bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of an In Vitro Integration Assay for theBacteroidesConjugative Transposon CTnDOT
Authors: Cheng Qi , Wesslund Neil , Shoemaker Nadja B. , Salyers Abigail A. , Gardner Jeffrey F. 
Published: October 2002

ABSTRACTIntegrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is theBacteroidesCTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by aBacteroidesextract or by purifiedEscherichia coliintegration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.
SipY Is the
Streptomyces lividans
Type I Signal Peptidase Exerting a Major Effect on Protein Secretion
Authors: Palacín Arantxa , Parro Víctor , Geukens Nick , Anné Jozef , Mellado Rafael P. 
Published: October 2002

ABSTRACT

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium
Streptomyces lividans
has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (
sipW
,
sipX
,
sipY
, and
sipZ
) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual
sip
mutants. None of the
sip
genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major
S. lividans
SPase, since the
sipY
mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.
Fe2+-Tetracycline-Mediated Cleavage of the Tn10Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7
Authors: McMurry Laura M. , Aldema-Ramos Mila L. , Levy Stuart B. 
Published: October 2002

ABSTRACTTetA specified by Tn10is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe2+cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe2+-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe2+occurred with Co2+, known to be cotransported with tetracycline. The known substrate Mg2+-tetracycline interfered with cleavage by Fe2+. These findings suggest that cleavage results from binding at a substrate-specific site. Fe2+is known to be able to cleave amide bonds in proteins at distances up to approximately 12 Å. We conclude that the α carbon of glutamine 225 is probably within 12 Å of the position of the Fe2+ion in the Fe2+-tetracycline complex bound to the protein.
Role for Phosphoglucomutase in
Vibrio fischeri-Euprymna scolopes
Symbiosis
Authors: DeLoney Cindy R. , Bartley Therese M. , Visick Karen L. 
Published: October 2002

ABSTRACT

Vibrio fischeri
, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid
Euprymna scolopes.
In a screen for
V. fischeri
colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type
V. fischeri
. Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization. We determined that the transposon in this mutant disrupted a gene with high sequence identity to the
pgm
(phosphoglucomutase) gene of
Escherichia coli
, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose. The
V. fischeri
mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between
E. coli
Pgm and the product of the
V. fischeri
gene, which was therefore designated
pgm
. In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents. Chromosomal complementation with the wild-type
pgm
allele restored the colonization ability to the mutant and also complemented the other noted defects. Unlike the
pgm
mutant, a galactose-utilization mutant (
galK
) of
V. fischeri
colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the
pgm
mutant is not due to an inability to catabolize galactose. Thus,
pgm
represents a new gene required for promoting colonization of
E. scolopes
by
V. fischeri
.
Staphylococcus aureusContains Two Low-Molecular-Mass Phosphotyrosine Protein Phosphatases
Authors: Soulat Didier , Vaganay Elisabeth , Duclos Bertrand , Genestier Anne-Laure , Etienne Jérôme , Cozzone Alain J. 
Published: October 2002

ABSTRACTThe analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacteriumStaphylococcus aureussuggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in anEscherichia colisystem. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40°C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors,N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases.
PCR-Based Method for Detecting Viral Penetration of Medical Exam Gloves
Authors: Broyles John M. , O'Connell Kevin P. , Korniewicz Denise M. 
Published: October 2002

ABSTRACTThe test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage φX174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated.
Evaluation of Performance of Four Genotypic Methods for Studying the Genetic Epidemiology of
Aspergillus fumigatus
Isolates
Authors: Lasker Brent A. 
Published: October 2002

