bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of DifferenttfdGenes for Degradation of Chloroaromatics byRalstonia eutrophaJMP134
Authors: Plumeier Iris , Pérez-Pantoja Danilo , Heim Sabina , González Bernardo , Pieper Dietmar H.
Published: July 2002

ThetfdCIDIEIFI,andtfdDIICIIEIIFIIgene modules of plasmid pJP4 ofRalstonia eutrophaJMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity oftfdI-encoded enzymes was usually higher than that oftfdII-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only onetfdgene module. ThetfdDII-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors ofR. eutrophastrains containing differenttfdgene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to formtrans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.
Cloning and Characterization of the Phosphatidylserine Synthase Gene of
Agrobacterium
sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1→3)-β-
d
-Glucan (Curdlan)
Authors: Karnezis Tara , Fisher Helen C. , Neumann Gregory M. , Stone Bruce A. , Stanisich Vilma A.
Published: July 2002

Genes involved in the production of the extracellular (1→3)-β-glucan, curdlan, by
Agrobacterium
sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology
9:
31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon Tn
phoA
was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene,

pss
AG

, encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this
Agrobacterium
strain is not solely dependent on PE in a pathway controlled by the Pss
AG
protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the

pss
AG

::Tn
phoA
mutation was complemented by the intact

pss
AG

gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.
Growth ofCampylobacter jejuniSupported by Respiration of Fumarate, Nitrate, Nitrite, Trimethylamine-N-Oxide, or Dimethyl Sulfoxide Requires Oxygen
Authors: Sellars Michael J. , Hall Stephen J. , Kelly David J.
Published: July 2002

The human gastrointestinal pathogenCampylobacter jejuniis a microaerophilic bacterium with a respiratory metabolism. The genome sequence ofC. jejunistrain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, andN- orS-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded byCj0264cwas shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth ofC. jejuniunder strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. TheC. jejunigenome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth ofC. jejuni. The results indicate that strict anaerobiosis is a stress condition forC. jejunibut that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.
Origins of the 2,4-Dinitrotoluene Pathway
Authors: Johnson Glenn R. , Jain Rakesh K. , Spain Jim C.
Published: July 2002

The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds. Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction. The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA). The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from
Burkholderia cepacia
R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source. Genes for the lower pathway in 2,4-DNT degradation were found downstream from
dntD
, the gene encoding the extradiol ring fission enzyme of the pathway. The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (
dntE
), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (
dntG
). Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that
dntD dntE
ORF13
dntG
composes an operon that encodes the lower pathway. Additional genes that were uncovered encode the 2,4-DNT dioxygenase (
dntAaAbAcAd
), methylnitrocatechol monooxygenase (
dntB
), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements. Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes. The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region.
pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism inEscherichia coli
Authors: Stancik Lauren M. , Stancik Dawn M. , Schmidt Brian , Barnhart D. Michael , Yoncheva Yuliya N. , Slonczewski Joan L.
Published: July 2002

Escherichia coligrows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and throughlacgene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locusalx, induced in extreme base, was identified asygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well aslacfusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in whichE. colimodulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.
Characterization of an Unusual Mycobacterium: a Possible Missing Link between
Mycobacterium marinum
and
Mycobacterium ulcerans
Authors: Chemlal K. , Huys G. , Laval F. , Vincent V. , Savage C. , Gutierrez C. , Laneelle M.-A. , Swings J. , Meyers W. M. , Daffe M. , Portaels F.
Published: July 2002

