bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively AlkaliphilicBacillus pseudofirmusOF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily
Authors: Gilmour Raymond , Messner Paul , Guffanti Arthur A. , Kent Rebecca , Scheberl Andrea , Kendrick Nancy , Krulwich Terry Ann
Published: July 2002

The large majority of proteins of alkaliphilicBacillus pseudofirmusOF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well.slpARNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. AnslpAmutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na+-replete or suboptimal Na+concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na+. By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na+-replete and suboptimal Na+conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na+-dependent transport of α-aminoisobutyric acid, or H+-dependent synthesis of ATP. However, the capacity for Na+-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.
BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in
Pseudomonas putida
Authors: Cowles Charles E. , Nichols Nancy N. , Harwood Caroline S.
Published: July 2002

Pseudomonas putida
converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded
meta
pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of
P. putida
to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of
pcaK
, the gene encoding the 4-HBA permease. Here we identified a gene,
benR
, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A
benR
mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to
benR
were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The
benABC
genes likely encode benzoate dioxygenase, and
benD
likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase.
benK
and
benF
were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene,
benE
, is not known.
benR
activated expression of a
benA-lacZ
reporter fusion in response to benzoate. It also activated expression of a
meta
cleavage operon promoter-
lacZ
fusion inserted in an
E. coli
chromosome. Third,
benR
was required for benzoate-mediated repression of
pcaK-lacZ
fusion expression. The
benA
promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the
meta
cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.
The Malonate Decarboxylase Operon of
Acinetobacter calcoaceticus
KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY
Authors: Koo Jae Hyung , Cho Ick Hyun , Kim Yu Sam
Published: July 2002

A regulatory gene-like open reading frame oriented oppositely to mdcL, coined
mdcY
, was found upstream from the structural genes of the
mdcLMACDEGBH
operon in
Acinetobacter calcoaceticus
KCCM 40902. To elucidate the function of this gene,
mdcY
was expressed in
Escherichia coli
, and the MdcY protein was purified to homogeneity. Its DNA binding activity and binding site were examined by gel retardation and footprinting assays in vitro and by site-directed mutagenesis of the binding sites in vivo. The regulator bound target DNA regardless of the presence of malonate, and the binding site was found centered at −65 relative to the
mdcL
transcriptional start site and contains a 12-bp palindromic structure (5′-ATTGTA/TACAAT-3′). Using a promoter fusion to the reporter gene
luc
, we found that the promoter P

mdcY

is negatively regulated by MdcY independent of malonate. However, the promoter P

mdcL

recovered its activity in the presence of malonate. When
mdcY
was introduced into
A. calcoaceticus
KCCM 40902 in which the gene is inactivated by an IS
3
family element, malonate decarboxylase was significantly repressed in cultures growing in acetate, succinate, or Luria-Bertani medium. However, in cells growing in malonate, malonate decarboxylase was induced, indicating that MdcY is a transcriptional repressor and that malonate or a product resulting from malonate metabolism should be the intracellular inducer of the
mdc
operon.
Flavodoxin Mutants of
Escherichia coli
K-12
Authors: Gaudu Philippe , Weiss Bernard
Published: July 2002

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of
Escherichia coli
, and its gene,
fldA
, is known to belong to the
soxRS
(superoxide response) oxidative stress regulon. An insertion mutation of
fldA
was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (
fldA
+
) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene,
fldB
. Unlike the
fldA
mutant, an
fldB
insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number
fldB
+
plasmid did not complement the
fldA
mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An
fldB-lacZ
fusion was not induced by H
2
O
2
and is therefore not a member of the
oxyR
regulon. However, it displayed a
soxS
-dependent induction by paraquat (methyl viologen), and the
fldB
gene is preceded by two overlapping regions that resemble known
soxS
binding sites. The
fldB
insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a
soxS-lacZ
fusion. Therefore,
fldB
is a new member of the
soxRS
(superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.
Osmoregulated Periplasmic Glucan Synthesis Is Required for
Erwinia chrysanthemi
Pathogenicity
Authors: Page Frederic , Altabe Silvia , Hugouvieux-Cotte-Pattat Nicole , Lacroix Jean-Marie , Robert-Baudouy Janine , Bohin Jean-Pierre
Published: July 2002

