bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of the Ferric Citrate Transport System of
Escherichia coli
: Mutations in Region 2.1 of the FecI Extracytoplasmic-Function Sigma Factor Suppress Mutations in the FecR Transmembrane Regulatory Protein
Authors: Stiefel Alfred , Mahren Susanne , Ochs Martina , Schindler Petra T. , Enz Sabine , Braun Volkmar
Published: July 2002

Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane of
Escherichia coli
K-12. Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active. In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of the
Pseudomonas aeruginosa
,
Pseudomonas putida
,
Bordetella pertussis
,
Bordetella bronchiseptica
, and
Caulobacter crescentus
genomes. The cytoplasmic portion of the FecR mutant proteins, FecR
1–85
, did not interact with wild-type FecI, in contrast to wild-type FecR
1–85
, which induced FecI-mediated
fecB
transport gene transcription. Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of the
fecR
mutants by ferric citrate. Region 2.1 of ς
70
is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI. In addition, missense mutations in the
fecI
promoter region resulted in a twofold increased transcription in
fecR
wild-type cells and a partial restoration of
fec
transport gene transcription in the
fecR
mutants. The mutations reduced binding of the Fe
2+
Fur repressor and as a consequence enhanced
fecI
transcription. The data reveal properties of the FecI ECF factor distinct from those of ς
70
and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.
Effects of Mutations in the
Pseudomonas putida miaA
Gene: Regulation of the
trpE
and
trpGDC
Operons in
P. putida
by Attenuation
Authors: Olekhnovich Igor , Gussin Gary N.
Published: July 2002

Tn
5
insertion mutants defective in regulation of the
Pseudomonas putida trpE
and
trpGDC
operons by tryptophan were found to contain insertions in the
P. putida miaA
gene, whose product (in
Escherichia coli
) modifies tRNA
Trp
and is required for attenuation. Nucleotide sequences upstream of
trpE
and
trpG
encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.
Identification, Cloning, Expression, and Characterization of the Extracellular Acarbose-Modifying Glycosyltransferase, AcbD, fromActinoplanessp. Strain SE50
Authors: Hemker Michael , Stratmann Ansgar , Goeke Klaus , Schröder Werner , Lenz Jürgen , Piepersberg Wolfgang , Pape Hermann
Published: July 2002

An extracellular enzyme activity in the culture supernatant of the acarbose producerActinoplanessp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene,acbD, in the putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose. TheacbDgene was cloned and heterologously produced inStreptomyces lividansTK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular α-glucosidases and/or transglycosylases of the α-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither α-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca2+ions for activity. The enzyme displayed maximal activity at 30°C and between pH 6.2 and 6.9. TheKmvalues of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 ofd-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced byActinoplanessp. strain SE50.
Genetic Studies ofmrp, a Locus Essential for Cellular Aggregation and Sporulation ofMyxococcus xanthus
Authors: Sun Hong , Shi Wenyuan
Published: July 2002

Under starvation conditions,Myxococcus xanthusundergoes a complex developmental process which includes cellular aggregation and sporulation. A transposon insertion mutant (the Tn5-Ω280 mutant) with defects in both aggregation and sporulation was analyzed in this study. The Tn5-Ω280 mutant was found to have a disrupted NtrC-like response regulator designatedMyxococcusregulatory protein B (mrpB). Further sequencing analyses revealed a histidine kinase homolog (mrpA) immediately upstream ofmrpBand a cyclic AMP receptor protein-like transcriptional regulator (mrpC) downstream ofmrpB. In-frame deletion analyses revealed that both themrpBandmrpCgenes were required for cellular aggregation and sporulation but that onlymrpAwas required for sporulation only. Site-specific mutagenesis of the putative phosphorylation site of MrpB, D58, showed that a D58A mutation caused defects in both aggregation and sporulation but that a D58E mutation resulted in only a sporulation defect. Further genetic and molecular analyses with reporter genes and reverse transcription-PCR indicated thatmrpAandmrpBare cotranscribed but thatmrpCis transcribed independently and that all of these genes are developmentally regulated. In addition, MrpB is essential for transcription ofmrpCand MrpC regulates its own transcription. These data indicate that Mrp proteins are important components required forM. xanthusdevelopment. The complicated interaction between Mrp proteins may play an important role in regulating developmental gene expression inM. xanthus.
Genetic Recombination in
Bacillus subtilis
168: Effect of Δ
helD
on DNA Repair and Homologous Recombination
Authors: Carrasco Begoña , Fernández Silvia , Petit Marie-Agnes , Alonso Juan C.
Published: July 2002

