator or repressor) but also by Mox4, accounting for the reciprocal regulation of the two groups of genes. Disruptions of
TIR1
,
TIR3
, or
TIR4
prevent anaerobic growth, indicating that each protein is essential for anaerobic adaptation. The Dan/Tir and Cwp proteins are homologous, with the greatest similarities shown within three subgroups: the Dan proteins, the Tip and Tir proteins, and, more distantly, the Cwp proteins. The clustering of homology corresponds to differences in expression: the Tip and Tir proteins are expressed during hypoxia and cold shock, the Dan proteins are more stringently repressed by oxygen and insensitive to cold shock, and the Cwp proteins are oppositely regulated by oxygen and temperature.
Competence Regulation by Oxygen Availability and by Nox Is Not Related to Specific Adjustment of Central Metabolism in
Streptococcus pneumoniae
Authors: Chapuy-Regaud Sabine , Duthoit Frédérique , Malfroy-Mastrorillo Laurence , Gourdon Pierre , Lindley Nic D. , Trombe Marie-Claude
Published: July 2002

In
Streptococcus pneumoniae
oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in
nox
, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase,
l
-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-
ldh
, which encodes the
l
-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.
Characterization of Two CrypticHelicobacter pyloriPlasmids: a Putative Source for Horizontal Gene Transfer and Gene Shuffling
Authors: Hofreuter Dirk , Haas Rainer
Published: July 2002

ManyHelicobacter pyloriisolates carry cryptic plasmids of extremely variable size. In this study we analyzed twoH. pyloriplasmids, pHel4 and pHel5, fromH. pyloristrains P8 and P29, respectively. Plasmid pHel4 consists of 10,970 bp, constituting 15 putative open reading frames (ORFs), whereas pHel5 consists of 18,291 bp, constituting 17 ORFs. The findings that both plasmids encode a conserved RepA protein and that both have an origin of replication containing an iteron place them in the group of theta plasmids. In pHel4, the products of the overlappingorf4C,orf4D,orf4E, andorf4Fsequences are homologous to MobA, MobB, MobC, and MobD, encoded by colicinogenic plasmids, suggesting that pHel4 might be mobilizable. A further putative operon consists oforf4Bandorf4A, the products of which are homologous to microcin C7 (MccC7) biosynthesis and secretion proteins MccB and MccC, respectively. Plasmid pHel5 carries putative genes encoding proteins with homology to an endonuclease and gene products of anH. pylorichromosomal plasticity zone. Both plasmids contain repeat sequences, such as the previously identified R2 repeat, which are considered preferred recombination sites. In pHel4, a new repeat sequence (R4 repeat), which seems to act as a hot spot for site-specific recombination, was identified. AllH. pyloriplasmids characterized so far have a modular structure. We suggest a model that explains the existing plasmids by insertions and deletions of genetic elements at the repeat sequences. A genetic exchange between plasmids and the bacterial chromosome, combined with plasmid mobilization, might add a novel mechanism to explain the high genetic macrodiversity within theH. pyloripopulation.
Oxygen-Mediated Regulation of Porphobilinogen Formation inRhodobacter capsulatus
Authors: Biel Alan J. , Canada Keith , Huang David , Indest Karl , Sullivan Karen
Published: July 2002

ARhodobacter capsulatus hemCmutant has been isolated and used to show that oxygen regulates the intracellular levels of porphobilinogen. Experiments using ahemB-catgene fusion demonstrated that oxygen does not transcriptionally regulatehemBtranscription. Porphobilinogen synthase activity is not regulated by oxygen nor is the enzyme feedback inhibited by hemin or protoporphyrin IX. It was demonstrated that less than 20% of [14C]aminolevulinate was incorporated into bacteriochlorophyll, suggesting that the majority of the aminolevulinate is diverted from the common tetrapyrrole pathway. Porphobilinogen oxygenase activity was not observed in this organism; however, an NADPH-linked aminolevulinate dehydrogenase activity was demonstrated. The specific activity of this enzyme increased with increasing oxygen tension. The results presented here suggest that carbon flow over the common tetrapyrrole pathway is regulated by a combination of feedback inhibition of aminolevulinate synthase and diversion of aminolevulinate from the pathway by aminolevulinate dehydrogenase.
Application of Virus-Specific Immunoglobulin M (IgM), IgG, and IgA Antibody Detection with a Polyantigenic Enzyme-Linked Immunosorbent Assay for Diagnosis of Epstein-Barr Virus Infections in Childhood
Authors: Schaade Lars , Kleines Michael , Häusler Martin
Published: July 2002

