bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (
lktCABD
) in
Mannheimia
(
Pasteurella
) haemolytica,
Mannheimia glucosida
, and
Pasteurella trehalosi
Authors: Davies Robert L. , Campbell Susan , Whittam Thomas S. 
Published: July 2002

ABSTRACT

The mosaic structure and molecular evolution of the leukotoxin operon (
lktCABD
) was investigated by nucleotide sequence comparison of the
lktC
,
lktB
, and
lktD
genes in 23
Mannheimia
(
Pasteurella
)
haemolytica
, 6
Mannheimia glucosida
, and 4
Pasteurella trehalosi
strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394–1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of
M. haemolytica
. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including
M. glucosida
and
P. trehalosi
. In contrast, the
lktD
gene is highly conserved in
M. haemolytica
. The
lktC
,
lktA
, and
lktB
genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of
M. haemolytica
. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5′ and 3′ ends of the operon are highly conserved in
M. haemolytica
, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the
lktA
and
lktB
genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.
Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II
Authors: Degen Olaf , Eitinger Thomas 
Published: July 2002

ABSTRACTHoxN, a high-affinity, nickel-specific permease ofRalstonia eutrophaH16, and NhlF, a nickel/cobalt permease ofRhodococcus rhodochrousJ1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+and Ni2+uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+and Co2+with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.
An Antimicrobial Peptide Is Produced by Extracellular Processing of a Protein from
Propionibacterium jensenii
Authors: Faye Therese , Brede Dag Anders , Langsrud Thor , Nes Ingolf F. , Holo Helge 
Published: July 2002

ABSTRACT

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of
Propionibacterium jensenii
LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (
pamA
) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of ∼4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.
Transcription Factor FnrP from
Paracoccus denitrificans
Contains an Iron-Sulfur Cluster and Is Activated by Anoxia: Identification of Essential Cysteine Residues
Authors: Hutchings Matthew I. , Crack Jason C. , Shearer Neil , Thompson Benjamin J. , Thomson Andrew J. , Spiro Stephen 
Published: July 2002

ABSTRACT

The
Paracoccus denitrificans
transcription factor FnrP has been characterized using artificial FNR-dependent promoter-
lacZ
fusion plasmids in
Escherichia coli
. FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at −41.5 with respect to the transcription start site. FnrP was found to be inactive in an
iscS
mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP. Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine. Thus, FnrP is a true orthologue of FNR from
E. coli
and switches on target genes in response to anoxia. Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster. Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo. Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation. The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system. Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO
+
) in defined growth media but are only moderately sensitive to those sources of NO that were tested.
Role of the RecBCD Recombination Pathway in
Salmonella
Virulence
Authors: Cano David A. , Pucciarelli M. Graciela , García-del Portillo Francisco , Casadesús Josep 
Published: July 2002

ABSTRACT

Mutants of
Salmonella enterica
lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933–936, 1993). The virulence-related defects of RecBC
−
mutants are not suppressed by
sbcB
and
sbcCD
mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by
S. enterica
may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during
Salmonella
infection.
Nonisotopic Detection of Human Papillomavirus DNA in Clinical Specimens Using a Consensus PCR and a Generic Probe Mix in an Enzyme-Linked Immunosorbent Assay Format
Authors: Kornegay J. R. , Shepard A. P. , Hankins C. , Franco E. , Lapointe N. , Richardson H. ,   The Canadian Women's HIV Study Group, Coutleé F. 
Published: July 2002

ABSTRACT
We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR–enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated β-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.
V3 Serological Subtyping of Human Immunodeficiency Virus Type 2 Infection Is Not Relevant
Authors: Plantier Jean-Christophe , Damond Florence , Souquières Sandrine , Brun-Vézinet Françoise , Simon François , Barin Francis 
Published: July 2002

