bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects Exerted by Transcriptional Regulator PcaU from
Acinetobacter
sp. Strain ADP1
Authors: Trautwein Gaby , Gerischer Ulrike
Published: July 2002

Protocatechuate degradation is accomplished in a multistep inducible catabolic pathway in
Acinetobacter
sp. strain ADP1. The induction is brought about by the transcriptional regulator PcaU in concert with the inducer protocatechuate. PcaU, a member of the new IclR family of transcriptional regulators, was shown to play a role in the activation of transcription at the promoter for the structural
pca
genes, leaving open the participation of additional activators. In this work we show that there is no PcaU-independent transcriptional activation at the
pca
gene promoter. The minimal inducer concentration leading to an induction response is 10
−5
M protocatechuate. The extent of expression of the
pca
genes was observed to depend on the nature of the inducing carbon source, and this is assumed to be caused by different internal levels of protocatechuate in the cells. The basal level of expression was shown to be comparatively high and to vary depending on the noninducing carbon source independent of PcaU. In addition to the activating function, in vivo results suggest a repressing function for PcaU at the
pca
gene promoter in the absence of an elevated inducer concentration. Expression at the
pcaU
gene promoter is independent of the growth condition but is subject to strong negative autoregulation. We propose a model in which PcaU exerts a repressor function both at its own promoter and at the structural gene promoter and in addition functions as an activator of transcription at the structural gene promoter at elevated inducer concentration.
Intergenic Suppression between the Flagellar MS Ring Protein FliF of
Salmonella
and FlhA, a Membrane Component of Its Export Apparatus
Authors: Kihara May , Minamino Tohru , Yamaguchi Shigeru , Macnab Robert M.
Published: July 2002

The MS ring of the flagellar basal body of
Salmonella
is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate
fliF
mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in
flhA
, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type
fliF
background, had no mutant phenotype. In the
fliF
mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the
fliF
mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.
Growth-Phase-Dependent Expression of the Cyclopeptide Antibiotic Microcin J25
Authors: Chiuchiolo Marı́a J. , Delgado Mónica A. , Farı́as Ricardo N. , Salomón Raúl A.
Published: July 2002

Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by
Escherichia coli.
We have isolated
lacZ
fusions to
mcjA
(encoding the 58-amino-acid microcin precursor) and
mcjB
and
mcjC
(which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The
mcjA
gene was found to be dramatically induced as cells entered the stationary phase. Expression of
mcjA
could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of
mcjA
expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced
mcjA
expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of
mcjA
is triggered by nutrient depletion. The
mcjB
and
mcjC
genes were also regulated by the growth phase of the culture, but in contrast to
mcjA
, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of
mcjA
,
mcjB
, and
mcjC
may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.
Involvement of Domain 3 in Oligomerization by the Protective Antigen Moiety of Anthrax Toxin
Authors: Mogridge Jeremy , Mourez Michael , Collier R. John
Published: July 2002

Protective antigen (PA), a component of anthrax toxin, binds receptors on mammalian cells and is activated by a cell surface protease. The resulting active fragment, PA
63
, forms ring-shaped heptamers, binds the enzymic moieties of the toxin, and translocates them to the cytosol. Of the four crystallographic domains of PA, domain 1 has been implicated in binding the enzymic moieties; domain 2 is involved in membrane insertion and oligomerization; and domain 4 binds receptor. To determine the function of domain 3, we developed a screen that allowed us to isolate random mutations that cause defects in the activity of PA. We identified several mutations in domain 3 that affect monomer-monomer interactions in the PA
63
heptamer, indicating that this may be the primary function of this domain.
Genes Involved in Copper Homeostasis in
Escherichia coli
Authors: Grass Gregor , Rensing Christopher
Published: July 2002

Recently, genes for two copper-responsive regulatory systems were identified in the
Escherichia coli
chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in
E. coli
.
An α/β-Type, Small, Acid-Soluble Spore Protein Which Has Very High Affinity for DNA Prevents Outgrowth of
Bacillus subtilis
Spores
Authors: Hayes Christopher S. , Setlow Peter
Published: July 2002