The
B. subtilis
Δ
helD
allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec
+
strain. The Δ
helD
allele was introduced into
rec
-deficient strains representative of the α (
recF
strain), β (
addA
addB
), γ (
recH
), ɛ (Δ
recU
), and ζ (Δ
recS
) epistatic groups. The Δ
helD
mutation increased the sensitivity to DNA-damaging agents of
addAB,
Δ
recU,
and Δ
recS
cells, did not affect the survival of
recH
cells, and decreased the sensitivity of
recF
cells. Δ
helD
also partially suppressed the DNA repair phenotype of other mutations classified within the α epistatic group, namely the
recL
, Δ
recO,
and
recR
mutations. The Δ
helD
allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the α, β, and γ epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of
recFLOR
and render
recFLOR
,
addAB
, and
recH
cells impaired in plasmid transformation.
The Genome of Archaeal Prophage ΨM100 Encodes the Lytic Enzyme Responsible for Autolysis of
Methanothermobacter wolfeii
Authors: Luo Yongneng , Pfister Peter , Leisinger Thomas , Wasserfallen Alain
Published: July 2002

Methanothermobacter wolfeii
(formerly
Methanobacterium wolfei
), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called ΨM100 on its chromosome. The nucleotide sequence of ΨM100 and its flanking regions was established and compared to that of the previously sequenced phage ΨM2 of
Methanothermobacter marburgensis
(formerly
Methanobacterium thermoautotrophicum
Marburg). The ΨM100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into ΨM100 but is absent in the genome of ΨM2. The remaining part of the ΨM100 genome showed 70.8% nucleotide sequence identity to the whole genome of ΨM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the ΨM100 genome. Comparison of ΨM100-encoded ORFs to those encoded by phage ΨM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of ΨM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by
M. wolfeii
.
Specificities of Eleven Different DNA Methyltransferases of
Helicobacter pylori
Strain 26695
Authors: Vitkute Jolanta , Stankevicius Kornelijus , Tamulaitiene Giedre , Maneliene Zita , Timinskas Albertas , Berg Douglas E. , Janulaitis Arvydas
Published: July 2002

Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of
Helicobacter pylori
strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m
4
C, m
5
C, and m
6
A.
An N-Terminally Truncated RpoS (σ
S
) Protein in
Escherichia coli
Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of
rpoS
Transcriptional and Translational Control Signals
Authors: Rajkumari K. , Gowrishankar J.
Published: July 2002

RpoS (σ
S
) in
Escherichia coli
is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial σ
70
family of proteins. Conserved domain 1.1 at the N-terminal end of σ
70
has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a σ
S
variant with a deletion of its N-terminal 50 amino acids (σ
S
Δ1-50), when expressed in vivo either from a chromosomal
rpoS
::IS
10
allele (in
rho
mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the
proU
P1 promoter; at three other σ
S
-dependent promoters that were tested (
osmY
,
katE
, and
csiD
), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the
csiD
promoter (which requires both σ
S
and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing σ
S
Δ1-50. The intracellular content of σ
S
Δ1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for σ
S
, even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type
rpoS
. Our results indicate that, unlike that in σ
70
, the N-terminal domain in σ
S
may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of σ
S
-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of σ
S
protein degradation.
Streptococcus pneumoniaeCapsule Biosynthesis Protein CpsB Is a Novel Manganese-Dependent Phosphotyrosine-Protein Phosphatase
Authors: Morona Judy K. , Morona Renato , Miller David C. , Paton James C.
Published: July 2002

