bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochromes
c
550
,
c
552
, and
c
1
in the Electron Transport Network of
Paracoccus denitrificans
: Redundant or Subtly Different in Function?
Authors: Otten Marijke F. , van der Oost John , Reijnders Willem N. M. , Westerhoff Hans V. , Ludwig Bernd , Van Spanning Rob J. M.
Published: July 2002

Paracoccus denitrificans
strains with mutations in the genes encoding the cytochrome
c
550
,
c
552
, or
c
1
and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome
c
mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome
bc
1
complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome
c
550
-deficient mutant did denitrify provided copper was present. The cytochrome
c
552
mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes
c
have multiple tasks in electron transfer. The cytochrome
bc
1
complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes
c
550
and
c
552
and to the
cbb
3
-type oxidase. Cytochrome
c
552
is an electron acceptor both of the cytochrome
bc
1
complex and of amicyanin, as well as a dedicated electron donor to the
aa
3
-type oxidase. Cytochrome
c
550
can accept electrons from the cytochrome
bc
1
complex and from amicyanin, whereas it is also the electron donor to both cytochrome
c
oxidases and to at least the nitrite reductase during denitrification. Deletion of the
c
-type cytochromes also affected the concentrations of remaining cytochromes
c
, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.
Use of Transposon Tn
5367
Mutagenesis and a Nitroimidazopyran-Based Selection System To Demonstrate a Requirement for
fbiA
and
fbiB
in Coenzyme F
420
Biosynthesis by
Mycobacterium bovis
BCG
Authors: Choi Kwang-Pil , Bair Thomas B. , Bae Young-Min , Daniels Lacy
Published: July 2002

Three transposon Tn
5367
mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of
Mycobacterium bovis
strain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F
420
was developed using the nitroimidazopyran-based antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defective in F
420
accumulation. Two mutants that could not make F
420
-5,6 but which made the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in two adjacent homologues of
Mycobacterium tuberculosis
genes, which we have named
fbiA
and
fbiB
for F
420
biosynthesis. Homologues of
fbiA
were found in all seven microorganisms that have been fully sequenced and annotated and that are known to make F
420
.
fbiB
homologues were found in all but one such organism. Complementation of the
fbiA
mutant with
fbiAB
and complementation of the
fbiB
mutant with
fbiB
both restored the F
420
-5,6 phenotype. Complementation of the
fbiA
mutant with
fbiA
or
fbiB
alone did not restore the F
420
-5,6 phenotype, but the
fbiA
mutant complemented with
fbiA
produced F
420
-2,3,4 at levels similar to F
420
-5,6 made by the wild-type strain, but produced much less F
420
-5. These data demonstrate that both genes are essential for normal F
420
-5,6 production and suggest that the
fbiA
mutation has a partial polar effect on
fbiB
. Reverse transcription-PCR data demonstrated that
fbiA
and
fbiB
constitute an operon. However, very low levels of
fbiB
mRNA are produced by the
fbiA
mutant, suggesting that a low-level alternative start site is located upstream of
fbiB
. The specific reactions catalyzed by FbiA and FbiB are unknown, but both function between FO and F
420
-5,6, since FO is made by both mutants.
New Regulatory Gene That Contributes to Control of
Bacteroides thetaiotaomicron
Starch Utilization Genes
Authors: Cho Kyu Hong , Cho Diedre , Wang Gui-Rong , Salyers Abigail A.
Published: July 2002

Bacteroides thetaiotaomicron
uses starch as a source of carbon and energy. Early steps in the pathway of starch utilization, such as starch binding and starch hydrolysis, are encoded by
sus
genes, which have been characterized previously. The
sus
structural genes are expressed only if cells are grown in medium containing maltose or higher oligomers of glucose. Regulation of the
sus
structural genes is mediated by SusR, an activator that is encoded by a gene located next to the
sus
structural genes. A strain with a disruption in
susR
cannot grow on starch but can still grow on maltose and maltotriose. A search for transposon-generated mutants that could not grow on maltose and maltotriose unexpectedly located a gene, designated
malR
, which regulates expression of an α-glucosidase not controlled by SusR. Although a disruption in
susR
did not affect expression of the
malR
controlled gene, a disruption in
malR
reduced expression of the
sus
structural genes. Thus, MalR appears to participate with SusR in regulation of the
sus
genes. Results of transcriptional fusion assays and reverse transcription-PCR experiments showed that
malR
is expressed constitutively. Moreover, multiple copies of
malR
provided on a plasmid (5 to 10 copies per cell) more than doubled the amount of α-glucosidase activity in cell extracts. Our results demonstrate that the starch utilization system of
B. thetaiotaomicron
is controlled on at least two levels by the regulatory proteins SusR and MalR.
Hyperactive Glycogen Synthase Mutants of
Saccharomyces cerevisiae
Suppress the
glc7-1
Protein Phosphatase Mutant
Authors: Anderson Catherine , Tatchell Kelly
Published: July 2002