ABSTRACT
V3 enzyme immunoassays have been shown to discriminate effectively between human immunodeficiency virus type 1 (HIV-1) subtypes. The aim of this study was to investigate the feasibility of V3 serotyping for HIV-2 infection. We serotyped 29 sera with three peptides, corresponding to the V3 loop of subtypes A, B, and D of HIV-2. Sera were collected from HIV-2-infected patients, whose infecting strains were sequenced and subjected to phylogenetic analysis. Our results indicate that HIV-2 serotyping using V3 peptides is not relevant. V3 serotyping data were not consistent with genotyping results. The V3-A and V3-D peptides displayed poor discrimination, and the V3-B peptide was not representative of circulating viruses. Comparison of amino acid sequences and serotype reactivities demonstrated the importance of positions 309 and 314, located on either side of the tip of the V3 loop, in antibody binding.
Use of Spoligotyping To Study the Evolution of the Direct Repeat Locus by IS
6110
Transposition in
Mycobacterium tuberculosis
Authors: Legrand Eric , Filliol Ingrid , Sola Christophe , Rastogi Nalin 
Published: July 2002

ABSTRACT

Based on the variability of 43 spacers within the direct repeat (DR) locus of
Mycobacterium tuberculosis
complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in
M. tuberculosis
clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS
6110
between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin. Microbiol. 38:1231–1234, 2000). In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS
6110
-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS
6110
associated with the DR locus and an additional double IS
6110
band of 1.4 kb. The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS
6110
fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS
6110
between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS
6110
preferential locus (
ipl
), permitting the insertion of two or more copies of IS
6110
, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS
6110
-RFLP and spoligotyping profiles.
Characterization of Human Cytomegalovirus Strains by Analysis of Short Tandem Repeat Polymorphisms
Authors: Walker Adrianne , Petheram Susan J. , Ballard Linda , Murph Jody R. , Demmler Gail J. , Bale James F. 
Published: July 2002

ABSTRACT
Human cytomegalovirus (HCMV) strains display genetic polymorphisms, and these polymorphisms can be analyzed to study viral transmission and pathogenesis. Recently, short tandem repeat (STR) length polymorphisms have been identified in the HCMV genome. We assessed the utility of STRs in characterizing HCMV strains and found that a multiplexed PCR assay using primers based upon these STRs accurately maps HCMV strains. Using primers for 10 microsatellite regions, the STR profiles of 44 wild-type and 2 laboratory strains of HCMV were characterized. The results of STR analysis were compared with those for strain characterization using nucleotide sequencing and restriction fragment length polymorphism analysis. In each instance, STR analysis accurately and specifically identified strains that were indistinguishable or distinct by conventional molecular analysis. Analysis of short tandem repeats also detected polymorphisms that supported simultaneous excretion of two HCMV strains. These results indicate that STR analysis allows rapid, precise molecular characterization of HCMV strains.
Identification of
Aspergillus fumigatus
and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions
Authors: Zhao Jun , Kong Fanrong , Li Ruoyu , Wang Xiaohong , Wan Zhe , Wang Duanli 
Published: July 2002

ABSTRACT

Aspergillus fumigatus
is the most common species that causes invasive aspergillosis. In order to identify
A. fumigatus
, partial ribosomal DNA (rDNA) from two to six strains of five different
Aspergillus
species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of
A. fumigatus
. A nested PCR method for identification of other
A. fumigatus
-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of
A. fumigatus
and variants, 8 isolates of
Aspergillus nidulans
, 7 isolates of
Aspergillus flavus
and variants, 8 isolates of
Aspergillus terreus
, 9 isolates of
Aspergillus niger
, 1 isolate each of
Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis
, and
Exophiala dermatitidis
, 4 isolates of
Candida
, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the
A. fumigatus
isolates and closely related species and showed a high degree of sensitivity. Additionally, four
A. fumigatus
strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of
A. fumigatus
and closely related species in culture and suggest further studies for the identification of
Aspergillus fumigatus
species in clinical specimens.
Serological Expression Cloning and Immunological Evaluation of MTB48, a NovelMycobacterium tuberculosisAntigen
Authors: Lodes Michael J. , Dillon Davin C. , Mohamath Raodoh , Day Craig H. , Benson Darin R. , Reynolds Lisa D. , McNeill Patricia , Sampaio Diana Pedral , Skeiky Yasir A. W. , Badaro Roberto , Persing David H. , Reed Steven G. , Houghton Raymond L. 
Published: July 2002