Erwinia chrysanthemi
is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the
opgGH
operon of
E. chrysanthemi
, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus
mdoGH
of
Escherichia coli
. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the
opgG
or
opgH
gene abolish OPG synthesis. The
opg
mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.
Evidence that an Additional Mutation Is Required To Tolerate Insertional Inactivation of theStreptomyces lividans recAGene
Authors: Vierling Silke , Weber Tilmann , Wohlleben Wolfgang , Muth Günther
Published: July 2002

In contrast torecAof other bacteria, therecAgene ofStreptomyces lividanshas been described as indispensable for viability (G. Muth, D. Frese, A. Kleber, and W. Wohlleben, Mol. Gen. Genet. 255:420–428, 1997.). Therefore, a closer analysis of this gene was performed to detect possible unique features distinguishing theStreptomycesRecA protein from the well-characterizedEscherichia coliRecA protein. TheS. lividans recAgene restored UV resistance and recombination activity of anE. coli recAmutant. Also, transcriptional regulation was similar to that ofE. coli recA. Gel retardation experiments showed thatS. lividans recAis also under control of theStreptomycesSOS repressor LexA. TheS. lividans recAgene could be replaced only by simultaneously expressing a plasmid encodedrecAcopy. Surprisingly, therecAexpression plasmid could subsequently be eliminated using an incompatible plasmid without the loss of viability. Besides being UV sensitive and recombination deficient, all the mutants were blocked in sporulation. Genetic complementation restored UV resistance and recombination activity but did not affect the sporulation defect. This indicated that all therecAmutants had suffered from an additional mutation, which might allow toleration of arecAdeficiency.
Respiration Capacity of the Fermenting Bacterium
Lactococcus lactis
and Its Positive Effects on Growth and Survival
Authors: Duwat Patrick , Sourice Sophie , Cesselin Bénédicte , Lamberet Gilles , Vido Karin , Gaudu Philippe , Le Loir Yves , Violet Florent , Loubière Pascal , Gruss Alexandra
Published: July 2002

Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe
Lactococcus lactis
, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to
L. lactis
if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of
L. lactis
to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The
cydA
gene, encoding cytochrome
d
oxidase, is required for respiration and plays a direct role in oxygen utilization.
cydA
expression is induced late in growth under respiration conditions. (ii) The
hemZ
gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in
L. lactis
and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.
Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process
Authors: Park Hee-Sung , Kim Hak-Sung
Published: July 2002

The aminophenol (AP) catabolic operon in
Pseudomonas putida
HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol
meta
cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene,
nbzR
, and the following nine structural genes,
nbzJCaCbDGFEIH
, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other
meta
cleavage operons led us to suggest that the regulatory unit (
nbzR
) was derived from the MarR family and that the structural unit (
nbzJCaCbDGFEIH
) has evolved from the ancestral
meta
cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.
Oxygen- and Growth Rate-Dependent Regulation of
Escherichia coli
Fumarase (FumA, FumB, and FumC) Activity
Authors: Tseng Ching-Ping , Yu Chin-Chu , Lin Hsiao-Hsien , Chang Chi-Yen , Kuo Jong-Tar
Published: July 2002

Escherichia coli
contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to
l
-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (
k
) was lowered from 1.2/h to 0.24/h at 1 and 21% O
2
, respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a
cya
mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (
k
= 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters.
Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin
Authors: Dairi Tohru , Hamano Yoshimitsu , Kuzuyama Tomohisa , Itoh Nobuya , Furihata Kazuo , Seto Haruo
Published: July 2002

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned fromStreptomyces griseolosporeusstrain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627–1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed inStreptomyces lividanstogether with the GGDP synthase gene under the control of theermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid,ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced inEscherichia coliand purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.
Enhanced Symbiotic Performance by
Rhizobium tropici
Glycogen Synthase Mutants
Authors: Marroquı́ Silvia , Zorreguieta Angeles , Santamarı́a Carmen , Temprano Francisco , Soberón Mario , Megı́as Manuel , Downie J. Allan
Published: July 2002