A yeast
glc7-1
mutant expressing a variant of protein phosphatase type 1 fails to accumulate glycogen. This defect is associated with hyperphosphorylated and inactive glycogen synthase, consistent with Glc7p acting directly to dephosphorylate and activate glycogen synthase. To characterize the glycogen synthesis defect of this mutant in more detail, we isolated 26 pseudorevertants of the
glc7-1
mutant. All pseudoreversion events were due to missense mutations in
GSY2
, the gene encoding the major isoform of glycogen synthase. A majority of the mutations responsible for the suppression were in the 3′ end of the gene, corresponding to the phosphorylated COOH terminus of Gsy2p. Phosphorylation of the mutant proteins was reduced, suggesting that they are poor substrates for glycogen synthase kinases. Suppressor mutations outside this domain did not decrease the phosphorylation of the resulting proteins, indicating that these proteins are immune to the regulatory effects of phosphorylation. Since no growth defect has been observed for strains with altered glycogen levels, the relative levels of fitness of
GSY2
mutants that fail to accumulate glycogen and that hyperaccumulate glycogen were assayed by cocultivation experiments. A wild-type strain outcompeted both hypo- and hyperaccumulating strains, suggesting that glycogen levels contribute substantially to the fitness of yeast.
Periplasmic Transit and Disulfide Bond Formation of the Autotransported
Shigella
Protein IcsA
Authors: Brandon Lauren D. , Goldberg Marcia B.
Published: July 2002

The
Shigella
outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal β domain forms a β barrel channel in the outer membrane through which the amino-terminal α domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by
Shigella
, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the
dsbB
gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.
Putative Sigma Factor SigI (YkoZ) of
Bacillus subtilis
Is Induced by Heat Shock
Authors: Zuber Ulrich , Drzewiecki Kathrin , Hecker Michael
Published: July 2002

A
Bacillus subtilis
disruption mutant with a mutation in
sigI
(formerly
ykoZ
) shows a temperature-sensitive growth on agar plates. The transcription of the
sigI
gene is heat shock induced in rich medium but not in minimal medium. Proteome studies revealed a reduced amount of GsiB protein in the
sigI
mutant under heat shock conditions.
Mobile Cytochrome
c
2
and Membrane-Anchored Cytochrome
c
y
Are Both Efficient Electron Donors to the
cbb
3
- and
aa
3
-Type Cytochrome
c
Oxidases during Respiratory Growth of
Rhodobacter sphaeroides
Authors: Daldal Fevzi , Mandaci Sevnur , Winterstein Christine , Myllykallio Hannu , Duyck Kristen , Zannoni Davide
Published: July 2002

We have recently established that the facultative phototrophic bacterium
Rhodobacter sphaeroides
, like the closely related
Rhodobacter capsulatus
species, contains both the previously characterized mobile electron carrier cytochrome
c
2
(cyt
c
2
) and the more recently discovered membrane-anchored cyt
c
y
. However,
R. sphaeroides
cyt
c
y
, unlike that of
R. capsulatus
, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt
bc
1
complex during photosynthetic growth. Nonetheless,
R. sphaeroides
cyt
c
y
can act at least in
R. capsulatus
as an electron carrier between the cyt
bc
1
complex and the
cbb
3
-type cyt
c
oxidase (
cbb
3
-C
ox
) to support respiratory growth. Since
R. sphaeroides
harbors both a
cbb
3
-C
ox
and an
aa
3
-type cyt
c
oxidase (
aa
3
-C
ox
), we examined whether
R. sphaeroides
cyt
c
y
can act as an electron carrier to either or both of these respiratory terminal oxidases.
R. sphaeroides
mutants which lacked either cyt
c
2
or cyt
c
y
and either the
aa
3
-C
ox
or the
cbb
3
-C
ox
were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt
bc
1
complex and the cyt
c
oxidases. They were characterized with respect to growth phenotypes, contents of
a
-,
b
-, and
c
-type cytochromes, cyt
c
oxidase activities, and kinetics of electron transfer mediated by cyt
c
2
or cyt
c
y
. The findings demonstrated that both cyt
c
2
and cyt
c
y
are able to carry electrons efficiently from the cyt
bc
1
complex to either the
cbb
3
-C
ox
or the
aa
3
-C
ox
. Thus, no dedicated electron carrier for either of the cyt
c
oxidases is present in
R. sphaeroides
. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt
bc
1
complex appears to be mediated via the cyt
c
2
-to-
cbb
3
-C
ox
and cyt

c
y

-to-
cbb
3
-C
ox
subbranches. The presence of multiple electron carriers and cyt
c
oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of
R. sphaeroides
are more complex than those of
R. capsulatus
.
Escherichia coli
Ribosome-Associated Protein SRA, Whose Copy Number Increases during Stationary Phase
Authors: Izutsu Kaori , Wada Chieko , Komine Yuriko , Sako Tomoyuki , Ueguchi Chiharu , Nakura Satomi , Wada Akira
Published: July 2002