ABSTRACT

In the present investigation, 49
Aspergillus fumigatus
isolates obtained from four nosocomial outbreaks were typed by
Afut1
restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP) analysis, and polymorphic microsatellite markers (PMM) analysis. The typing methods were evaluated with respect to discriminatory power (
D
), reproducibility, typeability, ease of use, and ease of interpretation to determine their performance and utility for outbreak and surveillance investigations.
Afut1
RFLP analysis detected 40 types. Thirty types were observed by RAPD analysis. PMM analysis detected 39 allelic types, but SSDP analysis detected only 14 types. All four methods demonstrated 100% typeability. PMM and RFLP analyses had comparable high degrees of discriminatory power (
D
= 0.989 and 0.988, respectively). The discriminatory power of RAPD analysis was slightly lower (
D
= 0.971), whereas SSDP analysis had the lowest discriminatory power (
D
= 0.889). Overall, SSDP analysis was the easiest method to interpret and perform. The profiles obtained by PMM analysis were easier to interpret than those obtained by RFLP or RAPD analysis. Bands that differed in staining intensity or that were of low intensity were observed by RAPD analysis, making interpretation more difficult. The reproducibilities with repeated runs of the same DNA preparation or with different DNA preparations of the same strain were high for all the methods. A high degree of genetic variation was observed in the test population, but isolates were not always similarly divided by each method. Interpretation of band profiles requires understanding of the molecular mechanisms responsible for genetic alternations. PMM analysis and
Afut1
RFLP analysis, or their combination, appear to provide the best overall discriminatory power, reproducibility, ease of interpretation, and ease of use. This investigation will aid in planning epidemiologic and surveillance studies of
A. fumigatus
.
Phenotypic and Genetic Diversity of Invasive Pneumococcal Isolates Recovered from French Children
Authors: Doit Catherine , Loukil Chawki , Geslin Pierre , Bingen Edouard 
Published: October 2002

ABSTRACT

We investigated the phenotypic and genetic diversity (by ribotyping) of
Streptococcus pneumoniae
isolates recovered from French children, by blood culture from meningitis-free patients (
n
= 244) and by cerebrospinal fluid culture from patients with meningitis (
n
= 154). Isolates belonging to serotypes associated with carriage and penicillin-resistant isolates were significantly more frequent in children under 2 years of age than in older children. The seven-valent pneumococcal conjugate vaccine covered 68% of strains associated with bacteremia and 61% of strains associated with meningitis in children under 2 years. Although some serotypes were recovered more frequently from children with bacteremia than from those with meningitis, no difference in the genetic backgrounds of the two groups of strains was found.
DNA Fingerprinting of
Pasteurella multocida
Recovered from Avian Sources
Authors: Amonsin Alongkorn , Wellehan James F. X. , Li Ling-Ling , Laber Judy , Kapur Vivek 
Published: October 2002

ABSTRACT

Repetitive sequence-based PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to characterize a sample of 43 field isolates and 4 attenuated vaccine strains of
Pasteurella multocida
recovered from multiple avian species. Both rep-PCR and AFLP assays were rapid and reproducible, with high indices of discrimination. Concordance analyses of rep-PCR and AFLP with somatic serotyping indicate that, in general, somatic serotyping is a poor indicator of genetic relatedness among isolates of
P. multocida
. In addition, the data provide evidence of host specificity of
P. multocida
clones. Overall, the results of our study indicate that the rep-PCR and AFLP techniques enable rapid fingerprinting of
P. multocida
isolates from multiple avian species and enhance the investigation of fowl cholera outbreaks.
Cross-Infection Due to Imipenem-Resistant
Bacteroides fragilis
Associated with a Totally Implantable Venous Port
Authors: Arpin Corinne , Dubois Véronique , Rogues Anne-Marie , Menard Fanny , Gavinet Anne-Marie , Maire Jean-Pierre , Gachie Jean-Pierre , Bezian Marie-Christine , Quentin Claudine 
Published: October 2002

ABSTRACT

Four patients in an oncology ward developed
Bacteroides fragilis
bacteremia over a 12-day period. Cross-infection between two of them, due to an imipenem-resistant strain, was demonstrated by epidemiological investigation and genotypic typing methods (arbitrarily primed PCR fingerprinting and nucleotide sequencing of the
cfiA
genes and upstream IS
1186/
IS
1168
elements).
Detection of Enteroaggregative
Escherichia coli
with Formalin-Preserved HEp-2 Cells
Authors: Miqdady Mohamad S. , Jiang Zhi-Dong , Nataro James P. , DuPont Herbert L. 
Published: October 2002

ABSTRACT

Formalin-stored HEp-2 cells were used to assay
Escherichia coli
for adherence. Cells refrigerated in formalin for up to 28 days and used in a wet assay format demonstrated an assay sensitivity ranging from 94 to 98% to detect enteroaggregative
E. coli
(EAEC). HEp-2 cells first fixed and stored with formalin and then stored dry in ambient conditions for 6 weeks demonstrated an assay sensitivity of 92% to detect EAEC. Using formalin-fixed HEp-2 cells will improve the efficiency of EAEC identification.
GenoType Mycobacterium Assay for Identification of Mycobacterial Species Isolated from Human Clinical Samples by Using Liquid Medium
Authors: Ruiz P. , Gutierrez J. , Zerolo F. J. , Casal M. 
Published: October 2002