The first four genes of the capsule locus (cps) ofStreptococcus pneumoniae(cpsAtocpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that anS. pneumoniaestrain with point mutations incpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which thecpsBgene had been deleted.
Identification of Virulence Genes in a Pathogenic Strain ofPseudomonas aeruginosaby Representational Difference Analysis
Authors: Choi Ji Young , Sifri Costi D. , Goumnerov Boyan C. , Rahme Laurence G. , Ausubel Frederick M. , Calderwood Stephen B.
Published: July 2002

Pseudomonas aeruginosais an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate ofP. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants,Caenorhabditis elegans, and the greater wax moth,Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification ofP. aeruginosavirulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain ofP. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences betweenP. aeruginosastrains PA14 and PAO1. We have found that thepilC,pilA, anduvrDgenes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to theybtQgene fromYersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of theybtQhomolog inP. aeruginosastrain PA14 significantly attenuates the virulence of this strain in bothG. mellonellaand a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence ofP. aeruginosastrain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.
Transposable Element IS
Hp608
of
Helicobacter pylori
: Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity
Authors: Kersulyte Dangeruta , Velapatiño Billie , Dailide Giedrius , Mukhopadhyay Asish K. , Ito Yoshiyuki , Cahuayme Lizbeth , Parkinson Alan J. , Gilman Robert H. , Berg Douglas E.
Published: July 2002

A new member of the IS
605
transposable element family, designated IS
Hp608
, was found by subtractive hybridization in
Helicobacter pylori
. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (
orfA
and
orfB
), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes;
orfB
is also related to the
Salmonella
virulence gene
gipA
. PCR and hybridization tests showed that IS
Hp608
is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. IS
Hp608
also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively;
P
= 0.04). Two IS
Hp608
types differing by ∼11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by ≤2% in DNA sequence, but several recombinant elements were also found. IS
Hp608
marked with a resistance gene was found to (i) transpose in
Escherichia coli
; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require
orfA
but not
orfB
for its transposition. IS
Hp608
represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.
The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in
Escherichia coli
Cells but Stimulates Its Activity
Authors: Morales Pilar , Rouviere-Yaniv Josette , Dreyfus Marc
Published: July 2002

In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA. Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation. We examine here whether the presence of HU, one of the major histone-like proteins in
Escherichia coli
cells (the genuine milieu for T7 RNAP) affects its activity. An engineered
lac
operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it. The flows of RNAP that enter and exit this operon were compared with regard to the content of HU. We found that the fraction of T7 RNAP molecules that do not reach the end of the
lac
operon (ca. 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it. However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the
hup
mutants, presumably reflecting changes in DNA supercoiling. Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect. Finally, our results suggest that HU can also influence mRNA stability in vivo.
Phenotypic Selection and Phase Variation Occur during Alfalfa Root Colonization by
Pseudomonas fluorescens
F113
Authors: Sánchez-Contreras María , Martín Marta , Villacieros Marta , O'Gara Fergal , Bonilla Ildefonso , Rivilla Rafael
Published: July 2002

During colonization of the alfalfa rhizosphere,
Pseudomonas fluorescens
F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the
fliC-
encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned
gacA
. Sequence analysis of the
gacA
alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the
gacA
or
gacS
gene. Mutation of the F113
sss
gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.
Differential Effects of Mutations intonB1on Intrinsic Multidrug Resistance and Iron Acquisition inPseudomonas aeruginosa
Authors: Zhao Qixun , Poole Keith
Published: July 2002

Loss oftonB1adversely affects iron acquisition and intrinsic multidrug resistance inPseudomonas aeruginosa. Several mutations intonB1compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only.
Conserved Serine/Threonine Kinase Encoded by
CBK1
Regulates Expression of Several Hypha-Associated Transcripts and Genes Encoding Cell Wall Proteins in
Candida albicans
Authors: McNemar Mark D. , Fonzi William A.
Published: July 2002