Protein D has previously been demonstrated to be associated with
Escherichia coli
ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the
E. coli
genetic map, between
ompC
and
sfcA
. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named
sra
. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of
sra
in the stationary phase.
AerR, a Second Aerobic Repressor of Photosynthesis Gene Expression in
Rhodobacter capsulatus
Authors: Dong Chen , Elsen Sylvie , Swem Lee R. , Bauer Carl E.
Published: July 2002

Open reading frame
orf192
, which is located immediately upstream of the aerobic repressor gene
crtJ
, was genetically and biochemically demonstrated to code for a second aerobic repressor (AerR) of photosynthesis gene expression in
Rhodobacter capsulatus
. Promoter-mapping studies indicate that
crtJ
has its own promoter but that a significant proportion of
crtJ
expression is promoted by read-through transcription of
orf192
(
aerR
) transcripts through
crtJ
. Disruption of
aerR
resulted in increased photopigment biosynthesis during aerobic growth to a level similar to that of disruption of
crtJ
. Like that reported for CrtJ, β-galactosidase assays of reporter gene expression indicated that disruption of
aerR
resulted in a two- to threefold increase in aerobic expression of the
crtI
and
pucB
operons. However, unlike CrtJ, AerR aerobically represses
puf
operon expression and does not aerobically repress
bchC
expression. Gel mobility shift analysis with purified AerR indicates that AerR does not bind to a
bchC
promoter probe but does bind to the
crtI
,
puc
, and
puf
promoter probes. These results indicate that AerR is a DNA-binding protein that targets genes partially overlapping a subset of genes that are also controlled by CrtJ. We also provide evidence for cooperative binding of AerR and CrtJ to the
puc
promoter region.
Coordination of Ubiquinol Oxidase and Cytochrome
cbb
3
Oxidase Expression by Multiple Regulators in
Rhodobacter capsulatus
Authors: Swem Danielle L. , Bauer Carl E.
Published: July 2002

Rhodobacter capsulatus
utilizes two terminal oxidases for aerobic respiration, cytochrome
cbb
3
and ubiquinol oxidase. To determine the transcription factors involved in terminal oxidase expression,
ccoN
-
lacZ
and
cydA
-
lacZ
protein fusions were assayed in a variety of regulatory mutants. The results of this and previous studies indicate that cytochrome
cbb
3
expression is controlled by
regB
-
regA
,
fnrL
, and
hvrA
and that ubiquinol oxidase expression is controlled by
regB
-
regA
,
fnrL
,
hvrA
,
crtJ
, and
aerR
.
Dam- and OxyR-Dependent Phase Variation of
agn43
: Essential Elements and Evidence for a New Role of DNA Methylation
Authors: Wallecha Anu , Munster Vincent , Correnti Jason , Chan Teresa , van der Woude Marjan
Published: July 2002

Phase variation of the outer membrane protein Ag43 in
E. coli
requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene,
agn43
, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of
agn43
transcription and its regulation. Transcription was initiated from a σ
70
-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of
agn′-lacZ
with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating
agn43
transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the
agn43
regulatory region from −16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the
agn43
regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation
Molecular Organization of Intrinsic Restriction and Modification Genes
Bsu
M of
Bacillus subtilis
Marburg
Authors: Ohshima Hideyuki , Matsuoka Satoshi , Asai Kei , Sadaie Yoshito
Published: July 2002

Transcriptional analysis and disruption of five open reading frames (ORFs),
ydiO
,
ydiP
,
ydiR
,
ydiS
, and
ydjA
, in the prophage 3 region of the chromosome of
Bacillus subtilis
Marburg revealed that they are component genes of the intrinsic
Bsu
M restriction and modification system of this organism. The classical mutant strain RM125, which lacks the restriction and modification system of
B. subtilis
Marburg, lacks the prophage 3 region carrying these five ORFs. These ORFs constitute two operons, the
ydiO-ydiP
operon and the
ydiR
-
ydiS
-
ydjA
operon, both of which are expressed during the logarithmic phase of growth. The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases. The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known. Disruption of the
ydiR-ydiS-ydjA
operon resulted in enhanced transformation by plasmid DNA carrying multiple
Bsu
M target sequences. Disruption of
ydiO
or
ydiP
function requires disruption of at least one of the following genes on the chromosome:
ydiR
,
ydiS
, and
ydjA
. The degrees of methylation of the
Bsu
M target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with
Xho
I (an isoschizomer of
Bsu
M) of DNAs extracted from the disruptant strains. Six
Xho
I (
Bsu
M) sites were examined.
Xho
I digested at the
Xho
I sites in the DNAs from disruptants with disruptions in both operons, while
Xho
I did not digest at the
Xho
I sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the
ydiR-ydiS-ydjA
operon. Therefore, the
ydiO-ydiP
operon and the
ydiR-ydiS-ydjA
operon are considered operons that are responsible for
Bsu
M modification and
Bsu
M restriction, respectively.
Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27
Authors: Lawley Trevor D. , Gilmour Matthew W. , Gunton James E. , Standeven Leah J. , Taylor Diane E.
Published: July 2002