The Enzygnost anti-Epstein-Barr virus enzyme-linked immunosorbent assay (ELISA) system, which is based on a defined antigen mixture and on detection of antibodies of the immunoglobulin G (IgG), IgM, and IgA classes, was evaluated for its reliability in diagnosing Epstein-Barr virus infections in childhood. With samples from 66 children, the Epstein-Barr virus status and the infection phase were defined by indirect immunofluorescence and anticomplement fluorescence assays: 11 children were seronegative, 8 had a primary infection, 20 had a recent primary or past infection, and in 27 a reactivated Epstein-Barr virus infection was diagnosed. When applying the Enzygnost ELISAs, 15 serum samples (22.7%) were not interpretable due to indeterminate results in at least one of the assays used and were therefore excluded from further evaluation. The respective sensitivities and specificities for the diagnosis of seronegativity were 100 and 100%, those for the diagnosis of primary infection were 100 and 97%, those for the diagnosis of recent primary or past infection were 100 and 52%, and those for the diagnosis of reactivated infection were 10 and 100%. This poor performance of the Enzygnost system with reactivated infections is due to the prerequisite of an IgG antibody value of >650 IU/ml for the diagnosis of viral activity, which was fulfilled in only two of the children. Despite the high rate of indeterminate results, the Enzygnost system is useful in diagnosing acute and past Epstein-Barr virus infection in childhood. For serological diagnosis of viral activity in childhood, a supplementary assay is necessary.
Comparison of Various Sample Preparation Methods for PCR Diagnosis of Visceral Leishmaniasis Using Peripheral Blood
Authors: Lachaud Laurence , Chabbert Elisabeth , Dubessay Pascal , Reynes Jacques , Lamothe Jacques , Bastien Patrick
Published: July 2002

We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (≥1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of ≤100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherapeutic follow-up.
Inoculum Standardization for Antifungal Susceptibility Testing of Filamentous Fungi Pathogenic for Humans
Authors: Petrikkou Eva , Rodrı́guez-Tudela Juan L. , Cuenca-Estrella Manuel , Gómez Alicia , Molleja Ana , Mellado Emilia
Published: July 2002

Two methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately.
High Degree of Interlaboratory Reproducibility of Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Sequencing of Plasma Samples from Heavily Treated Patients
Authors: Shafer Robert W. , Hertogs Kurt , Zolopa Andrew R. , Warford Ann , Bloor Stuart , Betts Bradley J. , Merigan Thomas C. , Harrigan Richard , Larder Brendon A.
Published: July 2002

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.
Secondary Typing of
Mycobacterium tuberculosis
Isolates with Matching IS
6110
Fingerprints from Different Geographic Regions of the United States
Authors: Yang Z. H. , Bates J. H. , Eisenach K. D. , Cave M. D.
Published: July 2002