We isolated a Tn
5
-induced
Rhizobium tropici
mutant that has enhanced capacity to oxidize
N,N
-dimethyl-
p
-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes
c
1
and CycM and a small increase in the amount of cytochrome
aa
3
. In plant tests, the mutant increased the dry weight of
Phaseolus vulgaris
plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from
Escherichia coli
: glycogen phosphorylase (
glgP
), glycogen branching enzyme (
glgB
), ADP glucose pyrophosphorylase (
glgC
), glycogen synthase (
glgA
), phosphoglucomutase (
pgm
), and glycogen debranching enzyme (
glgX
). All six genes are transcribed in the same direction, and analysis with
lacZ
gene fusions suggests that the first five genes are organized in one operon, although
pgm
appears to have an additional promoter;
glgX
is transcribed independently. Surprisingly, the
glgA
mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in
glgA
. This mutant had a phenotype similar to that of the Tn
5
mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of
glgA
and not to a polar effect on a downstream gene.
snr-1
Gene Is Required for Nitrate Reduction in
Pseudomonas aeruginosa
PAO1
Authors: Kerschen Edward J. , Irani Vida R. , Hassett Daniel J. , Rowe John J.
Published: July 2002

Pseudomonas aeruginosa
is able to use nitrate for both assimilation and anaerobic respiration. One set of genes, designated
snr
(for “shared nitrate reduction”), have been recently cloned and partially characterized. In this study, we demonstrate that the
snr-1
gene encodes a predicted 52.5-kDa protein that is 82% similar to a unique cytochrome
c
of
Desulfomonile tiedjei
DCB-1. Importantly, the Snr-1 protein sequence of
P. aeruginosa
differed from that of the cytochrome
c
of
D. tiedjei
primarily in the first 25 amino acids, which are required for membrane attachment in
D. tiedjei
. In
P. aeruginosa
, the Snr-1 protein hydropathy profile indicates that it is a soluble protein. An isogenic
snr-1
::Gm insertional mutant was unable to grow aerobically with nitrate as a sole nitrogen source or anaerobically with nitrate as an electron acceptor. Complementation of the
snr-1
::Gm mutant with the
snr-1
gene restored the wild-type phenotypes. Interestingly, anaerobic growth rates were significantly higher in the
snr-1
mutant harboring a multicopy plasmid containing
snr-1
. In contrast, aerobic growth rates of the restored mutant using nitrate as the sole nitrogen source were similar to those of the wild type. Transcriptional
lacZ
fusions demonstrated that
snr-1
was not regulated by molybdate, oxygen, or nitrate.
Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of
Aspergillus
Species
Authors: Meletiadis Joseph , Mouton Johan W. , Meis Jacques F. G. M. , Bouman Bianca A. , Donnelly Peter J. , Verweij Paul E. ,   EUROFUNG Network
Published: July 2002

The susceptibilities of 25 clinical isolates of various
Aspergillus
species (
Aspergillus fumigatus, A. flavus, A. terreus, A. ustus
, and
A. nidulans
) to itraconazole (ITC) and amphotericin B (AMB) were determined using the standard proposed by NCCLS for antifungal susceptibility testing of filamentous fungi, a modification of this method using spectrophotometric readings, and a colorimetric method using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT). Five MIC end points for ITC (MIC-0, no visible growth or ≤5% the growth control value [GC]; MIC-1, slight growth or 6 to 25% the GC; MIC-2, prominent reduction in growth or 26 to 50% the GC; MIC-3, slight reduction in growth or 51 to 75% the GC; and MIC-4, no reduction in growth or 76 to 100% the GC) and one for AMB (MIC-0) were determined visually by four observers and spectrophotometrically. The intraexperimental (between the observers) and interexperimental (between the experiments) levels of agreement of the NCCLS and XTT methods exceeded 95% for MIC-0 of AMB and MIC-0 and MIC-1 of ITC. The MIC-2 of ITC showed lower reproducibility, although spectrophotometric reading and/or incubation for 48 h increased the interexperimental reproducibility from 85 to >93%. Between visual and spectrophotometric readings, high levels of agreement were found for AMB (≈97%) and MIC-1 (≈92%) and MIC-2 (≈88%) of ITC. Poor agreement was found for MIC-0 of ITC (51% after 24 h), since the spectrophotometric readings resulted in higher MIC-0 values than the visual readings. The agreement was increased to 98% by shifting the threshold level of MIC-0 from 5 to 10% relative optical density and by establishing an optical density of greater than 0.1 for the GC as the validation criterion. No statistically significant differences were found between the NCCLS method and the XTT method, with the levels of agreement exceeding 97% for MIC-0 of AMB and 83% for MIC-0, MIC-1, and MIC-2 of ITC. The XTT method and spectrophotometric readings can increase the sensitivity and the precision, respectively, of in vitro susceptibility testing of
Aspergillus
species.
Lone Star Tick-Infecting Borreliae Are Most Closely Related to the Agent of Bovine Borreliosis
Authors: Rich Stephen M. , Armstrong Philip M. , Smith Ronald D. , Telford Sam R.
Published: July 2002