The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region betweentraHandtrhRwas cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of theoriTregion are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of theoriTregion are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.
Identification of a Contaminating
Mycobacterium tuberculosis
Strain with a Transposition of an IS
6110
Insertion Element Resulting in an Altered Spoligotype
Authors: Benjamin William H. , Lok Kerry H. , Harris Randall , Brook Nancy , Bond Lisa , Mulcahy Donna , Robinson Nancy , Pruitt Virginia , Kirkpatrick deNay P. , Kimerling Michael E. , Dunlap Nancy E.
Published: July 2002

Molecular fingerprinting with the IS
6110
insertion sequence is useful for tracking transmission of
Mycobacterium tuberculosis
within a population or confirming specimen contamination in the laboratory or through instrumentation. Secondary typing with other molecular methods yields additional information as to the relatedness of strains with similar IS
6110
fingerprints. Isolated, relatively rare, random events within the
M. tuberculosis
genome alter molecular fingerprinting patterns with any of the methods; therefore, strains which are different by two or more typing methods are usually not considered to be closely related. In this report, we describe two strains of
M. tuberculosis
, obtained from the same bronchoscope 2 days apart, that demonstrated unique molecular fingerprinting patterns by two different typing methods. They were closely linked through the bronchoscope by a traditional epidemiologic investigation. Genetic analysis of the two strains revealed that a single event, the transposition of an IS
6110
insertion sequence in one of the strains, accounted for both the differences in the IS
6110
pattern and the apparent deletion of a spacer in the spoligotype. This finding shows that a single event can change the molecular fingerprint of a strain in two different molecular typing systems, and thus, molecular typing cannot be the only means used to track transmission of this organism through a population. Traditional epidemiologic techniques are a necessary complement to molecular fingerprinting so that radical changes within the fingerprint pattern can be identified.
Unsatisfactory Performance of Flow Cytometer UF-100 and Urine Strips in Predicting Outcome of Urine Cultures
Authors: Zaman Zahur , Roggeman Sylvie , Verhaegen Jan
Published: July 2002

UF-100 flow cytometer and urine strip results were cross-interpreted to predict culture outcomes. The best negative predictive value was obtained with bacteria at ≥1,000/μl, white blood cells at ≥20/μl, or leukocyte esterase positivity. Nine of 24 false negatives were clinically significant. Thus, UF-100 and urine strip results do not accurately predict the outcome of cultures.
No Evidence of Infectious Retroviruses in Measles Virus Vaccines Produced in Chicken Embryo Cell Cultures
Authors: Shahabuddin Muhammad , Sears Johnna F. , Khan Arifa S.
Published: July 2002

All vaccines that are prepared in chicken embryo fibroblasts (CEFs) contain a low level of particle-associated reverse transcriptase (RT) activity, which is produced from the avian cell substrate. The RNAs present in the particles have sequence homology to viral DNAs belonging to the ancient endogenous avian virus (EAV) family or to the avian sarcoma-leukosis virus (ALV)-related subgroup E endogenous virus loci. Although no replication-competent retrovirus has been associated with the RT activity produced from CEFs, there have been some theoretical safety concerns regarding potential consequences of integration of EAV and ALV sequences in human DNA, which may result from nonproductive infection with replication-defective particles or infection with EAV and ALV pseudotypes bearing measles virus envelopes. To address these possibilities, we have analyzed EAV and ALV particles in a measles virus vaccine equivalent (MVVE) preparation, obtained from a U.S. manufacturer, for integration and for replication in human peripheral blood mononuclear cells (PBMCs). The results show the absence of EAV and ALV integrants in DNA prepared from MVVE-inoculated human cells by direct DNA PCR and
Alu
PCR assays and no propagation of retrovirus in 18-day cultures of MVVE-inoculated human PBMCs by a highly sensitive PCR-based RT assay. These results provide further confidence regarding the safety of chicken RT activity in live viral vaccines and support the continued use of chick-cell-derived vaccines in humans.
Nosocomial Spread of an Unusual Methicillin-Resistant
Staphylococcus aureus
Clone That Is Sensitive to All Non-β-Lactam Antibiotics, Including Tobramycin
Authors: Pournaras Spyros , Slavakis Aris , Polyzou Aleka , Sofianou Danai , Maniatis Antonios N. , Tsakris Athanassios
Published: July 2002

