bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Dephosphorylation of the TorR Response Regulator by the TorS Unorthodox Sensor in
Escherichia coli
Authors: Ansaldi Mireille , Jourlin-Castelli Cécile , Lepelletier Michèle , Théraulaz Laurence , Méjean Vincent 
Published: July 2002

ABSTRACT

Induction of the
torCAD
operon, encoding the trimethylamine
N
-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443→Asp723→His850→Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)→His850→Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of
tor
operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators.
IntI2 Integron Integrase in Tn7
Authors: Hansson Karin , Sundström Lars , Pelletier Alex , Roy Paul H. 
Published: July 2002

ABSTRACTIntegrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7and its relatives. We have completed the sequence of the Tn7integrase gene,intI2, which contains an internal stop codon. This codon was found to be conserved amongintI2genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7at different frequencies. The integrases from Tn21and Tn7showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21and Tn7. However, we did not observe a corresponding excision of theaadA1cassette from Tn21by IntI2*179E.
Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1
Authors: Sawitzke James A. , Li Yongfang , Sergueev Kirill , Youngren Brenda , Brendler Therese , Jones Kristine , Austin Stuart 
Published: July 2002

ABSTRACT

The partition site,
parS
, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The
parS
site was inserted into the
Escherichia coli
chromosome between the promoter and the structural gene for β-galactosidase,
lacZ
. There was little interference with
lacZ
expression when ParA and ParB were supplied in
trans
. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of
lacZ
was shut down. ParAM314I, ParB, and
parS
appear to form a nucleoprotein complex that blocks transcription. Mutations in
parA
and
parB
that relieved the
parA
M314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to
parA
and one to
parB
; all had a propagation-defective phenotype (Par
PD
) similar to that of
parA
M314I. Thus, whereas a null
par
mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-
parS
complex for partition to proceed but that the mutations block ParA dissociation. This “permanent” ParA-ParB-
parS
complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant
parA
or
parB
allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.
Application of a Simple Multiplex PCR To Aid in Routine Work of the Mycobacterium Reference Laboratory
Authors: Yeboah-Manu Dorothy , Yates Malcolm D. , Wilson Stuart Mark 
Published: July 2002

ABSTRACT

A PCR specific for spacer regions 33 and 34 of the direct repeat region of the
Mycobacterium tuberculosis
complex was developed to complement the biochemical differentiation of
M
.
tuberculosis
,
Mycobacterium bovis
,
M
.
bovis
BCG, and
Mycobacterium africanum
subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS
6110
and the 65-kDa antigen gene in order to differentiate members of the
M
.
tuberculosis
complex from atypical mycobacteria.
Clonal Relationships among
Shigella
Serotypes Suggested by Cryptic Flagellin Gene Polymorphism
Authors: Coimbra Roney S. , Lefevre Martine , Grimont Francine , Grimont Patrick A. D. 
Published: July 2002

ABSTRACT

The presence of cryptic
fliC
alleles in the genomes of 120 strains representative of the four
Shigella
species was investigated. One fragment was obtained by PCR amplification of
fliC
, with a size varying from 1.2 to 3.2 kbp, depending on the species or serotype. After digestion with endonuclease
Hha
I, the number of fragments in patterns varied from three to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this study. A unique pattern was observed for each the following serotypes:
Shigella dysenteriae
1, 2, 8, and 10 and
S. boydii
7, 13, 15, 16, and 17. On the contrary,
S. dysenteriae
serotype 13 and
S. sonnei
biotype e were each subdivided into two different F types.
S. flexneri
serotypes 3a and X could be distinguished from the cluster containing
S. flexneri
serotypes 1 to 5 and Y.
S. flexneri
serotype 6 clustered with
S. boydii
serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and
S. dysenteriae
serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising
S. dysenteriae
serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining
S. boydii
serotypes 5 and 9. None of the 17
fliC
patterns was found in the
fliC Hha
I pattern database previously described for
Escherichia coli
. Overall, this work supports the hypothesis that
Shigella
evolved from different ancestral strains of
E. coli
. Moreover, the method outlined here is a promising tool for the identification of some clinically important
Shigella
strains as well as for confirmation of atypical isolates as
Shigella
spp.
Multicenter Evaluation of the BDProbeTec ET System for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs
Authors: Van Der Pol Barbara , Ferrero Dennis V. , Buck-Barrington Linda , Hook Edward , Lenderman Connie , Quinn Thomas , Gaydos Charlotte A. , Lovchik Judith , Schachter Julius , Moncada Jeanne , Hall Geraldine , Tuohy Marion J. , Jones Robert B. 
Published: July 2002