The opportunistic fungal pathogen,
Candida albicans
, is reported to have several potential virulence factors. A potentially significant factor is the ability to undergo morphological transition from yeast to hypha. This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition. We have isolated a gene that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall. We have assigned the designation
CBK1
(cell wall biosynthesis kinase 1) to this gene. Mutants lacking
CBK1
form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation. Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall. The results suggest that the kinase encoded by
CBK1
plays a general role in the maintenance and alteration of the cell wall of
C. albicans
in all morphologies.
Rapid Identification of Clinically Relevant
Legionella
spp. by Analysis of Transfer DNA Intergenic Spacer Length Polymorphism
Authors: De Gheldre Yves , Maes Nicole , Presti François Lo , Etienne Jerome , Struelens Marc
Published: July 2002

Analysis of PCR-amplified transfer DNA (tDNA) intergenic spacers was evaluated as a rapid method for identification to the species level of 18 species of
Legionella
known as human pathogens. Type strains (
n
= 19), reference strains (
n
= 16), environmental strains (
n
= 31), and clinical strains (
n
= 32) were tested. PCR products using outwardly directed tDNA consensus primers were separated on polyacrylamide gels and analyzed with automated laser fluorescence. Test results were obtained in 8 h starting with 72-h-old bacterial growth on solid medium. Species-specific patterns were obtained for all 18
Legionella
species tested:
Legionella anisa
,
L. bozemanii
serogroups 1 and 2,
L. cincinnatiensis
,
L. dumoffii
,
L. feeleii
serogroups 1 and 2,
L. gormanii
,
L. hackeliae
serogroups 1 and 2,
L. jordanis
,
L. lansingensis
,
L. longbeachae
serogroups 1 and 2,
L. lytica
,
L. maceachernii
,
L. micdadei
,
L. oakridgensis
,
L. parisiensis
,
L. pneumophila
serogroups 1 to 14,
L. sainthelensi
serogroup 2,
L. tucsonensis
, and
L. wadsworthii
. Computer-assisted matching of tDNA-intergenic length polymorphism (ILP) patterns identified all 63 environmental and clinical strains to the species level and to serogroup for some strains. tDNA-ILP analysis is proposed as a routinely applicable method which allows rapid identification of environmental and clinical isolates of
Legionella
spp. associated with legionellosis.
Molecular Characterization of Rifampin-Resistant Isolates of
Mycobacterium tuberculosis
from Hungary by DNA Sequencing and the Line Probe Assay
Authors: Bártfai Zoltán , Somoskövi Ákos , Ködmön Csaba , Szabó Nóra , Puskás Erzsébet , Kosztolányi Lászlóné , Faragó Eszter , Mester Judit , Parsons Linda M. , Salfinger Max
Published: July 2002

Two regions of
rpoB
associated with rifampin resistance were sequenced in 29 rifampin-resistant (determined by the proportion method) isolates of
Mycobacterium tuberculosis
obtained from patients from three counties in Hungary. Of the 29 resistant strains, 27 had a mutation in either the 81-bp region (26 strains) or the N-terminal region (1 strain), while the other 2 strains had no mutations in either region. The locations and frequencies of the mutations differed from those previously reported. The most common mutation in this study, D516V, was found in 38% of the Hungarian strains, a frequency 2 to 10 times higher than that found in studies from other countries. These same 29 isolates were also evaluated with the Inno-LiPA Rif. TB test (LiPA), a reverse hybridization assay for the rapid detection of rifampin resistance. Although LiPA detected the presence of an
rpoB
mutation in 26 of the resistant isolates, the type of mutation could not be determined in 4 isolates because the mutations present were not among those included on the LiPA strip. In addition, a silent mutation in one of the rifampin-susceptible control strains was interpreted as rifampin resistant by LiPA. These findings demonstrate the importance of validating this rapid molecular test by comparison with DNA sequence results in each geographic location before incorporating the test into routine diagnostic work.
Longitudinal Variability of Human Immunodeficiency Virus Type 1 RNA Viral Load Measurements by Nucleic Acid Sequence-Based Amplification and NucliSens Assays in a Large Multicenter Study
Authors: Nowicki Marek J. , Benning Lorie , Bremer James W. , Meyer William A. , Hanson Carl , Brambilla Donald , Silver Sylvia , Kovacs Andrea ,   the Women's Interagency HIV-1 Study Collaborative Study Group
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) RNA measurements were evaluated within an externally controlled multilaboratory program. Three external standards (1.5 × 10
3
to 1.5 × 10
6
copies/ml) were included in 814 assay runs by four laboratories. Results indicate that HIV-1 RNA levels can be measured with a precision equal to that of the pre-highly active antiretroviral therapy era (standard deviations, ±0.16 to 0.25 log
10
units).
Genome Sequence-Based Fluorescent Amplified Fragment Length Polymorphism of
Campylobacter jejuni
, Its Relationship to Serotyping, and Its Implications for Epidemiological Analysis
Authors: Desai Meeta , Logan Julie M. J. , Frost Jennifer A. , Stanley John
Published: July 2002