In an attempt to characterize an unusual mycobacterial isolate from a 44-year-old patient living in France, we applied phenotypic characterizations and various previously described molecular methods for the taxonomic classification of mycobacteria. The results of the investigations were compared to those obtained in a previous study with a set of temporally and geographically diverse
Mycobacterium ulcerans
(
n
= 29) and
Mycobacterium marinum
(
n
= 29) isolates (K. Chemlal, G. Huys, P.-A. Fonteyne, V. Vincent, A. G. Lopez, L. Rigouts, J. Swings, W. M. Meyers, and F. Portaels, J. Clin. Microbiol. 39:3272-3278, 2001). The isolate, designated ITM 00-1026 (IPP 2000-372), is closely related to
M. marinum
according to its phenotypic properties, lipid pattern, and partial 16S rRNA sequence. Moreover, fingerprinting by amplified fragment length polymorphism (AFLP) analysis unequivocally classified this strain as a member of the species
M. marinum
, although it lacked two species-specific AFLP marker bands. However, PCR and restriction fragment length polymorphism analysis based on
M. ulcerans
-specific insertion sequence IS
2404
showed the presence of this element in a low copy number in isolate ITM 00-1026. In conclusion, the designation of this isolate as a transitional species further supports the recent claim by Stinear et al. (T. Stinear, G. Jenkin, P. D. Johnson, and J. K. Davies, J. Bacteriol. 182:6322-6330, 2000) that
M. ulcerans
represents a relatively recent phylogenetic derivative of
M. marinum
resulting from the systematic acquisition of foreign DNA fragments.
Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA
Authors: Sanchez Jason L. , Storch Gregory A.
Published: July 2002

We created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 106human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 101to 106copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.
Detection and Genotyping of Human Group A Rotaviruses by Oligonucleotide Microarray Hybridization
Authors: Chizhikov V. , Wagner M. , Ivshina A. , Hoshino Y. , Kapikian A. Z. , Chumakov K.
Published: July 2002

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5′-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.
Histologic and Genotypic Characterization of a Novel
Mycobacterium
Species Found in Three Cats
Authors: Appleyard Greg D. , Clark Edward G.
Published: July 2002

Three cases of feline atypical mycobacteriosis from different geographical regions in North America were characterized by large clusters of filamentous bacteria visible on hematoxylin-and-eosin-stained tissue sections. PCR amplification demonstrated the presence of
Mycobacterium
-specific nucleic acid in samples of skin lesions from these cases. PCR-assisted cloning and DNA sequence analysis of a 541-bp length of the
Mycobacterium
16S rRNA gene generated DNA sequences which were >95% identical, suggesting that the three isolates were closely related. Two of the sequences were 99% identical and may represent the same species. Alignment with comparable 16S rRNA gene sequences from 66
Mycobacterium
species and partially characterized isolates highlighted similarities (>94%) with
Mycobacterium bohemicum
,
Mycobacterium haemophilum
,
Mycobacterium ulcerans
,
Mycobacterium avium
subsp.
avium
, and isolate IWGMT 90242. Parsimony analysis of sequence data suggested relatedness to
M. leprae
. Significant molecular genetic and pathobiological differences between these three similar isolates and other known species of mycobacteria suggested that the organisms may not have been described previously and that these cases may represent a new form of mycobacterial disease in cats. We suggest the term “
Mycobacterium visibilis
” to describe the organism from which the two nearly identical sequences were obtained.
Seroprevalence ofBabesiaInfections in Humans Exposed to Ticks in Midwestern Germany
Authors: Hunfeld Klaus-Peter , Lambert Annette , Kampen Helge , Albert Sabine , Epe Christian , Brade Volker , Tenter Astrid M.
Published: July 2002