A derivative of SspC, a minor α/β-type, small, acid-soluble spore protein (SASP) from
Bacillus subtilis
, was generated that has a very high affinity for DNA. This protein (SspC
Δ11-D13K
) was able to confer UV resistance on spores lacking α/β-type SASP, and spores with SspC
Δ11-D13K
triggered germination normally. However, SspC
Δ11-D13K
blocked outgrowth of ≥90% of germinated spores, and SspC
Δ11-D13K
persisted in these germinated spores, whereas wild-type SspC was almost completely degraded. The outgrowth phenotype of spores with SspC
Δ11-D13K
is proposed to be due to the high stability of the SspC
Δ11-D13K
-DNA complex, which prevents rapid degradation of this α/β-type SASP early in germination. The persistence of this protein on spore DNA then interferes with transcription during spore outgrowth.
Specific Binding of Integrase to the Origin of Transfer (
oriT
) of the Conjugative Transposon Tn
916
Authors: Hinerfeld Douglas , Churchward Gordon
Published: July 2002

Purified integrase protein (Int) of the conjugative transposon Tn
916
was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon,
oriT
. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding to
oriT
required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to
oriT
may be part of a mechanism to prevent premature conjugal transfer of Tn
916
prior to excision from the donor DNA.
Overexpression of the PepF Oligopeptidase Inhibits Sporulation Initiation inBacillus subtilis
Authors: Kanamaru Kyoko , Stephenson Sophie , Perego Marta
Published: July 2002

TheyjbGgene encoding the homologue of the PepF1 and PepF2 oligoendopeptidases ofLactococcus lactis(Monnet et al., J. Biol. Chem. 269:32070–32076, 1994; Nardi et al., J. Bacteriol. 179:4164–4171, 1997) has been identified inBacillus subtilisas an inhibitor of sporulation initiation when present in the cells on a multicopy plasmid. Genetic analysis suggested that the inhibitory effect is due to hydrolysis of the PhrA peptide in a form as small as the pentapeptide (ARNQT). Inactivation of PhrA results in deregulation of the RapA phosphatase and thus dephosphorylation of the Spo0F∼∼P response regulator component of the phosphorelay for sporulation initiation. When overexpressed, theB. subtilisPepF is most likely hydrolyzing additional peptides of the Phr family, as is the case for PhrC involved in control of competence development. Chromosomal inactivation of theyjbG/pepFgene did not give rise to any detectable phenotype. The function of PepF inB. subtilisremains unknown. Limited experiments with ayjbGparalogue calledyusXindicated that a frameshift is present, making the corresponding gene product inactive.
BetS Is a Major Glycine Betaine/Proline Betaine Transporter Required for Early Osmotic Adjustment in
Sinorhizobium meliloti
Authors: Boscari Alexandre , Mandon Karine , Dupont Laurence , Poggi Marie-Christine , Le Rudulier Daniel
Published: July 2002

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb
Sinorhizobium meliloti
DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of
Escherichia coli
(34%) and to the glycine betaine transporter OpuD of
Bacillus subtilis
(30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of
betS
in
E. coli
mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with

K
m

s of 16 ± 2 μM and 56 ± 6 μM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na
+
driven. In an
S. meliloti betS
mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. β-Galactosidase activities of a
betS-lacZ
strain grown in various conditions showed that
betS
is constitutively expressed. Osmotic upshock experiments performed with wild-type and
betS
mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by
S. meliloti
subjected to osmotic upshock.
The PtlE Protein of
Bordetella pertussis
Has Peptidoglycanase Activity Required for Ptl-Mediated Pertussis Toxin Secretion
Authors: Rambow-Larsen Amy A. , Weiss Alison A.
Published: July 2002