ABSTRACT

The GenoType Mycobacterium assay was used to identify 98 mycobacteria isolates by using liquid cultures from positive BACTEC, MGIT, and ESP bottles. This system identifies 16 mycobacteria. There was complete agreement between the GenoType results and the laboratory identifications for
Mycobacterium tuberculosis
complex and other
Mycobacterium
spp. GenoType also identified mixed mycobacterial infections.
Serologic and Molecular Detection of
Ehrlichia chaffeensis
and
Anaplasma phagocytophila
(Human Granulocytic Ehrlichiosis Agent) in Korean Patients
Authors: Heo Eun-jeong , Park Jin-ho , Koo Ja-ryong , Park Man-suk , Park Mi-yeoun , Dumler J. Stephen , Chae Joon-seok 
Published: October 2002

ABSTRACT

Sera from 491 Korean patients with acute febrile diseases were tested for
Ehrlichia chaffeensis
and
Anaplasma phagocytophila
antibodies by indirect immunofluorescence assay (IFA), Western blotting, and TaqMan real-time PCR. Overall, 0.4% of sera reacted with
E. chaffeensis
, and 1.8% reacted with
A. phagocytophila
in IFAs. This is the first report of detection of antibodies to
A. phagocytophila
and
E. chaffeensis
in Korea and suggests the presence of
A. phagocytophila
and
E. chaffeensis
or antigenically similar species.
Alphavirus Minus-Strand RNA Synthesis: Identification of a Role for Arg183 of the nsP4 Polymerase
Authors: Fata Cori L. , Sawicki Stanley G. , Sawicki Dorothea L. 
Published: October 2002

ABSTRACT

A partially conserved region spanning amino acids 142 to 191 of the Sindbis virus (SIN) nsP4 core polymerase is implicated in host restriction, elongation, and promoter recognition. We extended the analysis of this region by substituting Ser, Ala, or Lys for a highly conserved Arg183 residue immediately preceding its absolutely conserved Ser184-Ala-Val-Pro-Ser188 sequence. In chicken cells, the nsP4 Arg183 mutants had a nonconditionally lethal, temperature-sensitive (
ts
) growth phenotype caused by a
ts
defect in minus-strand synthesis whose extent varied with the particular amino acid substituted (Ser>Ala>Lys). Plus-strand synthesis by nsP4 Arg183 mutant polymerases was unaffected when corrected for minus-strand numbers, although 26S mRNA synthesis was enhanced at the elevated temperature compared to wild type. The
ts
defect was not due to a failure to form or accumulate nsP4 at 40°C. In contrast to their growth in chicken cells, the nsP4 Arg183 mutants replicated equally poorly, if at all, in mosquito cells. We conclude that Arg183 within the Pro180-Asn-Ile-Arg-Ser184 sequence of the SIN nsP4 polymerase contributes to the efficient initiation of minus strands or the formation of its replicase and that a host factor(s) participates in this event.
The Metastatic Suppressor Nm23-H1 Interacts with EBNA3C at Sequences Located between the Glutamine- and Proline-Rich Domains and Can Cooperate in Activation of Transcription
Authors: Subramanian Chitra , Robertson Erle S. 
Published: October 2002

ABSTRACTEpstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. Recent studies have shown an in vitro and in vivo interaction between the EBV nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1, known to be downregulated in human invasive breast carcinoma. In this study, we have identified the domain of EBNA3C that specifically binds to Nm23-H1. This domain lies within the region comprising amino acids 637 to 675 of EBNA3C flanked by the proline- and glutamine-rich domains. Furthermore, we show that Nm23-H1 activates transcription when fused to the Gal4 DNA-binding domain and is coexpressed with a luciferase reporter construct containing the Gal4 binding sites upstream of a basal promoter. Gal4-Nm23-H1, when tethered to the promoter by binding to the Gal4 DNA binding sequences, consistently activated transcription. The level of activation increased when increasing amounts of Gal4-Nm23-H1 were introduced into the system. Moreover, EBNA3C when cotransfected with Gal4-Nm23-H1 enhanced the transcriptional activity. These results suggest that Nm23-H1 may have intrinsic transcription activities in EBV-infected cells and that this activity can be modulated in the presence of the essential latent antigen EBNA3C.
Down Regulation of the Interleukin-8 Promoter by Human Papillomavirus Type 16 E6 and E7 through Effects on CREB Binding Protein/p300 and P/CAF
Authors: Huang Shih-Min , McCance D. J. 
Published: October 2002