Babesiosis is considered to be an emerging tick-borne disease in humans worldwide. However, most studies on the epidemiology of human babesiosis to date have been carried out in North America, and there is little knowledge on the prevalence of infection and frequency of disease in other areas. The aim of this study was to investigate the prevalence ofBabesiainfections in a human population in Germany. A total of 467 sera collected between May and October 1999 from individuals living in the Rhein-Main area were tested for the presence of immunoglobulin G (IgG) and IgM antibodies to antigens ofBabesia microtiandBabesia divergensby indirect fluorescent-antibody (IFA) tests. These sera were derived from 84 Lyme borreliosis patients suffering from erythema migrans, 60 asymptomatic individuals with positive borreliosis serology, and 81 individuals with a history of tick bite. Cutoff values for discrimination between seronegative and seropositive results in the IFA tests were determined using sera from 120 healthy blood donors and 122 patients suffering from conditions other than tick-borne diseases (malaria,n= 40; toxoplasmosis,n= 22; syphilis,n= 20; Epstein-Barr virus infection,n= 20; and presence of antinuclear antibodies,n= 20). The overall specificities of the IFA tests forB. microtiandB. divergenswere estimated to be ≥97.5%. Positive IgG reactivity againstB. microtiantigen (titer, ≥1:64) orB. divergensantigen (titer, ≥1:128) was detected significantly more often (P< 0.05) in the group of patients exposed to ticks (26 of 225 individuals; 11.5%) than in the group of healthy blood donors (2 of 120 individuals; 1.7%). IgG antibody titers of ≥1:256 against at least one of the babesial antigens were found significantly more often (P< 0.05) in patients exposed to ticks (9 of 225) than in the control groups (1 of 242). In the human population investigated here, the overall seroprevalences forB. microtiandB. divergenswere 5.4% (25 of 467) and 3.6% (17 of 467), respectively. The results obtained here provide evidence for concurrent infections withBorrelia burgdorferiandBabesiaspecies in humans exposed to ticks in midwestern Germany. They also suggest that infections withBabesiaspecies in the German human population are more frequent than believed previously and should be considered in the differential diagnosis of febrile illness occurring after exposure to ticks or blood transfusions, in particular in immunocompromised patients.
Clostridium difficile
Genotyping Based on
slpA
Variable Region in S-Layer Gene Sequence: an Alternative to Serotyping
Authors: Karjalainen Tuomo , Saumier Nicolas , Barc Marie-Claude , Delmée Michel , Collignon Anne
Published: July 2002

Recent investigations of
Clostridium difficile
cell wall components have revealed the presence of an S-layer encoded by the
slpA
gene. The aim of this study was to determine whether
slpA
genotyping can be used as an alternative to serotyping. The variable regions of
slpA
were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups
.
These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the
slpA
variable region, could constitute an alternative typing method for determining
C. difficile
serotypes.
Genetic Diversity of Clinical and Environmental Strains of
Salmonella enterica
Serotype Weltevreden Isolated in Malaysia
Authors: Thong K. L. , Goh Y. L. , Radu S. , Noorzaleha S. , Yasin R. , Koh Y. T. , Lim V. K. E. , Rusul G. , Puthucheary S. D.
Published: July 2002

The incidence of food-borne salmonellosis due to
Salmonella enterica
serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of
Salmonella
serotype Weltevreden strains from humans and the environment. PFGE of
Xba
I-digested chromosomal DNA from 95 strains of
Salmonella
serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of
Salmonella
serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with
Salmonella
serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of
Salmonella
serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of
Salmonella
serotype Weltevreden in Malaysia.
Molecular Peculiarities of the
lytA
Gene Isolated from Clinical Pneumococcal Strains That Are Bile Insoluble
Authors: Obregón Virginia , García Pedro , García Ernesto , Fenoll Asunción , López Rubens , García José L.
Published: July 2002

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical
Streptococcus pneumoniae
) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent
lytA
alleles (pairwise evolutionary distances of about 20%) compared to the
lytA
alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to
S. pneumoniae
. The clinical importance of these findings is underlined by the role of the
lytA
gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the
Streptococcus mitis
group.
Sensitive Detection of HBsAg Mutants by a Gap Ligase Chain Reaction Assay
Authors: Osiowy Carla
Published: July 2002