ABSTRACT

The performance of the Becton Dickinson BDProbe Tec ET System
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Amplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for
C. trachomatis
and
N. gonorrhoeae
were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women.
C. trachomatis
and
N. gonorrhoeae
sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms.
Pneumocystis carinii
Carriage among Cystic Fibrosis Patients, as Detected by Nested PCR
Authors: Sing Andreas , Geiger Anna Maria , Hogardt Michael , Heesemann Jürgen 
Published: July 2002

ABSTRACT

A total of 137 sputa from 95 consecutive cystic fibrosis (CF) patients undergoing routine bacteriological surveillance were analyzed for
Pneumocystis carinii
colonization using nested PCR. Seven of 95 patients (7.4%) were PCR positive, suggesting that
P. carinii
carriage may exist among CF patients due to their underlying pulmonary disease.
Group G Beta-Hemolytic Streptococcal Bacteremia Characterized by 16S Ribosomal RNA Gene Sequencing
Authors: Woo Patrick C. Y. , Fung Ami M. Y. , Lau Susanna K. P. , Wong Samson S. Y. , Yuen Kwok-Yung 
Published: July 2002

ABSTRACT

Little is known about the relative importance of the four species of Lancefield group G beta-hemolytic streptococci in causing bacteremia and the factors that determine the outcome for patients with group G beta-hemolytic streptococcal bacteremia. From 1997 to 2000, 75 group G beta-hemolytic streptococcal strains were isolated from the blood cultures of 66 patients. Sequencing of the 16S rRNA genes of the group G beta-hemolytic streptococci showed that all 75 isolates were
Streptococcus dysgalactiae
subspecies
equisimilis
. The API system (20 STREP) and Vitek system (GPI) successfully identified 65 (98.5%) and 62 (93.9%) isolates, respectively, as
S. dysgalactiae
subspecies
equisimilis
with >95% confidence, whereas the ATB Expression system (ID32 STREP) only successfully identified 49 isolates (74.2%) as
S. dysgalactiae
subspecies
equisimilis
with >95% confidence. The median age of the patients was 76 years (range, 33 to 99 years). Fifty-six patients (85%) were over 60 years old. All patients had underlying diseases. No source of the bacteremia was identified (primary bacteremia) in 34 patients (52%), whereas 17 (26%) had cellulitis and 8 (12%) had bed sore or wound infections. Fifty-eight patients (88%) had community-acquired group G streptococcal bacteremia. Sixty-two patients (94%) had group G
Streptococcus
recovered in one blood culture, whereas 4 patients (6%) had it recovered in multiple blood cultures. Fifty-nine patients (89%) had group G
Streptococcus
as the only bacterium recovered in their blood cultures, whereas in 7 patients other bacteria were recovered concomitantly with the group G
Streptococcus
in the blood cultures (
Staphylococcus aureus
in 3,
Clostridium perfringens
in 2,
Citrobacter freundii
in 1, and
Bacteroides fragilis
in 1). Overall, 10 patients (15%) died. Male sex, diagnosis other than cellulitis, hospital-acquired bacteremia, and multiple positive blood cultures were associated with mortality {
P
< 0.005 (relative risk [RR] = 7.6),
P
< 0.05 (RR = 3.7),
P
< 0.005 (RR = 5.6), and
P
< 0.05 (RR = 5.6), respectively}. Unlike group C beta-hemolytic streptococcal bacteremia, group G beta-hemolytic streptococcal bacteremia is not a zoonotic infection in Hong Kong.
Application of Restriction Fragment Length Polymorphism Analysis of VP7-Encoding Genes: Fine Comparison of Irish and Global Rotavirus Isolates
Authors: O'Halloran F. , Lynch M. , Cryan B. , Fanning S. 
Published: July 2002

ABSTRACT

A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes (
Rsa
I,
Alu
I, and
Eco
RV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.
Genetic Profiling of Enterohemorrhagic
Escherichia coli
Strains in Relation to Clonality and Clinical Signs of Infection
Authors: Welinder-Olsson Christina , Badenfors Maria , Cheasty Tom , Kjellin Eva , Kaijser Bertil 
Published: July 2002