ABSTRACTPreviously, we reported that human papillomavirus (HPV) type 16 E6 binds to C/H1, C/H3, and the C-terminal domains of coactivators p300 and CBP, causing the modulation of the transcription of certain genes controlled by NF-κB (p65 orrelA) and p53. To establish the biological significance of these observations, we have focused on the transcriptional regulation of interleukin-8 (IL-8), a potent chemoattractant for T lymphocytes and neutrophils, which is also essential for the initiation of the local immune response. The IL-8 promoter is regulated by NF-κB/p65 in response to tumor necrosis factor alpha and requires the cooperation of the coactivators CBP/p300 and steroid receptor coactivator 1 (SRC-1) and the p300/CBP-associated factor (P/CAF) for optimal activation. Here we report that, in the presence of HPV-16 E6, the promoter activity of IL-8 was repressed. Moreover, from the mutational analysis of the IL-8 promoter, we found that E6 down-regulates the IL-8 promoter activity through the NF-κB/p65 binding site. This inhibition appears to result from the ability of HPV-16 E6 to compete with NF-κB/p65 and SRC-1 for binding to the N terminus and C terminus of CBP, respectively. Reporter data also showed that E7 represses IL-8 promoter activity, though to a lesser extent than E6 but, like E6, the repression by E7 is through the NF-κB/p65 binding site. E7 was shown for the first time to bind to P/CAF, and the binding was necessary for the down regulation of the IL-8 promoter. E6 and E7 together inhibited transcription of the IL-8 promoter to a greater extent than either alone. Finally, by RNase protection assay, we showed that the synthesis of endogenous IL-8 mRNA was repressed in keratinocytes stably expressing E6 and E7. Taken together, the results provide evidence that E6 and E7 can cooperatively disrupt IL-8 transcription through disruption of transcriptional active complexes, and this may have important consequences for immune responses in infected hosts.
Release of Macrophage Migration Inhibitory Factor and CXCL8/Interleukin-8 from Lung Epithelial Cells Rendered Necrotic by Influenza A Virus Infection
Authors: Arndt Ute , Wennemuth Gunther , Barth Peter , Nain Marianne , Al-Abed Yousef , Meinhardt Andreas , Gemsa Diethard , Bacher Michael 
Published: October 2002

ABSTRACTBronchiolar epithelial cells are the prime targets for influenza A virus infection. It still remains to be clarified which signals are generated from these cells to initiate an immune response. Among chemokines, viral infection of primary lung epithelial cells triggered exclusively the release of CXCL8/interleukin-8 (IL-8), which contrasts with our previous observation that influenza A virus induced in monocytes the expression of mononuclear-leukocyte-attracting chemokines and even suppressed the production of neutrophil-attracting chemokines. Therefore, we speculated that it may be advantageous for respiratory epithelial cells to release primarily neutrophil-attracting CXCL8/IL-8 since neutrophils rapidly remove necrotic debris and are the first line of defense against bacterial superinfections. This concept has also been supported by our finding that influenza A virus infection led to necrosis of lung epithelial cells. This is in striking contrast to previous studies where influenza A virus infection induced apoptosis in monocytes and epithelial cells from origins other than the lung. Thus, the cell type instead of the virus determines which death pathway will be followed. In addition to the release of CXCL8/IL-8, we obtained a massive release of macrophage migration inhibitory factor (MIF) from virus-infected lung cells. However, whereas the CXCL8/IL-8 secretion was accompanied by induced gene activation, the transcription rate of MIF remained unchanged during the infection course and the virus-induced MIF release was predominantly a discharge from intracellular stores, suggesting that MIF is passively released upon cell death. Despite virus induced necrosis, the passively liberated MIF remained bioactive. Considering the well-established immunostimulatory effects of MIF on different leukocyte subsets, is its very likely that enhanced levels of MIF may contribute to the host immune response during the acute phase of influenza A virus infection in humans.