The published genome sequence of
Campylobacter jejuni
strain NCTC 11168 was used to model an accurate and highly reproducible fluorescent amplified fragment length polymorphism (FAFLP) analysis. Predicted and experimentally observed amplified fragments (AFs) generated with the primer pair
Hin
dIII+A and
Hha
I+A were compared. All but one of the 61 predicted AFs were reproducibly detected, and no unpredicted fragments were amplified. This FAFLP analysis was used to genotype 74
C. jejuni
strains belonging to the nine heat-stable (HS) serotypes most prevalent in human disease in England and Wales. The 74
C. jejuni
strains exhibited 60 FAFLP profiles, and cluster analysis of them yielded a radial tree showing genetic relationships between and within 13 major clusters. Some clusters were related, and others were unrelated, to a single HS serotype. For example, all strains belonging to serotypes HS6 and HS19 grouped into corresponding single genotypic clusters, while strains of serotypes HS11 and HS18 each grouped into two genotypic clusters. Strains of HS50, the most prevalent serotype infecting humans, were found both in one large (multiserotype) cluster complex and dispersed throughout the tree. The strain genotypes within each FAFLP cluster were characterized by a particular combination of AFs, and among the cluster there were additional differential AFs. Identification of such AFs could act as a search tool to look for potential associations with disease or animal hosts, when applied to large number of human isolates. Genome-sequence based FAFLP, thus, has the potential to establish a genetic database for epidemiological investigations of
Campylobacter
.
Rotavirus Strains Bearing Genotype G9 or P[9] Recovered from Brazilian Children with Diarrhea from 1997 to 1999
Authors: Santos Norma , Volotão Eduardo M. , Soares Caroline C. , Albuquerque Maria Carolina M. , da Silva Fabiano M. , de Carvalho Tatiane R. B. , Pereira Cláudio F. A. , Chizhikov Vladimir , Hoshino Yasutaka
Published: July 2002

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (
n
= 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.
Nationwide German Multicenter Study on the Prevalence of Antibiotic Resistance in Streptococcal Blood Isolates from Neutropenic Patients and Comparative In Vitro Activities of Quinupristin-Dalfopristin and Eight Other Antimicrobials
Authors: Reinert Ralf René , von Eiff Christof , Kresken Michael , Brauers Johannes , Hafner Dieter , Al-Lahham Adnan , Schorn Holger , Lütticken Rudolf , Peters Georg ,   The Multicenter Study On Antibiotic Resistance In Staphylococci and Other Gram- Positive Cocci (Mars) Study Group
Published: July 2002