AlthoughBorrelia theileri, the agent of bovine borreliosis, was described at the turn of the century (in 1903), its relationship with borreliae causing Lyme disease or relapsing fever remains undescribed. We tested the previously published hypothesis that spirochetes infecting Lone Star ticks (Amblyomma americanum) may compriseB. theileriby analyzing the 16S ribosomal DNAs (rDNAs) and flagellin genes of these spirochetes.B. theileri, theAmblyommaagent, andB. miyamotoiformed a natural group or clade distinct from but most closely related to that of the relapsing fever spirochetes.B. theileriand theAmblyommaagent were 97 and 98% similar at the nucleotide level within the analyzed portions of the 16S rDNA and the flagellin gene respectively, suggesting a recent divergence. The agent of bovine borreliosis might be explored as a surrogate antigen for the as-yet-uncultivatableAmblyommaagent in studies designed to explore the etiology of a Lyme disease-like infection associated with Lone Star ticks.
Sequence Analysis of the Direct Repeat Region in
Mycobacterium bovis
Authors: Caimi Karina , Romano Maria I. , Alito Alicia , Zumarraga Martin , Bigi Fabiana , Cataldi Angel
Published: July 2002

Spoligotyping is a major tool for molecular typing of
Mycobacterium bovis
. This technique is based on the polymorphism of spacers that separate direct repeats (DRs) in the
M. tuberculosis
complex DR region. Numerous
M. bovis
strains show a lack of several spacers which appears as a gap in the spoligotyping pattern. To determine whether these gaps contain alternative spacers not included in the spoligotyping membrane, PCRs using primers that hybridize to the spacers adjacent to the gaps were performed. Comparing the sizes of products obtained by PCR with those deduced from spoligotyping patterns, fragments were selected and sequenced to look for alternative spacers. Upon analysis of the sequences, five alternative spacers were detected, although deletions of spacers are mainly responsible for the observed gaps. The alternative spacers, which are more frequent in
M. bovis
than in
M. tuberculosis
, may contribute to increased
M. bovis
differentiation.
Rapid Identification of
Candida glabrata
Based on Trehalose and Sucrose Assimilation Using Rosco Diagnostic Tablets
Authors: Lopez José , Dalle Frédéric , Mantelin Pierre , Moiroux Philippe , Nierlich Anne-Charlotte , Pacot Agnès , Cuisenier Bernadette , Vagner Odile , Bonnin Alain
Published: July 2002

We developed a simple method for the identification of
Candida glabrata
on the basis of the ability of this species to rapidly assimilate trehalose but not sucrose. After incubation of yeasts with Rosco diagnostic tablets containing sucrose or trehalose, identification of
C. glabrata
was achieved in 4 h with 100% sensitivity and specificity.
cagA
Status and Eradication Treatment Outcome of Anti-
Helicobacter pylori
Triple Therapies in Patients with Nonulcer Dyspepsia
Authors: Broutet Nathalie , Marais Armelle , Lamouliatte Hervé , de Mascarel Antoine , Samoyeau Roland , Salamon Roger , Mégraud Francis
Published: July 2002