ABSTRACT

Sixty-seven human strains of enterohemorrhagic
Escherichia coli
(EHEC) (from patients with more or less severe symptoms) were serogrouped and arranged according to pulsed-field gel electrophoresis (PFGE) patterns. We used PCR to investigate the strains according to known or putative virulence factors, and associations with disease were studied. All EHEC strains with the same PFGE pattern belonged to the same serogroup. On the contrary, two serogroups (O157 and O8) included strains with different PFGE patterns. We found several different combinations of chromosomal and plasmid-borne determinants, encoding the putative virulence factors, among the strains. As judged from clinical symptoms, there was no marked difference in pathogenicity among the strains and their combinations of virulence traits. All strains of O157 had the genes coding for verocytotoxin (VT) 2, intimin (eaeA),
E. coli
hemolysin (E-hly), and secreted serine protease (espP). Among EHEC non-O157 strains, the genes coding for VT1 and VT2 were equally dispersed. EaeA positivity was just as common among VT1- as VT2-positive strains. Among the plasmid-borne determinants, E-hly and espP were the most common and E-hly might be a pathogenicity marker among EHEC non-O157 strains. The conclusion is that PFGE is a very useful tool in epidemiological studies. The EHEC plasmids are heterogeneous in their gene composition, with the four plasmid-borne determinants found in many combinations. There was no reliable correlation between chromosomal and plasmid-borne virulence factors and human disease.
Six-Year Molecular Analysis of
Burkholderia cepacia
Complex Isolates among Cystic Fibrosis Patients at a Referral Center for Lung Transplantation
Authors: Heath David G. , Hohneker Kathy , Carriker Charlene , Smith Kelly , Routh Jonathan , LiPuma John J. , Aris Robert M. , Weber David , Gilligan Peter H. 
Published: July 2002

ABSTRACT

Over a 6-year period,
Burkholderia cepacia
complex species were isolated from cystic fibrosis (CF) patients receiving care at The University of North Carolina Hospitals (clinic CF patients) and from those referred from other treatment centers. Fifty-six isolates collected from 30 referred patients and 26 clinic CF patients were characterized by pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect the cable pilin gene,
cblA
. PFGE results indicated that six separate clusters (clusters A to F) were present among the 56 isolates and that three clusters (clusters A, B, and E) consisted only of isolates from referred patients infected with
B. cepacia
complex isolates prior to referral. However, one cluster (cluster C) consisted of isolates from four CF patients, and hospital records indicate that this cluster began with an isolate that came from a referred patient and that spread to three clinic CF patients. Cluster D consisted of two isolates from clinic CF patients, and hospitalization records are consistent with nosocomial, patient-to-patient spread.
cblA
was present in only 4 of the 56 isolates and included isolates in cluster E from the referred patients. Our results indicate a lack of spread of a previously characterized, transmissible clone from referred patients to our clinic CF population. Only two instances of nosocomial, patient-to-patient spread could be documented over the 6-year period. An additional spread of an isolate (cluster F) from a referred patient to a clinic patient could not be documented as nosocomial and may have been the result of spread in a nonhospitalized setting. The majority (36 of 56) of our
B. cepacia
complex-infected CF patients harbor isolates with unique genotypes, indicating that a diversity of sources account for infection. These data suggest that CF patients infected with
B. cepacia
complex and referred for lung transplantation evaluation were not a major source of
B. cepacia
complex strains that infected our resident CF clinic population.
The Surface Conformation of Sindbis Virus Glycoproteins E1 and E2 at Neutral and Low pH, as Determined by Mass Spectrometry-Based Mapping
Authors: Phinney Brett S. , Blackburn Kevin , Brown Dennis T. 
Published: July 2002

ABSTRACT
Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1–21 (region encompassing amino acids 1 to 21), 161–176, and 212–220, while the E2 regions that are accessible include domains 31–84, 134–148, 158–186, 231–260, 299–314, and 324–337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99–102 and 262–309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86–91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863–872, 1996). E1 273–287 and region 145–158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion.
In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity
Authors: Sedlik C. , Dadaglio G. , Saron M. F. , Deriaud E. , Rojas M. , Casal S. I. , Leclerc C. 
Published: July 2002

ABSTRACTMany approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.
Functional Heterogeneity and High Frequencies of Cytomegalovirus-Specific CD8+T Lymphocytes in Healthy Seropositive Donors
Authors: Gillespie Geraldine M. A. , Wills Mark R. , Appay Victor , O'Callaghan Chris , Murphy Mike , Smith Neil , Sissons Patrick , Rowland-Jones Sarah , Bell John I. , Moss Paul A. H. 
Published: July 2002

ABSTRACTHuman cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8+T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8+T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in “fresh” killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.