Fifty-nine isolates of
Mycobacterium tuberculosis
obtained from different states in the United States and representing 25 interstate clusters were investigated. These clusters were identified by computer-assisted analysis of DNA fingerprints submitted during 1996 and 1997 by different laboratories participating in the CDC National Genotyping and Surveillance Network. Isolates were fingerprinted with the IS
6110
right-hand probe (IS
6110
-3′), the IS
6110
left-hand probe (IS
6110
-5′), and the probe pTBN12, containing the polymorphic GC-rich sequence (PGRS). Spoligotyping based on the polymorphism in the 36-bp direct-repeat locus was also performed. As a control, 43
M. tuberculosis
isolates in 17 clusters obtained from patients in Arkansas during the study period were analyzed. Of the 25 interstate clusters, 19 were confirmed as correctly clustered when all the isolates were analyzed on the same gel using the IS
6110
-3′ probe. Of the 19 true IS
6110
-3′ clusters, 10 (53%) were subdivided by one or more secondary typing methods. Clustering of the control group was virtually identical by all methods. Of the three different secondary typing methods, spoligotyping was the least discriminating. IS
6110
-5′ fingerprinting was as discriminating as PGRS fingerprinting. The data indicate that the IS
6110
-5′ probe not only is a useful secondary typing method but also probably would prove to be a more useful primary typing method for a genotyping network which involves isolates from different geographic regions.
Mycolic Acid Index Susceptibility Method for
Mycobacterium tuberculosis
Authors: Viader-Salvadó José M. , Garza-González Elvira , Valdez-Leal Ramón , de los Angeles del Bosque-Moncayo M. , Tijerina-Menchaca Rolando , Guerrero-Olazarán Martha
Published: July 2002

A rapid drug susceptibility test to measure the susceptibility of
Mycobacterium tuberculosis
to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of
M. tuberculosis
were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods. Overall, there was agreement between the two methods for 398 (99.5%) of the 400 total tests. Specifically, the sensitivity of the MAI susceptibility method for INH and RIF was 97.6 and 100%, respectively. The specificity and positive predictive value were 100% for both drugs, and the negative predictive value for INH and RIF was 98.3 and 100%, respectively. In conclusion, the MAI susceptibility method described here can be used for rapid drug susceptibility testing of
M. tuberculosis
clinical isolates within 5 days after clinical isolates are incubated in the presence or absence of an antituberculosis drug.
Variation in Restriction Fragment Length Polymorphisms among Serial Isolates from Patients with
Trichophyton rubrum
Infection
Authors: Gupta Aditya K. , Kohli Yatika , Summerbell Richard C.
Published: July 2002

Molecular genotyping of strains of
Trichophyton rubrum
and
T. mentagrophytes
from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of
T. rubrum
and 11 strains of
T. mentagrophytes
were obtained from 20 patients (16 patients with
T. rubrum
, 4 with
T. mentagrophytes
) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of
Eco
RI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of
T. rubrum
. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among
T. rubrum, T. mentagrophytes
, and
T. tonsurans
, intraspecific RFLP variation was observed for
T. rubrum
and
T. mentagrophytes
strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in
Candida
species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection.
Clinical and Virological Aspects of Blood Donors Infected with Hepatitis B Virus Genotypes B and C
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn
Published: July 2002

Pathogenic and therapeutic differences among hepatitis B virus (HBV) genotypes have been documented. However, the association of virological characteristics with clinical differences among HBV genotypes remains unclear. We therefore studied the clinical and virological characteristics of Taiwanese volunteer blood donors infected with HBV genotypes B and C. HBV genotypes were determined in 300 candidate blood donors positive for HBV surface antigen (HBsAg), and sequences of the precore gene of the HBV genome were determined in 50 HBV e antigen (HBeAg)-positive and 50 HBeAg-negative blood donors. Of 300 HBsAg-positive blood donors, 10% had elevated serum aminotransferase levels and 27% were positive for HBeAg. HBV genotype distribution in 264 viremic carriers was as follows: B, 221 (83.7%); C, 39 (14.8%); F, 1 (0.4%); and mixed infection, 3 (1.1%). Blood donors with genotype C infection tended to have a higher frequency of HBeAg positivity and a higher serum HBV DNA level than those with genotype B infection. The frequency of precore stop codon mutation was significantly higher in HBeAg-negative blood donors than HBeAg-positive ones, irrespective of HBV genotypes. Meanwhile, only 5% of blood donors with genotype C infection had C-1858 strains. In conclusion, mixed infection of HBV genotypes indeed occurs, and genotype C has a higher serum HBV DNA level than genotype B. Precore stop codon mutation is common in HBeAg-negative HBV carriers, irrespective of HBV genotypes. In contrast, precore C-1858 strains are rarely identified in Taiwanese HBV genotype C.
Human Immunodeficiency Virus Type 1 Group M Protease in Cameroon: Genetic Diversity and Protease Inhibitor Mutational Features
Authors: Fonjungo Peter N. , Mpoudi Eitel N. , Torimiro Judith N. , Alemnji George A. , Eno Laura T. , Lyonga Esther J. , Nkengasong John N. , Lal Renu B. , Rayfield Mark , Kalish Marcia L. , Folks Thomas M. , Pieniazek Danuta
Published: July 2002