The differences in eradication rates reported in clinical trials aiming to cure
Helicobacter pylori
infection cannot be entirely explained by the type of regimen, bacterial resistance, or lack of compliance. Using data from a clinical trial, a logistic regression model was constructed to determine whether
cagA
status, assessed by PCR, affects the outcome of eradication. Resistance to clarithromycin (10% of the strains) predicted failure perfectly. In the model (
n
= 156), a
cagA
-lacking strain (odds ratio [OR] = 2.2; 95% confidence interval [CI], (1.1 to 4.7), tobacco smoking OR = 3.1; 95% CI, 1.3 to 7.0), and a double dose of proton pump inhibitor in the treatment regimen (OR = 0.3; 95% CI, 0.2 to 0.7) were associated with the treatment outcome. The exact role of
cagA
in the outcome of
H. pylori
eradication therapy has not been explored. However, the type of histological lesions which it causes in the gastric mucosa may be implicated. Regardless of the mechanism involved,
cagA
status is a good predictive marker of eradication outcome.
Hepatitis C Virus Genotyping Based on 5′ Noncoding Sequence Analysis (Trugene)
Authors: Halfon P. , Trimoulet P. , Bourliere M. , Khiri H. , de Lédinghen V. , Couzigou P. , Feryn J. M. , Alcaraz P. , Renou C. , Fleury H. J. A. , Ouzan D.
Published: July 2002

Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5′NC genotyping kit, based on sequence analysis of the 5′ noncoding region (5′ NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5′ NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.
Sensitivity and Specificity of Human Immunodeficiency Virus Rapid Serologic Assays and Testing Algorithms in an Antenatal Clinic in Abidjan, Ivory Coast
Authors: Koblavi-Dème Stéphania , Maurice Chantal , Yavo Daniel , Sibailly Toussaint S. , N′guessan Kabran , Kamelan-Tano Yvonne , Wiktor Stefan Z. , Roels Thierry H. , Chorba Terence , Nkengasong John N.
Published: July 2002

To evaluate serologic testing algorithms for human immunodeficiency virus (HIV) based on a combination of rapid assays among persons with HIV-1 (non-B subtypes) infection, HIV-2 infection, and HIV-1–HIV-2 dual infections in Abidjan, Ivory Coast, a total of 1,216 sera with known HIV serologic status were used to evaluate the sensitivity and specificity of four rapid assays: Determine HIV-1/2, Capillus HIV-1/HIV-2, HIV-SPOT, and Genie II HIV-1/HIV-2. Two serum panels obtained from patients recently infected with HIV-1 subtypes B and non-B were also included. Based on sensitivity and specificity, three of the four rapid assays were evaluated prospectively in parallel (serum samples tested by two simultaneous rapid assays) and serial (serum samples tested by two consecutive rapid assays) testing algorithms. All assays were 100% sensitive, and specificities ranged from 99.4 to 100%. In the prospective evaluation, both the parallel and serial algorithms were 100% sensitive and specific. Our results suggest that rapid assays have high sensitivity and specificity and, when used in parallel or serial testing algorithms, yield results similar to those of enzyme-linked immunosorbent assay-based testing strategies. HIV serodiagnosis based on rapid assays may be a valuable alternative in implementing HIV prevention and surveillance programs in areas where sophisticated laboratories are difficult to establish.
Rapid Pulsed-Field Gel Electrophoresis Protocol for Subtyping of
Campylobacter jejuni
Authors: Ribot Efrain M. , Fitzgerald Collette , Kubota Kristy , Swaminathan Bala , Barrett Timothy J.
Published: July 2002

We developed a rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping
Campylobacter
isolates based on the standardized protocols used by PulseNet laboratories for the subtyping of other food-borne bacterial pathogens. Various combinations of buffers, reagents, reaction conditions (e.g., cell suspension concentration, lysis time, lysis temperature, and restriction enzyme concentration), and electrophoretic parameters were evaluated in an effort to devise a protocol that is simple, rapid, and robust. PFGE analysis of
Campylobacter
isolates can be completed in 24 to 30 h using this protocol, whereas the most widely used current protocols require 3 to 4 days to complete. Comparison of PFGE patterns obtained in six laboratories showed that subtyping results obtained using this protocol are highly reproducible.
Outcome of Cephalosporin Treatment for Serious Infections Due to Apparently Susceptible Organisms Producing Extended-Spectrum β-Lactamases: Implications for the Clinical Microbiology Laboratory
Authors: Paterson David L. , Ko Wen-Chien , Von Gottberg Anne , Casellas Jose Maria , Mulazimoglu Lutfiye , Klugman Keith P. , Bonomo Robert A. , Rice Louis B. , McCormack Joseph G. , Yu Victor L.
Published: July 2002