In a prospective multicenter study (1996 to 1999), 156 episodes of bacteremic streptococcal infections of neutropenic patients were evaluated.
Streptococcus oralis
(26.3%),
S. pneumoniae
(26.3%),
S. agalactiae
(11.5%),
S. mitis
(9%), and
S. pyogenes
(5.8%) were the predominant species. Four strains (2.6%) were found to be intermediately resistant to penicillin. One strain (0.6%) was found to be highly resistant to penicillin (MIC, 8 mg/liter). Reduced susceptibility to penicillin was detected among
S. oralis
(14.6%),
S. mitis
(7.1%), and
S. pneumoniae
(4.9%) isolates but was not recorded among
S. agalactiae
and
S. pyogenes
. Resistance rates and intermediate resistance rates for other antimicrobials were as follows (all species): amoxicillin, 1.3 and 3.2%; erythromycin, 16 and 2.6%; clindamycin, 5.8 and 0%; ciprofloxacin, 1.9 and 7.7%. Quinupristin-dalfopristin showed good in vitro activity against most streptococcal isolates (MIC at which 50% of the isolates were inhibited [MIC
50
], 0.5 mg/liter; MIC
90
, 1 mg/liter, MIC range, 0.25 to 4 mg/liter).
Enzyme-Linked Immunosorbent Assay for Detecting Herpesvirus Exposure in Mediterranean Tortoises (Spur-Thighed Tortoise [Testudo graeca] and Hermann's Tortoise [Testudo hermanni])
Authors: Origgi F. C. , Klein P. A. , Mathes K. , Blahak S. , Marschang R. E. , Tucker S. J. , Jacobson E. R.
Published: July 2002

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to a herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (Testudo hermanni)]. This serodiagnostic test was validated through a hyperimmunization study. The mean of theA405readings of the plasma samples collected at time zero of the hyperimmunization study plus three times the standard deviation was used as the cutoff for seropositivity in tortoises. ELISA results were compared to serum neutralization (SN) values for the same samples by using the McNemar test. The results obtained by SN and ELISA were not significantly different (P> 0.05). This new ELISA could be used as an important diagnostic tool for screening wild populations and private and zoo collections of Mediterranean tortoises.
Antifungal Effects of Lysozyme and Lactoferrin against Genetically Similar, Sequential
Candida albicans
Isolates from a Human Immunodeficiency Virus-Infected Southern Chinese Cohort
Authors: Samaranayake Y. H. , Samaranayake L. P. , Pow E. H. N. , Beena V. T. , Yeung K. W. S.
Published: July 2002

A variety of innate defense factors in saliva such as lysozyme and lactoferrin contribute to mucosal protection and modulate
Candida
populations in the oral cavity. It is also known that in human immunodeficiency virus (HIV)-infected individuals significant variations in the concentrations of lysozyme and lactoferrin in saliva occur during disease progression. Therefore, the aim of this study was to determine the in vitro susceptibility to human lactoferrin and hen egg white lysozyme of genotypically similar oral
Candida albicans
isolates obtained from six HIV-infected ethnic Chinese during sequential visits over a 12-month period. The similarity of the genotypes (50 in total) was evaluated using a randomly amplified polymorphic DNA assay. A blastospore viability assay was performed to evaluate the sensitivity of the organisms to lysozyme and lactoferrin. Exposure to physiological concentrations of either lysozyme (30 μg/ml) or lactoferrin (20 μg/ml) caused a rapid loss of viability among all isolates to a varying extent. None of the sequential
C
.
albicans
isolates demonstrated significant differences in sensitivity to either protein from one visit to the next; similar results were noted when the different genotypes from the same individual were compared. On Spearman correlation analysis of two genotypes that were sequentially isolated from a single patient, a significant negative correlation between lysozyme (
r
= −0.88;
P
< 0.02) (but not lactoferrin) resistance and the duration of HIV disease was seen. These results imply that a minority of
C
.
albicans
isolates that persist intraorally in individuals with HIV disease develop progressive resistance to innate salivary antifungal defenses such as lysozyme, possibly as an adaptive response. However, the vast majority of the
Candida
isolates appear to succumb to these nonspecific host immune mediators abundantly present in the oral environment.
Comparative Analysis of Two Commercial Phenotypic Assays for Drug Susceptibility Testing of Human Immunodeficiency Virus Type 1
Authors: Qari Shoukat H. , Respess Richard , Weinstock Hillard , Beltrami Elise M. , Hertogs Kurt , Larder Brendan A. , Petropoulos Christos J. , Hellmann Nicholas , Heneine Walid
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) isolates from 50 plasma specimens were analyzed for phenotypic susceptibility to licensed reverse transcriptase inhibitors and protease inhibitors by the Antivirogram and PhenoSense HIV assays. Twenty of these specimens were from recently seroconverted drug-naïve persons, and 30 were from patients who were the sources of occupational exposures to HIV-1; 16 of the specimens in the latter group were from drug-experienced patients. The phenotypic results of the Antivirogram and PhenoSense HIV assays were categorized as sensitive or reduced susceptibility on the basis of the cutoff values established by the manufacturers of each assay. Data for 12 to 15 drugs were available by both assays for 38 specimens and represented a total of 529 pairs of results. The two data sets had a 91.5% concordance by phenotypic category. The discordant results (
n
= 45) were distributed randomly among 26 specimens and included 28 results (62.2%) which were within a twofold difference of the assay cutoff values. None of the discordant results were associated with primary resistance mutations that predicted high-level (>20-fold) resistance. Discordant results were distributed equally among specimens from drug-experienced and drug-naïve individuals and were slightly higher for protease inhibitors than for nonnucleoside reverse transcriptase inhibitors or nucleoside reverse transcriptase inhibitors. The findings of the present study demonstrate that the results of the Antivirogram and PhenoSense HIV assays correlate well, despite the use of different testing strategies.
Detection of Meningococcal Carriage by Culture and PCR of Throat Swabs and Mouth Gargles
Authors: Jordens J. Zoe , Williams Jeannette N. , Jones Graeme R. , Heckels John E.
Published: July 2002