Hepatitis B virus (HBV) surface gene variants have been associated with diagnostic escape and immune escape following vaccination. The most common mutation observed in these variants is a glycine-to-arginine substitution at amino acid 145 (G145R). In order to sensitively detect the presence of this mutant in serum, a new molecular detection system was developed; in this new system, a gap ligase chain reaction (gLCR) assay was coupled with electrochemiluminescence detection of reaction products. The gLCR assay could detect approximately 10 copies of mutant DNA and could discriminate low levels of mutant DNA in the presence of excess wild-type DNA. Detection of the G145R mutant in clinical specimens was evaluated by testing 56 suspect serum specimens. The G145R mutation was observed in 18 of 28 HBV-DNA-positive samples. The approximate percentage of mutant present in each specimen was calculated by comparison with a standard curve of an increasing ratio of mutant DNA to wild-type DNA. Most samples contained a very low percentage of mutant virus (approximately 5%), with an observed range of approximately 3 to 74%. The G145R mutation was most frequently observed in specimens producing a diagnostic anomaly or from transplant patients but was also observed in specimens from vaccinated individuals and specimens in which HBsAg diagnostic escape was suspected. Therefore, the gLCR assay is a sensitive and specific method for detection of G145R mutants, which could be modified to include the detection of other HBV mutants.
Genetic Diversity of the Outer Surface Protein C Gene of SouthernBorreliaIsolates and Its Possible Epidemiological, Clinical, and Pathogenetic Implications
Authors: Lin Tao , Oliver, James H. , Gao Lihui
Published: July 2002

TheospCgenes of 20 southernBorreliastrains were sequenced. The strains consisted ofB. burgdorferisensu stricto,B. andersonii,B. bissettii, one undescribed genospecies, MI-8, and one probably newBorreliaspecies, TXW-1. A high degree of similarity exists betweenB. burgdorferisensu stricto andB. bissettiiand betweenB. bissettiiandB. andersonii. Lateral transfers of theospCgene probably occurred betweenB. burgdorferisensu stricto andB. bissettiiand betweenB. bissettiiandB. andersonii. Internal gene recombination appears to occur among them. The highest degree of genetic diversity among them was observed in the two variable domains (V1 and V2), semivariable domain (SV), and the species-specific epitopes (between amino acids 28 and 31). Differences inospCsequences among southern strains reflect diversity at the strain and genospecies levels. MI-8, which was recognized as an undescribed genospecies in our previous reports, remains distinguishable in our current analysis ofospCgenes and is distinct fromB. burgdorferisensu stricto. Interestingly, another undescribed southern isolate, TXW-1, was not amplified under various PCR conditions. Compared to EuropeanB. burgdorferisensu stricto strains, AmericanB. burgdorferisensu stricto strains show greater genetic heterogeneity. SouthernB. burgdorferisensu stricto,B. andersonii, andB. bissettiiisolates were intermixed with each other in the phylogenetic trees. In the derived trees in our work, at least one southeastern strain ofB. burgdorferi, MI-2, most closely aligns with a so-called invasive cluster that possesses many proven human-invasive strains. Transmission experiments show that MI-2 and the strains in this group of southern spirochetes are able to infect mice and hamsters and that the typical vector of Lyme disease,Ixodes scapularis, can acquire the spirochetes from infected mammals. Currently, strain MI-2 appears to be the only southern isolate among the 20 we analyzed that clusters with an OspC invasive group and thus might be invasive for humans.
Accuracy of Results Obtained by Performing a Second Ligase Chain Reaction Assay and PCR Analysis on Urine Samples with Positive or Near-Cutoff Results in the LCx Test for
Chlamydia trachomatis
Authors: Castriciano S. , Luinstra K. , Jang D. , Patel J. , Mahony J. , Kapala J. , Chernesky M.
Published: July 2002

Nucleic acid amplification assays such as the ligase chain reaction and PCR have encountered reproducibility problems. The initial extract and a newly extracted aliquot of urine specimens (
n
= 120) which had signal-to-cutoff (S/CO) ratios above 0.80 by the LCx Chlamydia assay were retested. Nucleic acid was extracted from an additional urine sample for testing by the AMPLICOR PCR Chlamydia assay. Fifteen percent (18 of 120) of the urine specimens were negative by all repeat tests (initial mean S/CO ratio by the LCx Chlamydia assay, 0.93; S/CO ratio range, 0.80 to 3.30). Repeat testing of the 102 specimens with possible positive results by the LCx Chlamydia assay by use of the initially extracted aliquot confirmed the results for 95 (93.1%) of the specimens; repeat testing of a newly extracted aliquot confirmed the results for 87 (85.3%) of the specimens. Twenty specimens had discordant results by the two repeat LCx Chlamydia assays. A total of 78 of 102 (76.5%) of the specimens were positive by the AMPLICOR PCR, and the AMPLICOR PCR confirmed the results for 82.1% (78 of 95) and 89.6% (78 of 87) of the specimens positive by the two repeat LCx Chlamydia assays, respectively. Some of the discrepancies observed by multiple repeat tests may have been due to specimen mislabeling or contamination during performance of the procedure rather than to the LCx Chlamydia assay. Both assays suffered from a lack of reproducibility on repeat testing with a small proportion of specimens, probably due to the presence of low levels of DNA, the presence of variable amounts of amplification inhibitors, and the loss of DNA during extraction.
Native Valve Endocarditis Due to
Enterococcus hirae
Authors: Poyart Claire , Lambert Thierry , Morand Philippe , Abassade Philippe , Quesne Gilles , Baudouy Yves , Trieu-Cuot Patrick
Published: July 2002