Between January and December 1999, in Hippokration General Hospital, Thessaloniki, Greece, a large proportion of the methicillin-resistant
Staphylococcus aureus
isolates (34.4%) exhibited susceptibility to virtually all alternative non-β-lactam antibiotics, including tobramycin. Twenty-five of them were selected randomly for further testing; all belonged to a unique genotype and were characterized as heterogeneously resistant to oxacillin. The
aadD
gene, encoding tobramycin resistance, failed to be amplified in all cases, indicating absence of the gene or the entire plasmid pUB110 from the
mec
DNA.
Heterogeneity of
Pneumocystis
Sterol Profiles of Samples from Different Sites in the Same Pair of Lungs Suggests Coinfection by Distinct Organism Populations
Authors: Amit Zunika , Kaneshiro Edna S.
Published: July 2002

Sterol profiles of samples taken from different sites of a
Pneumocystis
-infected human lung showed large variations in pneumocysterol similar to those that occur among samples from different patients. Thus, the influence of diet or drugs on pneumocysterol accumulation was ruled out, suggesting distinct phenotypic populations as the basis for the heterogeneity.
Phage Types and Genotypes of Shiga Toxin-Producing
Escherichia coli
O157 in Finland
Authors: Saari Marjut , Cheasty Thomas , Leino Kirsikka , Siitonen Anja
Published: July 2002

This study examined Shiga toxin-producing
Escherichia coli
(STEC) O157, using phage typing, pulsed-field gel electrophoresis, and typing of Shiga toxin variant genes by PCR with restriction fragment length polymorphism in an epidemiological survey of STEC O157 isolated from humans in Finland between 1990 and 1999.
Novel Mycobacterium Related to
Mycobacterium triplex
as a Cause of Cervical Lymphadenitis
Authors: Hazra Rohan , Floyd Margaret M. , Sloutsky Alexander , Husson Robert N.
Published: July 2002

The
Mycobacterium avium
complex (MAC) is an important cause of cervical lymphadenitis in children, and its incidence appears to be increasing in the United States and elsewhere. In areas where
Mycobacterium tuberculosis
is not prevalent,
M. avium
causes the vast majority of cases of mycobacterial lymphadenitis, although several other nontuberculous mycobacterial species have been reported as etiologic agents. This report describes the case of a child with cervical lymphadenitis caused by a nontuberculous mycobacterium that could not be identified using standard methods, including biochemical reactions and genetic probes. Direct 16S ribosomal DNA sequencing showed greater than 99% homology with
Mycobacterium triplex
, but sequence analysis of the 283-bp 16S-23S internal transcribed spacer (ITS) sequence showed only 95% identity, suggesting that it is a novel species or subspecies within a complex of organisms that includes
M. triplex
. Mycolic acid high-performance liquid chromatography analysis also identified this isolate as distinct from
M. triplex
, and differences in susceptibility to streptomycin and rifampin between this strain and
M. triplex
were also observed. These data support the value of further testing of clinical isolates that test negative with the MAC nucleic acid probes and suggest that standard methods used for the identification of mycobacteria may underestimate the complexity of the genus
Mycobacterium
. ITS sequence analysis may be useful in this setting because it is easy to perform and is able to distinguish closely related species and subspecies. This level of discrimination may have significant clinical ramifications, as closely related organisms may have different antibiotic susceptibility patterns.
A Combination of Two Genetic Markers Is Sufficient for Restriction Fragment Length Polymorphism Typing of
Mycobacterium tuberculosis
Complex in Areas with a High Incidence of Tuberculosis
Authors: Rasolofo-Razanamparany Voahangy , Ramarokoto Herimanana , Aurégan Guy , Gicquel Brigitte , Chanteau Suzanne
Published: July 2002

The incidence of tuberculosis (TB) in Madagascar is 150 cases per 100,000 people. Because of this endemicity, we studied the genetic diversity of
Mycobacterium tuberculosis
strains isolated in four big cities in 1994 to 1995 with the aim of monitoring TB transmission. Isolates from 316 cases of pulmonary TB (PTM
+
) were typed by Southern hybridization with genetic markers IS
6110
and DR. Of the 316 PTM
+
strains, 66 (20.8%) had a single IS
6110
band and were differentiated by the DR marker into 33 profiles. Using both markers, 37.7% (119) of the patients were clustered, a proportion similar to that in countries with a high prevalence of TB. There was no significant difference between clustered and nonclustered patients in age, sex,
Mycobacterium bovis
BCG status, and drug susceptibility of strains. Clustering was significantly greater in the capital, Antananarivo, than in the other cities, suggesting a higher rate of transmission. However, most of the patients in clusters were living in different areas, and, within a distance of 0.7 km, we did not find epidemiologically unrelated strains with the same restriction fragment length polymorphism profile. Despite an apparently low polymorphism, genetic markers such as IS
6110
are potentially valuable for monitoring TB transmission. However, the high proportion of Malagasy isolates with a single IS
6110
copy makes this marker alone unsuitable for typing. Additional markers such as DR are necessary for the differentiation of the isolates and for epidemiological surveys.
Random Amplified Polymorphic DNA Assay as a Rapid Tool in Screening for
Neisseria meningitidis
Serogroup C Isolates of Electrophoretic Type 24
Authors: Schmink Susanna , Reeves Michael W. , Plikaytis Brian , Popovic Tanja
Published: July 2002