Pertussis toxin of
Bordetella pertussis
is secreted by a type IV secretion system comprised of the products of the nine
ptl
(pertussis toxin liberation) genes. These proteins are believed to form a complex spanning both the inner and outer membranes and passing through the peptidoglycan layer. Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules. Assembled pertussis toxin and the secretion component proteins PtlC through PtlH are too large to diffuse through intact peptidoglycan. Therefore, we hypothesized that the Ptl system contains a peptidoglycanase activity. The PtlE protein was found to exhibit a sequence match to the active site of glycohydrolase enzymes. An N-terminally polyhistidine-tagged PtlE fusion protein, constructed and expressed in
Escherichia coli
and in
B. pertussis
, exhibited peptidoglycanase activity on activity gels. A fusion protein with alanine substitutions at the putative active site residues (aspartic acid at position 53 and glutamic acid at position 62) lacked peptidoglycanase activity.
B. pertussis
strains with the amino acid substitutions were deficient for pertussis toxin secretion. Based on these results, we concluded that PtlE is a peptidoglycanase responsible for the local removal or rearrangement of the peptidoglycan layer during Ptl secretion complex assembly.
High Frequency of Mutator Strains among Human Uropathogenic
Escherichia coli
Isolates
Authors: Denamur Erick , Bonacorsi Stéphane , Giraud Antoine , Duriez Patrick , Hilali Farida , Amorin Christine , Bingen Edouard , Andremont Antoine , Picard Bertrand , Taddei François , Matic Ivan
Published: July 2002

By using a panel of 603 commensal and pathogenic
Escherichia coli
and
Shigella
isolates, we showed that mutation rates of strains vary considerably among different ecotypes. Uropathogenic strains had the highest frequency of mutators, while strains from patients with bacteremia had the lowest mutation rates. No correlation between the mutation rates and antibiotic resistance was observed among the studied strains.
Laribacter
hongkongensis
gen. nov., sp. nov., a Novel Gram-Negative Bacterium Isolated from a Cirrhotic Patient with Bacteremia and Empyema
Authors: Yuen Kwok-Yung , Woo Patrick C. Y. , Teng Jade L. L. , Leung Kit-Wah , Wong Michelle K. M. , Lau Susanna K. P.
Published: July 2002

A bacterium was isolated from the blood and empyema of a cirrhotic patient. The cells were facultatively anaerobic, nonsporulating, gram-negative, seagull shaped or spiral rods. The bacterium grows on sheep blood agar as nonhemolytic, gray colonies 1 mm in diameter after 24 h of incubation at 37°C in ambient air. Growth also occurs on MacConkey agar and at 25 and 42°C but not at 4, 44, and 50°C. The bacterium can grow in 1 or 2% but not 3, 4, or 5% NaCl. No enhancement of growth is observed with 5% CO
2
. The organism is aflagellated and nonmotile at both 25 and 37°C. It is oxidase, catalase, urease, and arginine dihydrolase positive, and it reduces nitrate. It does not ferment, oxidize, or assimilate any sugar tested. 16S rRNA gene sequencing showed that there are 91 base differences (6.2%), 112 base differences (7.7%), and 116 base differences (8.2%) between the bacterium and
Microvirgula
aerodenitrificans
,
Vogesella
indigofera
, and
Chromobacterium
species, respectively. The G+C content (mean and standard deviation) is 68.0% ± 2.43%, and the genomic size is about 3 Mb. Based on phylogenetic affiliation, the bacterium belongs to the
Neisseriaceae
family of the β-subclass of
Proteobacteria
. For these reasons, a new genus and species,
Laribacter
hongkongensis
gen. nov., sp. nov., is proposed, for which HKU1 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging pathogen.
Nationwide Survey Shows that Methicillin-Resistant
Staphylococcus aureus
Strains Heterogeneously and Intermediately Resistant to Vancomycin Are Not Disseminated throughout Japanese Hospitals
Authors: Ike Yasuyoshi , Arakawa Yoshichika , Ma Xinghua , Tatewaki Kenichi , Nagasawa Mitsuaki , Tomita Haruyoshi , Tanimoto Koichi , Fujimoto Shuhei
Published: July 2002