Although extended-spectrum beta-lactamases (ESBLs) hydrolyze cephalosporin antibiotics, some ESBL-producing organisms are not resistant to all cephalosporins when tested in vitro. Some authors have suggested that screening klebsiellae or
Escherichia coli
for ESBL production is not clinically necessary, and when most recently surveyed the majority of American clinical microbiology laboratories did not make efforts to detect ESBLs. We performed a prospective, multinational study of
Klebsiella pneumoniae
bacteremia and identified 10 patients who were treated for ESBL-producing
K. pneumoniae
bacteremia with cephalosporins and whose infecting organisms were not resistant in vitro to the utilized cephalosporin. In addition, we reviewed 26 similar cases of severe infections which had previously been reported. Of these 36 patients, 4 had to be excluded from analysis. Of the remaining 32 patients, 100% (4 of 4) patients experienced clinical failure when MICs of the cephalosporin used for treatment were in the intermediate range and 54% (15 of 28) experienced failure when MICs of the cephalosporin used for treatment were in the susceptible range. Thus, it is clinically important to detect ESBL production by klebsiellae or
E. coli
even when cephalosporin MICs are in the susceptible range (≤ 8 μg/ml) and to report ESBL-producing organisms as resistant to aztreonam and all cephalosporins (with the exception of cephamycins).
Sensitive Detection of
Borrelia burgdorferi
Sensu Lato DNA and Differentiation of
Borrelia
Species by LightCycler PCR
Authors: Mommert Susanne , Gutzmer Ralf , Kapp Alexander , Werfel Thomas
Published: July 2002

In order to differentiate species within the
Borrelia burgdorferi
sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the
p66
gene and the
recA
gene, were performed. It was demonstrated that nested LightCycler PCR amplification of
p66
is more sensitive in the detection of borrelia DNA than amplification of the
recA
gene.
B. burgdorferi
sensu stricto could be differentiated from
Borrelia garinii
and
Borrelia afzelii
by melting-curve analysis of the
p66
gene amplicon.
B. garinii
could be differentiated from
B. afzelii
and
B. burgdorferi
sensu stricto by melting-curve analysis of the
recA
gene amplicon. Therefore, the PCRs complement each other in subtyping different
Borrelia
species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of
B. burgdorferi
sensu lato.
Pulmonary Infection Caused by an Unusual, Slowly Growing Nontuberculous Mycobacterium
Authors: Suomalainen Sini , Koukila-Kähkölä Pirkko , Brander Eljas , Katila Marja-Leena , Piilonen Anneli , Paulin Lars , Mattson Karin
Published: July 2002

Mycobacterium triplex
, a recently described slowly growing nontuberculous mycobacterium, was isolated from a Finnish patient with pulmonary mycobacteriosis. The disease was successfully treated with antimycobacterial drugs. The strain isolated, which was similar to the type strain but differed slightly from the species description, was regarded as a variant of
M. triplex
sensu stricto. According to present knowledge this variant of the species has never been isolated before.
PCR Detection and Molecular Identification of
Chlamydiaceae
Species
Authors: Hartley J. C. , Kaye S. , Stevenson S. , Bennett J. , Ridgway G.
Published: July 2002

Recent taxonomic developments, based on 16s and 23s rRNA gene sequences, have divided the family
Chlamydiaceae
into two genera and nine species, of which five have been found to infect humans. Few simple methods are available to detect and identify all species sensitively and specifically. In this study the suitability of the
omp2
gene as a target for molecular identification of
Chlamydiaceae
is demonstrated. Phylogenetic analysis of partial
omp2
gene sequences from all nine species agrees with the recently published taxonomic changes based on the ribosomal genes. The use of a family-specific PCR primer pair, which is able to amplify the 5′ end of the
omp2
gene from all
Chlamydiaceae
except some
Chlamydophila pecorum
strains, is described. Identification of all nine species was achieved using restriction fragment length polymorphism analysis with a single enzyme,
Alu
I, confirmed by DNA sequencing. A PCR enzyme-linked oligonucleotide assay was developed which can detect a single chlamydial genome and may be applied to DNA extracts from any specimen or culture for the detection of single or mixed human chlamydial infection.
Six-Year Study of the Incidence of Herpes in Genital and Nongenital Cultures in a Central Kentucky Medical Center Patient Population
Authors: Ribes Julie A. , Steele Anchalee D. , Seabolt John Pat , Baker Doris J.
Published: July 2002