ABSTRACTImproved diagnostics are needed for the detection ofMycobacterium tuberculosis,especially for patients with smear-negative disease. To address this problem, we have screenedM. tuberculosis(H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity withM. tuberculosislysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of theM. tuberculosisH37Rv isolate and showed 99% identity to theMycobacterium bovisandM. bovisBCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing theesat-6gene that is deleted in theM. bovisBCG isolate. Themtb481,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in theM. tuberculosisandM. bovisisolates tested but not in other mycobacterial species tested, includingMycobacterium lepraeandMycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and theM. tuberculosis38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity forM. tuberculosisinfection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.
Clarithromycin-Susceptible and -Resistant
Helicobacter pylori
Isolates with Identical Randomly Amplified Polymorphic DNA-PCR Genotypes Cultured from Single Gastric Biopsy Specimens Prior to Antibiotic Therapy
Authors: van der Ende A. , van Doorn L.-J. , Rooijakkers S. , Feller M. , Tytgat G. N. J. , Dankert J. 
Published: July 2002

ABSTRACT

Of the
Helicobacter pylori
populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous
H. pylori
population, resistant
H. pylori
colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible
H. pylori
colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous
H. pylori
populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary
H. pylori
population is used to test for clarithromycin susceptibility.
Antibody-Secreting Cell Responses to Rotavirus Proteins in Gnotobiotic Pigs Inoculated with Attenuated or Virulent Human Rotavirus
Authors: Chang K. O. , Vandal O. H. , Yuan L. , Hodgins D. C. , Saif L. J. 
Published: July 2002

ABSTRACT

Because of their similarities to infants in mucosal immune responses and their susceptibility to human rotavirus (HRV) diarrhea, gnotobiotic pigs provide a useful model for rotaviral disease. In this study, we performed quantitative enzyme-linked immunospot (ELISPOT) assays to measure local and systemic isotype-specific antibody-secreting cell (ASC) responses to individual structural (VP4, VP6, and VP7) and nonstructural (NSP3 and NSP4) proteins of Wa HRV. The
Spodoptera frugiperda
cells expressing each recombinant baculovirus HRV protein were formalin fixed and used as antigen for ELISPOT assays. Neonatal gnotobiotic pigs were orally inoculated once with virulent Wa (WaV) or three times with attenuated Wa (WaA) HRV or mock inoculated (Mock) and then were challenged with virulent Wa (WaV/PC) 28 days after the first inoculation. The ASCs from intestinal and systemic lymphoid tissues of pigs from each group were quantitated by ELISPOT assay at the day of challenge, at postinoculation day 28 (WaV, WaA, and Mock) or at postchallenge day (PCD) 7 (WaV+WaV/PC, WaA+WaV/PC, and Mock+WaV/PC). In all virus-inoculated pigs, regardless of the inoculum, lymphoid tissue, or isotype, VP6 induced the highest numbers of ASCs, followed by VP4; ASCs specific for VP7, NSP3, and NSP4 were less numerous. At challenge, total HRV- and HRV protein-specific immunoglobulin A (IgA) and IgG ASCs in intestinal lymphoid tissues were significantly greater in WaV- than in WaA-inoculated pigs, and WaV pigs were fully protected against diarrhea postchallenge, whereas the WaA pigs were partially protected. At PCD 7, there were no significant differences in ASC numbers for any HRV proteins between the WaV+WaV/PC and WaA+WaV/PC groups.
Sequence Typing Confirms that
Campylobacter jejuni
Strains Associated with Guillain-Barré and Miller-Fisher Syndromes Are of Diverse Genetic Lineage, Serotype, and Flagella Type
Authors: Dingle K. E. , Van Den Braak N. , Colles F. M. , Price Lawrence J. , Woodward David L. , Rodgers Frank G. , Endtz H. P. , Van Belkum A. , Maiden M. C. J. 
Published: July 2002

ABSTRACT

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by
Campylobacter jejuni
in up to 40% of cases. Nucleotide sequence-based typing of 25
C. jejuni
isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800
C. jejuni
isolates not associated with neuropathy. A total of 13 genetic lineages and 20
flaA
short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the
flaA
short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.