A total of 6,625 methicillin-resistant
Staphylococcus aureus
(MRSA) clinical isolates obtained from 278 hospitals throughout Japan were obtained between November and December 1997 and were examined for their sensitivities to vancomycin using Mueller Hinton (MH), brain heart infusion (BHI), agar plates, or the broth microdilution method. A concentrated inoculum of an MRSA strain or the use of highly enriched medium, such as BHI medium, allows an individual cell to grow on agar plates containing a vancomycin concentration greater than the MIC for the parent strain. However, cells of the colonies which grew on BHI agar plates containing the higher vancomycin concentrations did not acquire a level of vancomycin resistance greater than that of the parent strain and were not subpopulations of heterogeneously vancomycin-resistant MRSA. There was no significance in the fact that these colonies grew on the higher concentration of vancomycin: none showed stable resistance to vancomycin at a concentration above the MIC for the parent strain, and no cell from these colonies showed a relationship between the MIC and the ability of these colonies to grow on higher concentrations of vancomycin. The vancomycin MIC was not above 2 μg/ml for any of the cells originating from these colonies. No Mu3-type heterogeneously resistant MRSA strains, which constitutively produce subpopulations from MRSA clinical isolates with intermediate vancomycin resistance at a high frequency, were detected. There was a unipolar distribution of the MICs ranging from 0.25 to 2 μg of vancomycin/ml among the 6,625 MRSA clinical isolates, indicating that there was no Mu50-type intermediately vancomycin-resistant MRSA (MIC, 8 μg/ml by National Committee for Clinical Laboratory Standards criteria) among the clinical isolates, and there was no evidence of dissemination of Mu3-type MRSA heteroresistant to vancomycin.
Identification, Classification, and Phylogeny of the Pathogenic SpeciesExophiala jeanselmeiand Related Species by Mitochondrial CytochromebGene Analysis
Authors: Wang Li , Yokoyama Koji , Miyaji Makoto , Nishimura Kazuko
Published: July 2002

We analyzed a 402-bp sequence of the mitochondrial cytochromebgene of 34 strains ofExophiala jeanselmeiand 16 strains representing 12 related species. The strains ofE.jeanselmeiwere classified into 20 DNA types and 17 amino acid types. The differences between these strains were found in 1 to 60 nucleotides and 1 to 17 amino acids. On the basis of the identities and similarities of nucleotide and amino acid sequences, some strains were reidentified: i.e., two strains ofE.jeanselmeivar.hetermorphaand one strain ofE.castellaniiasE.dermatitidis(including the type strain), three strains ofE.jeanselmeiasE.jeanselmeivar.lecanii-corni(including the type strain), three strains ofE.jeanselmeiasE.bergeri(including the type strain), seven strains ofE.jeanselmeiasE.pisciphila(including the type strain), seven strains ofE.jeanselmeiasE.jeanselmeivar.jeanselmei(including the type strain), one strain ofE.jeanselmeiasFonsecaea pedrosoi(including the type strain), and one strain ofE.jeanselmeiasE.spinifera(including the type strain). SomeE.jeanselmeistrains showed distinct nucleotide and amino acid sequences. The amino-acid-based UPGMA (unweighted pair group method with the arithmetic mean) tree exhibited nearly the same topology as those of the DNA-based trees obtained by neighbor joining, maximum parsimony, and maximum likelihood methods.
Maxillary Sinusitis Caused by
Actinomucor elegans
Authors: Davel Graciela , Featherston Patricia , Fernández Anibal , Abrantes Ruben , Canteros Cristina , Rodero Laura , Sztern Carlos , Perrotta Diego
Published: July 2002