The standard method for detecting meningococcal carriage is culture of throat swabs on selective media, but the levels of carriage determined depend heavily on the skills of the individuals taking the swab and interpreting the cultures. This study aimed to determine the most sensitive detection method for meningococcal carriage. Throat swabs and saline mouth gargles, obtained from 89 university students, were processed in parallel by conventional culture and TaqMan
ctrA
PCR. Carriage of meningococci, as detected by the combined methods, was 20%. The sensitivities of throat swab culture, throat swab PCR, gargle culture, and gargle PCR were 72, 56, 56, and 50%, respectively, and the probabilities that these techniques would correctly identify the absence of carriage (negative predictive value [NPV]) were 93.4, 89.9, 89.9, and 88.8%. Culturing both throat swabs and gargles increased the NPV to 98.6%. The further addition of throat swab PCR increased this to 100%. Testing gargles by both culture and PCR was as sensitive as testing throat swabs by both methods, suggesting that gargles may be a suitable alternative for large-scale screening studies when throat swabs are difficult to obtain, although they required more lengthy laboratory processing. PCR was a useful adjunct to culture for detecting nasopharyngeal carriage, but it failed to detect some nongroupable strains. For maximum sensitivity, a combination of techniques was required. This study indicates the confidence with which health care professionals involved in meningococcal screening can regard laboratory results.
Tiamulin Activity against Fastidious and Nonfastidious Veterinary and Human Bacterial Isolates: Initial Development of In Vitro Susceptibility Test Methods
Authors: Jones Ronald N. , Pfaller Michael A. , Rhomberg Paul R. , Walter Donald H.
Published: July 2002