Enterococcus hirae
is a rare isolate in clinical specimens. We describe a case of native aortic-valve endocarditis that was caused by
Enterococcus hirae
in a 72-year-old man. This is the first reported case of endocarditis due to this organism.
Fatal
Saccharomyces cerevisiae
Aortic Graft Infection
Authors: Smith Davey , Metzgar David , Wills Christopher , Fierer Joshua
Published: July 2002

Saccharomyces cerevisiae
is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults (J. N. Aucott, J. Fayan, H. Grossnicklas, A. Morrissey, M. M. Lederman, and R. A. Salata, Rev. Infect. Dis. 12:406-411, 1990). We present a case of
S. cerevisiae
fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of
S. cerevisiae
fungemia where the identity of the pathogen was confirmed by rRNA sequencing.
Neutralizing Anti-F Glycoprotein and Anti-Substance P Antibody Treatment Effectively Reduces Infection and Inflammation Associated with Respiratory Syncytial Virus Infection
Authors: Haynes Lia M. , Tonkin Joelyn , Anderson Larry J. , Tripp Ralph A.
Published: July 2002

Respiratory syncytial virus (RSV) is the most important virus mediating lower respiratory tract illness in infants and young children. RSV infection is associated with pulmonary inflammation and increased levels of substance P (SP), making the airways and leukocytes that express SP receptors susceptible to the proinflammatory effects of this peptide. This study examines combining neutralizing anti-F glycoprotein and anti-SP antibody treatment of RSV-infected BALB/c mice to inhibit RSV replication and inflammation associated with infection. BALB/c mice were prophylactically treated with antibody prior to RSV infection or were therapeutically treated at day 2 or 6 post-RSV infection. Prophylactic or therapeutic treatment with anti-SP antibodies promptly reduced pulmonary inflammatory cell infiltration and decreased the number of cells expressing proinflammatory cytokines, while anti-F antibody treatment reduced virus titers. The results suggest that combined anti-viral and anti-SP antibody treatment may be effective in treating RSV disease.
Complementary Roles of Multiple Nuclear Targeting Signals in the Capsid Proteins of the Parvovirus Minute Virus of Mice during Assembly and Onset of Infection
Authors: Lombardo Eleuterio , Ramírez Juan C. , Garcia Javier , Almendral José M.
Published: July 2002

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T=1 icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.
Immune Control of the Number and Reactivation Phenotype of Cells Latently Infected with a Gammaherpesvirus
Authors: Tibbetts Scott A. , van Dyk Linda F. , Speck Samuel H. , Virgin Herbert W.
Published: July 2002

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8+T cells regulate both latency and persistent replication and demonstrate for the first time that CD8+T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-γ) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-γ are site specific, with IFN-γ being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-γ and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-γ acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.
Importance of the Cytoplasmic Tails of the Measles Virus Glycoproteins for Fusogenic Activity and the Generation of Recombinant Measles Viruses
Authors: Moll Markus , Klenk Hans-Dieter , Maisner Andrea
Published: July 2002