Herpes infections are among the most common sexually transmitted diseases and are the most common cause of genital ulcer disease in the United States. This study addresses the changing distribution of herpes simplex virus type 1 (HSV-1) and HSV-2 in patients presenting for evaluation of herpetic infections. Viral culture results from the University of Kentucky Clinical Microbiology Laboratory were reviewed for a 6-year period (1994 through 1999). Data were collected on patient sex, site of culture, and culture result. These data were analyzed statistically to identify yearly trends. Of the 4,498 cultures analyzed, nearly equal proportions of HSV-1 (13.3%) and HSV-2 (12.0%) were detected for an overall culture positivity rate of 25.3%. Approximately two-thirds of all positive cultures were from women. Although HSV-2 remained the predominant type of genital herpes, over the 6-year span of this study, there was a trend toward increasing proportions of HSV-1 genitalis, with 31.8% of male patients and 44.8% of female patients demonstrating HSV-1 genitalis by 1999. The majority of patients with HSV in nongenital sites grew HSV-1. Although there was significant yearly variation, HSV-2 was isolated from only 9.4% of patients with nongenital HSV for the entire 6-year period. This study therefore concludes that HSV-2 remains primarily a genital pathogen, while HSV-1 is taking on an increasingly important role in causing genital ulcer disease in addition to being the primary nongenital HSV.
Recombinant Assay for Serodiagnosis of Lyme Disease Regardless of OspA Vaccination Status
Authors: Gomes-Solecki Maria J. C. , Wormser Gary P. , Schriefer Martin , Neuman Glenn , Hannafey Laura , Glass John D. , Dattwyler Raymond J.
Published: July 2002

All current seroassays using culturedBorrelia burgdorferias their antigen source have been rendered obsolete by the recombinant OspA Lyme disease vaccine. OspA is the major outer surface protein expressed in culturedB. burgdorferi, and any seroassay that uses whole organisms as its antigen source cannot differentiate between subjects who received the vaccine and those who were naturally infected. We developed a new sensitive and specific enzyme-linked immunosorbent assay (ELISA) utilizing recombinant chimeric borrelia proteins devoid of OspA (rNon-OspA) that can be used to detect antibodies to diagnostically importantB.burgdorferiantigens in both OspA-vaccinated and nonvaccinated individuals. We tested sera from patients with Lyme disease and with conditions associated with false-positive serologies, OspA-vaccinated individuals, and healthy high-risk workers from an area of endemicity and normal sera from individuals from areas of nonendemicity. The rNon-OspA test was compared with two commercially available whole-cell immunoassays. The rNon-OspA assay is as sensitive and specific as the whole-cell assay (P> 0.05) for detection of anti-B.burgdorferiantibodies. However, the rNon-OspA assay can differentiate between populations comprised of naturally infected and OspA-vaccinated individuals (P< 0.05). Our data demonstrate that this new sensitive rNon-OspA ELISA can be used for the laboratory detection ofB.burgdorferiantibodies regardless of vaccination status and could replace existing serologic assays for Lyme disease.
Predominance of
Trypanosoma cruzi
Lineage I in Mexico
Authors: Bosseno Marie-France , Barnabé Christian , Magallón Gastélum Ezequiel , Lozano Kasten Felipe , Ramsey Janine , Espinoza Bertha , Brenière Simone Frédérique
Published: July 2002

Randomly amplified polymorphic DNA (RAPD) has emerged as an effective genetic marker for analysis of
Trypanosoma cruzi
population variability. This method has been used to study the genetic variability of Mexican
T. cruzi
stocks and to relate these results to previous classifications. High clonal diversity was observed among the Mexican populations: 24 RAPD types were scored among 56 stocks analyzed. Only two stocks (3.6%) belonged to the
T. cruzi
II lineage, while all others belonged to
T. cruzi
I. The robustness of these clusters was statistically highly significant. Mexican
T. cruzi
I stocks formed a homogeneous group with reduced genetic distances among its members. Parasites from this group were isolated from both domestic and sylvatic cycles over a broad geographic area in Mexico. The two Mexican stocks classified as
T. cruzi
II (isolated from sylvatic cycles) were of the same RAPD type, although they were not closely related to the three reference
T. cruzi
II stocks circulating in domestic cycles in Argentina, Brazil, Bolivia, and Chile. These stocks were also unrelated to the formerly named Zymodeme III.
Genetic Characterization of H1N2 Influenza A Viruses Isolated from Pigs throughout the United States
Authors: Karasin Alexander I. , Landgraf John , Swenson Sabrina , Erickson Gene , Goyal Sagar , Woodruff Mary , Scherba Gail , Anderson Gary , Olsen Christopher W.
Published: July 2002