We report the first case of maxillary sinusitis caused by
Actinomucor elegans
in an 11-year-old patient. Histopathological and mycological examinations of surgical maxillary sinuses samples showed coenocytic hyphae characteristic of mucoraceous fungi. The fungi recovered had stolons and rhizoids, nonapophyseal and globose sporangia, and whorled branched sporangiophores and was identified as
A. elegans
. After surgical cleaning and chemotherapy with amphotericin B administered intravenously and by irrigation, the patient became asymptomatic and the mycological study results were negative.
Vancomycin-Resistant
Enterococcus faecium
Strain Carrying the
vanB2
Gene Variant in a Polish Hospital
Authors: Kawalec Magdalena , Gniadkowski Marek , Zielińska Urszula , Kłos Waldemar , Hryniewicz Waleria
Published: July 2002

About 2.5 years after the first isolation of the VanA phenotype of vancomycin-resistant
Enterococcus faecium
(VREM) in Poland, the first VREM strains with the VanB phenotype have emerged independently in two different Warsaw hospitals. In one of these the VREM strain was selected during the long-term antimicrobial treatment of a patient with a wide variety of infection risk factors who died after 3 months of hospitalization. The strain was found to contain the transferable
vanB2
gene cluster variant of the polymorphic type that was identified earlier in vancomycin-resistant enterococci from several different countries. In the course of infection the strain underwent genetic diversification due to DNA recombination.
Clinical Significance and Taxonomy ofActinobacillus hominis
Authors: Friis-Møller Alice , Christensen Jens Jørgen , Fussing Vivian , Hesselbjerg Annemarie , Christiansen Jytte , Bruun Brita
Published: July 2002

Clinical findings in 36 immunosuppressed patients with lower respiratory tract infection or bacteremia withActinobacillus hominisare described. Animal contact was only recorded for three patients; nine patients died despite appropriate antimicrobial treatment. Although infections with this microorganism seem to be rare, the fact that 37 of 46 strains characterized in this study have been found in Copenhagen indicates that under-reporting may occur.A. hominisis phenotypically relatively homogeneous but can be difficult to differentiate from otherActinobacillusspecies unless extensive biochemical testing is performed. Mannose-positive strains ofA. hominisare especially difficult to differentiate fromA. equuli. Attempts to identifyA. hominisby automatic identification systems may lead to misidentifications. Ribotyping and DNA-DNA hybridization data show thatA. hominisis a homogeneous species clearly separated from other species within the genusActinobacillus.
Performance of the Applied Biosystems ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Sequence-Based Analysis of HIV-1 in Pediatric Plasma Samples
Authors: Cunningham Shawn , Ank Bonnie , Lewis Dan , Lu Wei , Wantman Michael , Dileanis JoAnn , Jackson J. Brooks , Palumbo Paul , Krogstad Paul , Eshleman Susan H.
Published: July 2002

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.
Prevalence of CongenitalToxoplasma gondiiInfection among Newborns from the Poznań Region of Poland: Validation of a New Combined Enzyme Immunoassay forToxoplasma gondii-Specific Immunoglobulin A and Immunoglobulin M Antibodies
Authors: Paul Małgorzata , Petersen Eskild , Szczapa Jerzy
Published: July 2002