Tiamulin is a pleuromutilin derivative used in veterinary practice for the control and specific therapy of infections in swine. This report summarizes studies to establish standardized susceptibility testing methods, interpretive criteria, and reagent details for use in veterinary methods recently developed by the National Committee for Clinical Laboratory Standards (NCCLS) (standards M31-A and M37-A, NCCLS, Wayne, Pa., 1999). A total of 636 fastidious and nonfastidious animal and human pathogens were processed by using media and procedures described by the NCCLS. Tiamulin disk diffusion tests used a 30-μg disk concentration, and the proposed MIC breakpoints corresponding to levels achievable in animal target tissues (lung) were ≤4 μg/ml for susceptibility and ≥32 μg/ml for resistance. Correlate zone diameters for specific nonfastidious species were as follows: for
Pasteurella multocida
and staphylococci tested on Mueller-Hinton agar, susceptibility at ≥19 mm and resistance at ≤11 mm, and for
Actinobacillus suis
,
Erysipelothrix rhusiopathiae
, and
Streptococcus suis
tested on enriched chocolate Mueller-Hinton agar, susceptibility at ≥16 mm and resistance at ≤8 mm. When
Actinobacillus pleuropneumoniae
was tested, a susceptibility breakpoint of ≤16 μg/ml (≥9 mm) was suggested for veterinary fastidious medium broth and enriched chocolate Mueller-Hinton agar. Absolute categorical agreement between NCCLS dilution and disk diffusion test results with these criteria ranged from 90.5 to 96.2%. Tiamulin susceptibility testing methods appear to be accurate in their categorical classification for indicated species, and their availability will allow immediate testing of animal isolates to guide therapy via appropriate levels of dosing and to monitor the development of resistance for agents in this unique class.
Epidemic Strains of
Shigella sonnei
Biotype g Carrying Integrons
Authors: McIver Christopher J. , White Peter A. , Jones Louisa A. , Karagiannis Tom , Harkness Jock , Marriott Deborah , Rawlinson William D.
Published: July 2002

Class 2 integrons (Tn
7
) were found in all randomly selected epidemic (
n
= 27) and preepidemic (
n
= 13) strains of multiresistant
Shigella sonnei
biotype g. A class 1 integron was also found in two epidemic strains. Gene cassettes within these integrons account for resistance to commonly used therapeutic agents.
Evaluation of the VERSANT HCV RNA 3.0 Assay for Quantification of Hepatitis C Virus RNA in Serum
Authors: Trimoulet Pascale , Halfon Philippe , Pohier Eric , Khiri Hacène , Chêne Geneviève , Fleury Hervé
Published: July 2002

We assessed the performance of a new assay (VERSANT HCV RNA 3.0 [bDNA 3.0] assay [Bayer Diagnostics]) to quantitate HCV RNA levels and compared the results of the bDNA 3.0 assay to results of the Quantiplex HCV RNA 2.0 (bDNA 2.0) assay. Samples used in this study included 211 serum specimens from hepatitis C virus (HCV)-infected persons from two sites (Bordeaux and Marseille, France) with different genotypes; 383 serum specimens from HCV antibody-negative, HCV RNA-negative persons; and serial dilutions of World Health Organization (WHO) HCV RNA standard at a titer of 100,000 IU/ml. The specificity of the bDNA 3.0 assay was 98.2%. A high correlation was observed between expected and observed values in all dilutions of WHO standard (
r
= 0.9982), in serial dilutions of pooled samples (
r
= 0.9996), and in diluted sera from different HCV genotypes (
r
= 0.9930 to 0.9995). The standard deviations (SD) for the within-run and between-run reproducibility of the bDNA 3.0 assay were ≤0.2 and ≤0.14, respectively. The intersite SD ranged from 0.03 to 0.14. The bDNA 3.0 assay results were positively correlated with the bDNA 2.0 assay results (
r
= 0.9533). Taking in account the overall performance, this assay could be used as a routine tool for the HCV RNA quantification.
Distinct Mechanisms of Entry by Envelope Glycoproteins of Marburg and Ebola (Zaire) Viruses
Authors: Chan Stephen Y. , Speck Roberto F. , Ma Melissa C. , Goldsmith Mark A.
Published: July 2002

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.
Mutants of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase Resistant to Nonnucleoside Reverse Transcriptase Inhibitors Demonstrate Altered Rates of RNase H Cleavage That Correlate with HIV-1 Replication Fitness in Cell Culture
Authors: Archer Richard H. , Dykes Carrie , Gerondelis Peter , Lloyd Amanda , Fay Philip , Reichman Richard C. , Bambara Robert A. , Demeter Lisa M.
Published: July 2002

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3′-end-directed and RNA 5′-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803–5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3′-end- and RNA 5′-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.