Neisseria meningitidis
serogroup C (NMSC) isolates of electrophoretic type 24 (ET-24), as identified by multilocus enzyme electrophoresis, are the main cause of serogroup C meningococcal disease outbreaks and sporadic meningococcal disease in the United States. We evaluated a random amplified polymorphic DNA assay as a rapid tool to screen for isolates of ET-24 by testing 199 NMSC isolates of 51 different ETs. A sensitivity of 88% and a specificity of 87% was achieved in identification of ET-24 isolates when the patterns obtained by two primers, P1 and P5, were analyzed together.
PCR-Hybridization Assay for
Mycobacterium avium
Complex: Optimization of Detection in Peripheral Blood from Humans
Authors: Ferrario Giulio , Gori Andrea , Rossi Agostino , Catozzi Lidia , Molteni Chiara , Marchetti Giulia , Bandera Alessandra , Rossi Maria Cristina , Esposti Anna Degli , Franzetti Fabio
Published: July 2002

We evaluated the sensitivity of a DNA amplification test for the detection of
Mycobacterium avium
in blood samples using different blood components and different DNA extraction methods.
M. avium
-inoculated blood samples were processed to obtain separate blood components: peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNCs), and whole-blood sodium dodecyl sulfate (SDS)-lysate pellets. The sensitivity for the detection of the lowest mycobacterial load (1 CFU/ml) was significantly greater (
P
< 0.01) with DNA extracted from SDS-lysate pellets than with DNA extracted from PBMCs or PMNCs. Subsequently, DNA extraction methods based on guanidine NaOH, and proteinase were compared. The sensitivity of the guanidine-based method was significantly greater (
P
< 0.01) than those of the others.
One-Step Purification of
Enterocytozoon bieneusi
Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption
Authors: Accoceberry Isabelle , Thellier Marc , Datry Annick , Desportes-Livage Isabelle , Biligui Sylvestre , Danis Martin , Santarelli Xavier
Published: July 2002

An original, reliable, and reproducible method for the purification of
Enterocytozoon bieneusi
spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of
E. bieneusi
spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous
E. bieneusi
spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT).
E. bieneusi
spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10
7
spores were purified per run. This technique for isolating
E. bieneusi
spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.
Development of an
env
gp41-Based Heteroduplex Mobility Assay for Rapid Human Immunodeficiency Virus Type 1 Subtyping
Authors: Agwale S. M. , Robbins K. E. , Odama L. , Saekhou A. , Zeh C. , Edubio A. , Njoku O. M. , Sani-Gwarzo N. , Gboun M. F. , Gao F. , Reitz M. , Hone D. , Folks T. M. , Pieniazek D. , Wambebe C. , Kalish M. L.
Published: July 2002

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (
env
) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an
env
gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.
Amplified Fragment Length Polymorphism Fingerprinting for Identification of a Core Group of
Neisseria gonorrhoeae
Transmitters in the Population Attending a Clinic for Treatment of Sexually Transmitted Diseases in Amsterdam, The Netherlands
Authors: Spaargaren Joke , Stoof Jeroen , Fennema Han , Coutinho Roel , Savelkoul Paul
Published: July 2002

Amplified fragment length polymorphism analysis seems well suited for studying the epidemiology of isolates of
Neisseria gonorrhoeae
obtained from patients attending the Sexually Transmitted Disease Outpatient Clinic in Amsterdam, The Netherlands. It shows potential to identify the core group of transmitters.
Genetic Diversity and Biochemical Characteristics of
Trichosporon asahii
Isolated from Clinical Specimens, Houses of Patients with Summer-Type-Hypersensitivity Pneumonitis, and Environmental Materials
Authors: Sugita Takashi , Ichikawa Tomoe , Matsukura Manami , Sueda Mika , Takashima Masako , Ikeda Reiko , Nishikawa Akemi , Shinoda Takako
Published: July 2002