We determined the value of a new serological assay detectingToxoplasma-specific immunoglobulin M (IgM) and IgA antibodies at birth for use in mass neonatal screening. The incidence of congenital infection in newborns was compared with data from an epidemiological investigation on the seroprevalence ofToxoplasmain the studied population. Peripheral blood was collected on Guthrie cards during the first 3 days of life and tested for anti-ToxoplasmaIgA and IgM using a noncommercial immunocapture enzyme-linked immunosorbent assay (ELISA). When the screening assay was positive, serum samples from the child and the mother were collected for use in Western blotting comparative immunological profile analysis and traditional serological tests for determination of specific IgG, IgM, and IgA antibodies. From December 1998 to April 2000, 17,653 filter paper samples from live-born neonates were successively screened. CongenitalT. gondiiinfection was finally confirmed in 19 newborns. In traditional assays, 13 of 19 infants were IgM and IgA positive using filter paper eluates at birth, 1 child was positive only for IgM, 1 patient was positive for IgM and borderline for IgA, 1 had an equivocal level of IgA, and 3 cases were confirmed only by the Western blot assay. The prevalence ofToxoplasma-specific IgA and/or IgM in filter paper samples at birth was 1 per 929 live-born neonates (1.08/1,000) or about 1 per 523 children (1.9/1,000) born to nonimmune women with a potential risk of primaryT. gondiiinfection during pregnancy, compared to the actual seropositivity rate of 43.7%. The diagnostic sensitivity of the combined IgA-IgM ELISA using neonatal filter paper specimens was not more than 95%, the positive predictive value of the test was 82.6%, and the diagnostic specificity was calculated to be 99.9%. The combined IgA-IgM ELISA is a valuable method for the diagnosis of congenital toxoplasmosis at birth and fulfills criteria for neonatal screening programs. The method showed a good diagnostic sensitivity in neonates untreated prenatally who were born in an area of high seroprevalence ofT. gondiiinfection.
Fulminant Japanese Spotted Fever Associated with Hypercytokinemia
Authors: Iwasaki Hiromichi , Mahara Fumihiko , Takada Nobuhiro , Fujita Hiromi , Ueda Takanori
Published: July 2002

We report a patient with Japanese spotted fever caused by
Rickettsia japonica
who developed shock associated with hypercytokinemia. Elevated levels of cytokines (macrophage colony-stimulating factor, interleukin 1 beta, interleukin 10, and gamma interferon) decreased rapidly after a combination treatment using an antibiotic (minocycline hydrochloride [MINO]) and methylprednisolone; however, tumor necrosis factor alpha levels were increased. The patient's fever relapsed and was resolved only after the addition of ciprofloxacin hydrochloride. The administration of new quinolones alone may be another useful form of treatment to eradicate
R. japonica
even if the symptoms of hypercytokinemia appear to improve with the administration of MINO and methylprednisolone.
Molecular Identification of a
Dietzia maris
Hip Prosthesis Infection Isolate
Authors: Pidoux O. , Argenson J.-N. , Jacomo V. , Drancourt M.
Published: July 2002

Dietzia maris
, an environmental actinomycete, has been implicated only once in human disease. We herein report the first
D. maris
isolate from a bone biopsy specimen in a patient hospitalized for a total hip prosthesis replacement. Cell wall fatty acid analysis and 16S ribosomal DNA gene sequencing were utilized to achieve its definite identification. This case report illustrates the usefulness of such methods for the accurate identification of actinomycetes.
Identification of Aminoglycoside-Modifying Enzymes by Susceptibility Testing: Epidemiology of Methicillin-Resistant
Staphylococcus aureus
in Japan
Authors: Ida Takashi , Okamoto Ryoichi , Shimauchi Chieko , Okubo Toyoji , Kuga Akio , Inoue Matsuhisa
Published: July 2002

A multiple-primer PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 381 clinical isolates of methicillin-resistant
Staphylococcus aureus
(MRSA). The technique used three sets of primers delineating specific DNA fragments of the
aph(3′)-III, ant(4′)-I
, and
aac(6′)-aph(2")
genes, which influence the MICs of gentamicin, tobramycin, and lividomycin. Isolates with none of the three genes detected were susceptible to all three agents. Isolates with the
aph(3′)-III
gene showed resistance to lividomycin (MIC > 1,024 μg/ml), and those with the
ant(4′)-I
gene were resistant to tobramycin (MIC ≥ 8 μg/ml). Isolates with only the
aac(6′)-aph(2")
gene were resistant to gentamicin (MIC ≥ 8 μg/ml) and tobramycin in decreasing order; those with both the
ant(4′)-I
and
aac(6′)-aph(2")
genes also were resistant to gentamicin and tobramycin, but in increasing order. Susceptibility testing, then, could detect specific genes. In 381 Japanese MRSA isolates, the
ant(4′)-I, aac(6′)-aph(2")
, and
aph(3′)-III
genes were prevalent in 84.5, 61.7, and 8.9%, respectively. Isolates with only the
ant(4′)-I
gene had coagulase type II or III, but isolates with both the
ant(4′)-I
and
aac(6′)-aph(2")
genes included all coagulase types. Most isolates with coagulase type IV or VII carried the
aac(6′)-aph(2")
gene. Of the MRSA isolates with
ant(4′)-I
and/or
aac(6′)-aph(2")
genes, 97% were resistant to aminoglycosides in clinical use, but a new aminoglycoside, arbekacin, had excellent activity against these isolates.
Community-Acquired
Acinetobacter baumannii
Bacteremia in Adult Patients in Taiwan
Authors: Wang Jann-Tay , McDonald Lawrence Clifford , Chang Shan-Chwen , Ho Monto
Published: July 2002