To establish a baseline for monitoring resistance to protease inhibitors (PIs) and examining the efficacy of their use among persons in Cameroon infected with human immunodeficiency virus type 1 (HIV-1), we analyzed genetic variability and PI resistance-associated substitutions in PCR-amplified protease (PR) sequences in strains isolated from 110 HIV-1-infected, drug-naïve Cameroonians. Of the 110 strains, 85 were classified into six HIV-1 PR subtypes, A (
n
= 1), B (
n
= 1), F (
n
= 4), G (
n
= 7), H (
n
= 1), and J (
n
= 7), and a circulating recombinant form, CRF02-AG (
n
= 64). PR genes from the remaining 25 (23%) specimens were unclassifiable, whereas 2% (7 of 301) unclassifiable PR sequences were reported for a global collection. Two major PI resistance-associated mutations, 20M and 24I, were detected in strains from only two specimens, whereas secondary mutations were found in strains from all samples except one strain of subtype B and two strains of CRF02-AG. The secondary mutations showed the typical PI resistance-associated pattern for non-subtype B viruses in both classifiable and unclassifiable PR genes, with 36I being the predominant (99%) mutation, followed by 63P (18%), 20R (15%), 77I (13%), and 10I or 10V (11%). Of these mutations, dual and triple PI resistance-associated substitutions were found in 38% of all the Cameroonian strains. Compared with classifiable PR sequences, unclassifiable sequences had significantly more dual and triple substitutions (64% versus 30%;
P
= 0.004). Phenotypic and clinical evaluations are needed to estimate whether PI resistance during antiretroviral drug treatment occurs more rapidly in individuals infected with HIV-1 strains harboring multiple PI resistance-associated substitutions. This information may be important for determination of appropriate drug therapies for HIV-1-infected persons in Cameroon, where more than one-third of HIV-1 strains were found to carry dual and triple minor PI resistance-associated mutations.
Burkholderia cepacia
Complex Bacteria from Clinical and Environmental Sources in Italy: Genomovar Status and Distribution of Traits Related to Virulence and Transmissibility
Authors: Bevivino Annamaria , Dalmastri Claudia , Tabacchioni Silvia , Chiarini Luigi , Belli Maria L. , Piana Sandra , Materazzo Alberto , Vandamme Peter , Manno Graziana
Published: July 2002

Sixty-eight
Burkholderia cepacia
complex isolates recovered from the sputum of 53 cystic fibrosis patients and 75 isolates collected from the maize rhizosphere were compared to each other to assess their genomovar status as well as some traits related to virulence such as antibiotic susceptibility, proteolytic and hemolytic activities, and transmissibility, in which transmissibility is determined by detection of the
esmR
and
cblA
genes. Among the clinical isolates,
B. cepacia
genomovar III comprised the majority of isolates examined and only a very few isolates were assigned to
B. cepacia
genomovar I,
B. stabilis
, and
B. pyrrocinia
; among the environmental isolates a prevalence of
B. cepacia
genomovar III and
B. ambifaria
was observed, whereas few environmental isolates belonging to
B. cepacia
genomovar I and
B. pyrrocinia
were found. Antibiotic resistance analysis revealed a certain degree of differentiation between clinical and environmental isolates. Proteolytic activity and onion tissue maceration ability were found to be spread equally among both clinical and environmental isolates, whereas larger percentages of environmental isolates than clinical isolates had hemolytic activity. The
esmR
gene was found exclusively among isolates belonging to
B. cepacia
genomovar III, with a marked prevalence in clinical isolates, whereas only one clinical isolate belonging to
B. cepacia
genomovar III was found to bear the
cblA
gene. In conclusion, the results of the present study show that the species compositions of the clinical and environmental
B. cepacia
complex populations examined are quite different and that some of the candidate determinants related to virulence and transmissibility are not confined solely to clinical isolates but are also spread among environmental isolates belonging to different species of the
B. cepacia
complex.
Comparison of Four Antiseptic Preparations for Skin in the Prevention of Contamination of Percutaneously Drawn Blood Cultures: a Randomized Trial
Authors: Calfee David P. , Farr Barry M.
Published: July 2002