The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcΔ30), as well as an MV with an H tail of 14 residues (rMV-HcΔ20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.
CD4 T-Cell-Mediated Demyelination Is Increased in the Absence of Gamma Interferon in Mice Infected with Mouse Hepatitis Virus
Authors: Pewe Lecia , Haring Jodie , Perlman Stanley
Published: July 2002

Mice infected with the murine coronavirus, mouse hepatitis virus, strain JHM (MHV) develop an immune-mediated demyelinating encephalomyelitis. Adoptive transfer of MHV-immune splenocytes depleted of either CD4 or CD8 T cells to infected mice deficient in recombination activation gene 1 resulted in demyelination. We showed previously that the process of CD8 T-cell-mediated demyelination was strongly dependent on the expression of gamma interferon (IFN-γ) by donor cells. In this report, we show, in contrast, that demyelination and lymphocyte infiltration were increased in recipients of IFN-γ−/−CD4 T cells when compared to levels in mice receiving C57BL/6 CD4 T cells.
Matrix Metalloproteinase Expression Correlates with Virulence following Neurotropic Mouse Hepatitis Virus Infection
Authors: Zhou Jiehao , Stohlman Stephen A. , Atkinson Roscoe , Hinton David R. , Marten Norman W.
Published: July 2002

The relationship(s) between viral virulence and matrix metalloproteinase (MMP) expression in the central nervous system (CNS) of mice undergoing lethal and sublethal infections with neurotropic mouse hepatitis virus was investigated. Lethal infection induced increased levels of MMP-3 and MMP-12 mRNAs as well as that of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) compared to sublethal infection. Increased induction of MMP, TIMP, and chemokine expression correlated with increased virus replication but not with inflammatory cell infiltration. Infection of immunosuppressed mice suggested that expression of most MMP, TIMP, and chemokine mRNA was induced primarily in CNS-resident cells. By contrast, MMP-9 protein activity was associated with the infiltration of neutrophils into the CNS. These data indicate an association between the magnitude of inflammatory gene expression within the CNS and viral virulence.
Avian and Mammalian Hepadnaviruses Have Distinct Transcription Factor Requirements for Viral Replication
Authors: Tang Hong , McLachlan Alan
Published: July 2002

Hepadnavirus replication occurs in hepatocytes in vivo and in hepatoma cell lines in cell culture. Hepatitis B virus (HBV) replication can occur in nonhepatoma cells when pregenomic RNA synthesis from viral DNA is activated by the expression of the nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X receptor α (RXRα) plus peroxisome proliferator-activated receptor α (PPARα) heterodimer. Nuclear hormone receptor-dependent HBV replication is inhibited by hepatocyte nuclear factor 3 (HNF3). In contrast, HNF3 and HNF4 support duck hepatitis B virus (DHBV) replication in nonhepatoma cells, whereas the RXRα-PPARα heterodimer inhibits HNF4-dependent DHBV replication. HNF3 and HNF4 synergistically activate DHBV pregenomic RNA synthesis and viral replication. The conditions that support HBV or DHBV replication in nonhepatoma cells are not able to support woodchuck hepatitis virus replication. These observations indicate that avian and mammalian hepadnaviruses have distinct transcription factor requirements for viral replication.
Zinc Finger Domain of Murine Leukemia Virus Nucleocapsid Protein Enhances the Rate of Viral DNA Synthesis in Vivo
Authors: Zhang Wen-hui , Hwang Carey K. , Hu Wei-Shau , Gorelick Robert J. , Pathak Vinay K.
Published: July 2002

In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A
trans
-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in
trans
. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.
Reassessing the Role of Region A in Pit1-Mediated Viral Entry
Authors: Farrell Karen B. , Russ Jill L. , Murthy Ravi K. , Eiden Maribeth V.
Published: July 2002

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.
Contribution of Protein p40 to Hypovirus-Mediated Modulation of Fungal Host Phenotype and Viral RNA Accumulation
Authors: Suzuki Nobuhiro , Nuss Donald L.
Published: July 2002

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus
Cryphonectria parasitica
, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Δp40 mutants retained the ability to confer hypovirulence, Δp40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Δp29 mutant virus. As observed for Δp29-infected colonies, pigment production was significantly increased in Δp40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.