We determined the clinical and microbiologic characteristics of community-acquired
Acinetobacter baumannii
bacteremia in 19 adult patients. We found that malignancy was the most frequent underlying disease. The overall mortality rate was 58%. All 14 available isolates were identified as genomic species 2 (
A. baumannii
) by 16S ribosomal DNA sequencing and were found to be genetically distinct by pulsed-field gel electrophoresis.
Multilocus Sequence Typing for Characterization of Clinical and Environmental
Salmonella
Strains
Authors: Kotetishvili Mamuka , Stine O. Colin , Kreger Arnold , Morris, J. Glenn , Sulakvelidze Alexander
Published: July 2002

Multilocus sequence typing (MLST) based on the 16S RNA,
pduF
,
glnA
, and
manB
genes was developed for
Salmonella
, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231
Salmonella
isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of
Salmonella
is better than that of serotyping and/or PFGE typing.
manB
-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose
manB
gene sequences were analyzed. The G+C contents and Splitstree analysis of the
manB
,
glnA
, and
pduF
genes of
Salmonella
indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.
Evaluation of an Immunofluorescent-Antibody Test Using Monoclonal Antibodies Directed against
Enterocytozoon bieneusi
and
Encephalitozoon intestinalis
for Diagnosis of Intestinal Microsporidiosis in Bamako (Mali)
Authors: Alfa Cisse O. , Ouattara A. , Thellier M. , Accoceberry I. , Biligui S. , Minta D. , Doumbo O. , Desportes-Livage I. , Thera M. A. , Danis M. , Datry A.
Published: July 2002

A 2-month study was carried out in Mali to evaluate an immunofluorescent-antibody test (IFAT) using monoclonal probes specific for
Enterocytozoon bieneusi
or
Encephalitozoon intestinalis
. Sixty-one human immunodeficiency virus (HIV)-seropositive adult patients and 71 immunocompetent children were enrolled. Microsporidia were detected in stools from 8 of 61 patients (13.1%) seropositive for HIV. A single species,
E. bieneusi
, was identified. All the children were negative for microsporidia. The sensitivity and specificity of IFAT were 100% compared with those of PCR, which was used as the “gold standard.” Moreover, species identification by IFAT was more rapid and less expensive than that by PCR. These results show the suitability of IFAT for detection of microsporidia in developing countries.
Parameters of Human Immunodeficiency Virus Infection of Human Cervical Tissue and Inhibition by Vaginal Virucides
Authors: Greenhead Peter , Hayes Peter , Watts Patricia S. , Laing Ken G. , Griffin George E. , Shattock Robin J.
Published: July 2002

Heterosexual transmission of human immunodeficiency virus (HIV) is the most frequent mode of infection worldwide. However, the immediate events between exposure to infectious virus and establishment of infection are still poorly understood. This study investigates parameters of HIV infection of human female genital tissue in vitro using an explant culture model. In particular, we investigated the role of the epithelium and virucidal agents in protection against HIV infection. We have demonstrated that the major target cells of infection reside below the genital epithelium, and thus HIV must cross this barrier to establish infection. Immune activation enhanced HIV infection of such subepithelial cells. Furthermore, our data suggest that genital epithelial cells were not susceptible to HIV infection, appear to play no part in the transfer of infectious virus across the epithelium, and thus may provide a barrier to infection. In addition, experiments using a panel of virucidal agents demonstrated differential efficiency to block HIV infection of subepithelial cells from partial to complete inhibition. This is the first demonstration that virucidal agents designed for topical vaginal use block HIV infection of genital tissue. Such agents have major implications for world health, as they will provide women with a mechanism of personal and covert protection from HIV infection.
Soluble Receptor-Induced Retroviral Infection of Receptor-Deficient Cells
Authors: Damico Rachel , Bates Paul
Published: July 2002