A number of skin antiseptics have been used to prevent the contamination of blood cultures, but the comparative efficacies of these agents have not been extensively evaluated. We therefore sought to compare the efficacy of four skin antiseptics in preventing blood culture contamination in a randomized, crossover, investigator-blinded study conducted in an emergency department and the inpatient wards of a university hospital. The patient group included all patients from whom blood samples were obtained percutaneously for culture. Skin antisepsis was performed with 10% povidone-iodine, 70% isopropyl alcohol, tincture of iodine, or povidone-iodine with 70% ethyl alcohol (i.e., Persist). The blood culture contamination rate associated with each antiseptic was then determined. A total of 333 (2.62%) of 12,692 blood cultures were contaminated during the study period compared to 413 (3.21%) of 12,859 blood cultures obtained during the previous 12-month period (relative risk = 0.82; 95% confidence interval, 0.71 to 0.94;
P
= 0.006). During the study, the contamination rates were determined to be 2.93% with povidone-iodine, 2.58% with tincture of iodine, 2.50% with isopropyl alcohol, and 2.46% with Persist (
P
= 0.62). We detected no significant differences in the blood culture contamination rates among these four antiseptics, although there was some evidence suggesting greater efficacy among the alcohol-containing antiseptics. Among the evaluated antiseptics, isopropyl alcohol may be the optimal antiseptic for use prior to obtaining blood for culture, given its convenience, low cost, and tolerability.
Molecular Subtyping Methods for Detection of
Salmonella enterica
Serovar Oranienburg Outbreaks
Authors: Kumao Toshio , Ba-Thein William , Hayashi Hideo
Published: July 2002

This study involved 82
Salmonella enterica
serovar Oranienburg isolates from patients with gastroenteritis and/or focal infections, healthy carriers, and cuttlefish chips which were epidemiologically linked to a major outbreak that had affected 1,505 people in Japan between 1998 and 1999. We concurrently investigated four different molecular subtyping methods using human salmonellosis-associated
Salmonella
serovars and their applicability in detection of serovar Oranienburg in an outbreak. Pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic sequence PCR (ERIC2-PCR), or 16S/23S rRNA ribotyping provided a high degree of interserovar discrimination for most of the serovars, with PFGE being the most discriminatory. For intraserovar typing of serovar Oranienburg, ERIC2-PCR was found to be the most sensitive. Native plasmid profiling, however, revealed nine different subgroups among epidemiologically and genetically related outbreak strains. Using these methods, a link was confirmed between food (cuttlefish chips) and patients in the serovar Oranienburg outbreak. This study underscores the limitations of chromosome-based and plasmid-based typing methods.
Genetic Structure ofCandida glabrataPopulations in AIDS and Non-AIDS Patients
Authors: de Meeûs T. , Renaud F. , Mouveroux E. , Reynes J. , Galeazzi G. , Mallié M. , Bastide J. M.
Published: July 2002

The genotypes of 63 strains (11 reference strains and 52 strains from hospitalized patients) of the haploid yeastCandida glabratawere determined from 33 putative gene enzymatic loci. This enabled the characterization of 26 different multilocus genotypes. Genetic differentiation was found between distant hospitals (located in Montpellier and Paris, France) but not for other parameters (anatomic origins or human immunodeficiency virus-positive [HIV+] and HIV−patients). Strong nonrandom association between loci could be seen. Such statistical linkages were confirmed upon comparing the patterns of 14 RAPD [random(ly) amplified polymorphic DNA] primers from 20 of these strains to results obtained from multilocus enzyme electrophoresis analysis. This finding suggests a mainly clonal mode of reproduction ofC. glabrata. The consequences of the clonality displayed byC. glabratapopulations on the epidemiology of this yeast are also discussed.