Trichosporon asahii
, which is distributed in the environment, is the major causative agent of the opportunistic infection trichosporonosis, and it also causes summer-type hypersensitivity pneumonitis (SHP). Random amplification of polymorphic DNA analysis was used to determine the intraspecies diversity of 39
T. asahii
isolates from clinical specimens, SHP patients' houses, and environmental materials. The three primers used revealed 46 polymorphic bands. A phenogram was generated by the unweighted pair-group method with arithmetic mean. Clinical isolates formed a cluster, characterized by a 90% matching coefficient, but they did not cluster with strains isolated from SHP patients' houses or environmental sources. In addition, the biochemical characteristics of 86 strains from three sources were examined with 31 compounds using an ID32C kit, and a phenogram was constructed. The phenogram consisted of three major clusters. Cluster I included most of the clinical SHP isolates, and cluster II included most of the environmental isolates. Cluster III contained only one strain. A remarkable difference was found in the abilities of the strains belonging to clusters I and II to utilize six compounds. These results suggest that the genetic diversity and biochemical characteristics of
T. asahii
seem to be related to the source of the isolate. We also found a specific DNA fragment for the clinical isolates and strains isolated from SHP patients' houses.
Utility of 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenyl-Amino)Carbonyl]-2
H
-Tetrazolium Hydroxide (XTT) and Minimum Effective Concentration Assays in the Determination of Antifungal Susceptibility of
Aspergillus fumigatus
to the Lipopeptide Class of Compounds
Authors: Hawser S. P. , Jessup C. , Vitullo J. , Ghannoum M. A.
Published: July 2002

The susceptibility of
Aspergillus fumigatus
to mulundocandin, an echinocandin-like compound, and other antifungal agents was assessed by the National Committee for Clinical Laboratory Standards (NCCLS) M38-P method, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2
H
-tetrazolium hydroxide (XTT)-based colorimetric assay, and determination of morphologic alterations by microscopy. In contrast to the NCCLS M38-P method, which does not predict the activity in vivo, the XTT-based assay showed that
A. fumigatus
is susceptible to mulundocandin. Thus, the XTT-based assay might be useful for determination of the susceptibilities of molds to echinocandins. Further evaluation is warranted.
First Report of Human Infection Due to the Fungus
Triadelphia pulvinata
Authors: Al-Hedaithy Saleh S. A.
Published: July 2002

Triadelphia pulvinata
, a soil hyphomycete, was found to be the cause of eczematoid, scaly, grey lesions on the skin of both eyelids of a 30-year-old Indian male living in Saudi Arabia. Repeated KOH preparations of the skin scrapings showed presence of sclerotic, branched, septate hyphae. When cultured, skin scrapings from the lesion grew the dematiaceous fungus
T. pulvinata
. Treatment with topical clotrimazole cured the infection, and no recurrence of the infection was noted in a 5-year follow-up.
Phylogenetic Origin of Hepatitis B Virus Strains with Precore C-1858 Variant
Authors: Alestig Erik , Hannoun Charles , Horal Peter , Lindh Magnus
Published: July 2002

Mutations that prevent the expression of the hepatitis B e antigen frequently emerge in the immunoreactive phase of infection. The predominant mutation, the precore G→A-1896 mutation, is restricted by the variability at position 1858 and is rare in strains with cytosine at nucleotide 1858. The C-1858 variant is characteristic of genotype A. It also occurs in genotypes C and F, but not in B, D, or E, explaining the geographical variation in the prevalence of precore mutants. C-1858 strains have been frequently observed in southeast Asia, but have not been phylogenetically characterized. By sequencing eight complete hepatitis B virus genomes, C-1858 variants of east Asian origin were found to constitute a phylogenetic entity within genotype C that probably diverged several hundred years ago. Further study of the distribution of this variant is warranted.
Performance Characteristics of the COBAS Amplicor Hepatitis C Virus (HCV) Monitor, Version 2.0, International Unit Assay and the National Genetics Institute HCV Superquant Assay
Authors: Konnick Eric Q. , Erali Maria , Ashwood Edward R. , Hillyard David R.
Published: July 2002

The COBAS Amplicor Hepatitis C Virus (HCV) Monitor assay, version 2.0, which reports in international units per milliliter, was compared to the assay reported in copies per milliliter by analyzing dilution series and clinical plasma samples by both methods. In addition, the Amplicor international unit assay was compared to the National Genetics Institute HCV Superquant assay. The dilution series ranged from <100 to 5,000,000 HCV RNA copies/ml and consisted of 32 points, assayed in triplicate in each assay. Thirty clinical samples ranging from 1,000 to 1,000,000 HCV RNA copies/ml were assayed in duplicate. Deming regression analysis comparing the Amplicor HCV RNA international units-per-milliliter and copies-per-milliliter assays was calculated as follows: (Amplicor international units per milliliter) = 1.030(Amplicor copies per milliliter) − 0.392;
R
2
= 0.981;
n
= 28;

S
y/x

(standard error of the estimate) = 0.129. The linearity of the Amplicor international units-per-milliliter assay was as follows: observed = 0.886(expected) + 0.437;
R
2
= 0.983;
n
= 30. The linearity of the Superquant assay was as follows: observed= 0.918 (expected) + 0.436;
R
2
= 0.986;
n
= 32. Deming regression analysis comparing the Amplicor and Superquant assays was calculated as follows: Superquant = 1.066(Amplicor) − 0.0197;
R
2
= 0.908;