An H1N2 influenza A virus was isolated from a pig in the United States for the first time in 1999 (A. I. Karasin, G. A. Anderson, and C. W. Olsen, J. Clin. Microbiol. 38:2453-2456, 2000). H1N2 viruses have been isolated subsequently from pigs in many states. Phylogenetic analyses of eight such viruses isolated from pigs in Indiana, Illinois, Minnesota, Ohio, Iowa, and North Carolina during 2000 to 2001 showed that these viruses are all of the same reassortant genotype as that of the initial H1N2 isolate from 1999.
A Positive-Strand RNA Virus with Three Very Different Subgenomic RNA Promoters
Authors: Koev Gennadiy , Miller W. Allen
Published: July 2002

Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3′-proximal genes. A major step in control of viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. We used barley yellow dwarf virus (BYDV) as a model system to study transcriptional control in a virus with multiple sgRNAs. BYDV generates three sgRNAs during infection. The sgRNA1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structures. It was determined that sgRNA1 is not required for BYDV RNA replication in oat protoplasts. In this study, we show that neither sgRNA2 nor sgRNA3 is required for BYDV RNA replication. The promoters for sgRNA2 and sgRNA3 synthesis were mapped by using deletion mutagenesis. The minimal sgRNA2 promoter is approximately 143 nt long (nt 4810 to 4952) and is located immediately downstream of the putative sgRNA2 start site (nt 4809). The minimal sgRNA3 core promoter is 44 nt long (nt 5345 to 5388), with most of the sequence located downstream of sgRNA3 start site (nt 5348). For both promoters, additional sequences upstream of the start site enhanced sgRNA promoter activity. These promoters contrast to the sgRNA1 promoter, in which almost all of the promoter is located upstream of the transcription initiation site. Comparison of RNA sequences and computer-predicted secondary structures revealed little or no homology between the three sgRNA promoter elements. Thus, a small RNA virus with multiple sgRNAs can have very different subgenomic promoters, which implies a complex system for promoter recognition and regulation of subgenomic RNA synthesis.
The Majority of Duck Hepatitis B Virus Reverse Transcriptase in Cells Is Nonencapsidated and Is Bound to a Cytoplasmic Structure
Authors: Yao Ermei , Gong Yunhao , Chen Nanhai , Tavis John E.
Published: July 2002

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.
Is the 135S Poliovirus Particle an Intermediate during Cell Entry?
Authors: Huang Yan , Hogle James M. , Chow Marie
Published: July 2002

Poliovirus binding to its receptor (PVR) on the cell surface induces a conformational transition which generates an altered particle with a sedimentation value of 135S versus the 160S of the native virion. A number of lines of evidence suggest that the 135S particle is a cell entry intermediate. However, the low infection efficiencies of the 135S particle and the absence of detectable 135S particles during infection at 26°C by the cold-adapted mutants argue against a role for the 135S particle during the cell entry process. We show here that binding of 135S-antibody complexes to the Fc receptor (CDw32) increases the infectivity of these particles by 2 to 3 orders of magnitude. Thus, the low efficiency of infection by 135S particles is due in part to the low binding affinity of these particles. In addition, we show that there is an additional stage in the entry process that is associated with RNA release. This stage occurs after formation of the 135S particle, is rate limiting during infection at 37°C, but not at 26°C, and is PVR independent. The data also demonstrate that during infection at 26°C, the rate-limiting step is the PVR-mediated conversion of wild-type 160S particles to 135S particles. This suggests that during infection at 26°C by the cold-adapted viruses, 135S particles are formed, but they fail to accumulate to detectable levels because the subsequent post-135S particle events occur at a significantly faster rate than the initial conversion of 160S to 135S particles. These data support a model in which the 135S particle is an intermediate during poliovirus entry.