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demonstration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), induces conformational changes in the viral envelope protein. These changes include conversion of the envelope protein to an active, membrane-binding state likely representing a fusogenic conformation. To determine whether binding of the soluble Tva (sTva) receptor was sufficient to activate fully the fusogenic potential of the ASLV-A envelope protein, we have evaluated the ability of ASLV-A to infect receptor-deficient cell lines in the presence of sTva. Soluble receptor efficiently mediated infection of cells devoid of endogenous Tva in a dose-dependent manner, and this infection was dependent absolutely on the addition of sTva. The infectivity of the virus was enhanced dramatically in the presence of the polycationic polymer Polybrene or when centrifugal forces were applied during inoculation, resulting in viral titers comparable to those achieved on cells expressing endogenous receptor. sTva functioned to mediate infection at low concentrations, approaching the estimated binding constant of the receptor and viral envelope protein. These results demonstrate that receptor binding can activate the ASLV-A envelope protein and convert it to a fusogenic conformation competent to mediate the fusion of the viral and cellular membranes.
Phosphorylation of Human Immunodeficiency Virus Type 1 Vpr Regulates Cell Cycle Arrest
Authors: Zhou Yi , Ratner Lee
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) Vpr regulates nuclear transport of the viral preintegration complex, G
2
cell cycle arrest, and transcriptional transactivation. We asked whether phosphorylation could affect Vpr activity. Vpr was found to be phosphorylated on serine residues in transiently transfected and infected cells. Residues 79, 94, and 96 were all found to be phosphorylated, as assessed by alanine mutations. Mutation of Ser-79 to Ala abrogated effects of Vpr on cell cycle progression, whereas mutation of Ser-94 and Ser-96 had no effect. Simultaneous mutation of all three Vpr serine residues attenuated HIV-1 replication in macrophages, whereas single and double Ser mutations had no effect.
Origin of Hepatitis Delta Virus mRNA
Authors: Gudima Severin , Wu Shwu-Yuan , Chiang Cheng-Ming , Moraleda Gloria , Taylor John
Published: July 2002

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5′ end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5′-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5′ ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5′ end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3′-end addition to the template. These new findings support the interpretation that the 5′ end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.
Mapping and Characterization of the N-Terminal I Domain of Human Immunodeficiency Virus Type 1 Pr55
Gag
Authors: Sandefur Stephanie , Smith Rita M. , Varthakavi Vasundhara , Spearman Paul
Published: July 2002

Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55
Gag
molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55
Gag
. The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.
Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism
Authors: Wu Pei , Xiao Wu , Conlon Thomas , Hughes Jeffrey , Agbandje-McKenna Mavis , Ferkol Thomas , Flotte Terence , Muzyczka Nicholas
Published: July 2002

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.
Persistent Virus Infection despite Chronic Cytotoxic T-Lymphocyte Activation in Gamma Interferon-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus
Authors: Bartholdy Christina , Christensen Jan Pravsgaard , Wodarz Dominik , Thomsen Allan Randrup
Published: July 2002

The role of gamma interferon (IFN-γ) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-γ-deficient (IFN-γ −/−) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-γ −/− mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-γ −/− mice. The prolonged effector phase in infected IFN-γ −/− mice was associated with elevated numbers of CD8+T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8+T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-γ −/− mice than in wild-type mice. These findings indicate that in the absence of IFN-γ, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-γ-mediated antiviral activity on the dynamics between virus replication and CTL activity.