S
y/x

= 0.308;
n
= 28. The Amplicor and Superquant assays were linear through the range of 600 to 600,000 IU of HCV RNA/ml and ∼300 to 5,000,000 HCV RNA copies/ml, respectively. The narrow range of the Amplicor assay means that some samples will require dilution and retesting for accurate quantification above 600,000 IU of HCV RNA/ml. The Amplicor and Superquant assays agreed well within the range of 600 to 600,000 IU of HCV RNA/ml (∼1,000 to ∼1,000,000 HCV RNA copies/ml). Overall, the Amplicor and Superquant assays agree well, and results obtained in one assay could be expected to compare well with results from the other when reported in copies per milliliter.
Detection of Neutralizing Antibodies against Human Papillomaviruses (HPV) by Inhibition of Gene Transfer Mediated by HPV Pseudovirions
Authors: Bousarghin Latifa , Combita-Rojas Alba-Lucia , Touzé Antoine , El Mehdaoui Slimane , Sizaret Pierre-Yves , Bravo Maria-Mercedes , Coursaget Pierre
Published: July 2002

The goal of this study was to develop a human papillomavirus (HPV) neutralization assay using HPV pseudovirions generated in vitro. For this purpose, gene transfer efficiency of HPV virus-like particles (VLPs) was improved by using direct interaction between a reporter plasmid and the VLPs. Electron microscopic observation of the interaction between DNA molecules and VLPs revealed that VLPs always interact with a single DNA molecule and that VLPs bind to the end of linearized DNA molecules. An 100-fold improvement in the gene transfer was obtained by simple interaction between a linearized DNA molecule and VLPs. Moreover, direct interaction methods offer the possibility of transferring plasmids a size higher than that of the papillomavirus genome. The approach that we developed to generate HPV-16 and HPV-31 pseudovirions proved to be suitable for testing neutralizing antibodies in human sera both after immunization and after natural infection.
Multilocus Sequence Typing of
Bordetella pertussis
Based on Surface Protein Genes
Authors: van Loo Inge H. M. , Heuvelman Kees J. , King Audrey J. , Mooi Frits R.
Published: July 2002

Despite more than 50 years of vaccination,
Bordetella pertussis
has remained endemic in The Netherlands, causing epidemic outbreaks every 3 to 5 years. Strain variation may play a role in the persistence of
B. pertussis
and was studied by sequencing 15 genes coding for surface proteins, including genes for all five components of acellular pertussis vaccines: pertussis toxin (Ptx), pertactin (Prn), filamentous hemagglutinin, and fimbriae (Fim2 and Fim3). A low level of allelic variation was observed, confirming a recent evolutionary origin of
B. pertussis
. In modern isolates, polymorphism was observed only in
prn
,
ptxS1
,
ptxS3
, and
tcfA
. Polymorphism in
ptxS1
,
ptxS3
, and
tcfA
was used to categorize isolates in multilocus sequence types (MLSTs). Analysis of Dutch isolates from 1949 to 1999 revealed five MLSTs, which showed a highly dynamic temporal behavior. We observed significant changes in the MLSTs after the introduction of pertussis vaccination in The Netherlands. Epidemic years were found to be associated with the expansion of MLST-4 or MLST-5. MLST-5 showed a remarkable expansion from 10% in 1997 to 80% in 1999. The MLST analysis was extended to a number of widely separated geographic regions: Finland, Italy, Japan, and the United States. MLST-4 and MLST-5 were found to dominate in Italy and the United States. In Finland and Japan, MLST-3 and MLST-2, respectively, were predominant. Thus, although each region showed distinctive MLST frequencies, in three of the five regions MLST-4 and MLST-5 were predominant. These types may represent newly emerged, successful clones. The identification of highly successful clones may shed light on the question of how
B. pertussis
is able to maintain itself in vaccinated populations.
Fatal Endocarditis in a Neonate Caused by the Dematiaceous FungusPhialemonium obovatum: Case Report and Review of the Literature
Authors: Gavin Patrick J. , Sutton Deanna A. , Katz Ben Z.
Published: July 2002

Phialemoniumspecies are grouped by most authorities among the dematiaceous fungi. Like several other darkly pigmented fungi, they appear to be an emerging cause of human disease, especially in the immunocompromised host. As numbers of immunocompromised patients increase, the trend of unusual fungi emerging as new pathogens is set to continue. Previous experience suggests that reports of rare fungal infections among selected patients often heralds the emergence of infection in a wider population of patients. We report a case of fatal endocarditis caused byPhialemonium obovatumin a premature neonate. To our knowledge this is the first documented case of native valve endocarditis due to this species, and we review the literature on invasive disease caused by the species. Unfamiliar fungal isolates are frequently misidentified or mistaken for environmental contaminants, and because of their relative rarity, data upon which to base antifungal treatment are limited. Thus, the diagnosis and treatment of unusual fungal pathogens present a significant challenge to clinicians and microbiologists alike. Early referral of such isolates to a specialist reference laboratory is advisable.