bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterologous Inducible Expression of
Enterococcus faecalis
pCF10 Aggregation Substance Asc10 in
Lactococcus lactis
and
Streptococcus gordonii
Contributes to Cell Hydrophobicity and Adhesion to Fibrin
Authors: Hirt Helmut , Erlandsen Stanley L. , Dunny Gary M. 
Published: July 2002

ABSTRACT

Aggregation substance proteins encoded by the sex pheromone plasmid family of
Enterococcus faecalis
have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further,
prgB
, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible
nisA
promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of
E. faecalis
and the heterologous bacteria
Lactococcus lactis
and
Streptococcus gordonii
was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of
E. faecalis
and
L. lactis
but not on that of
S. gordonii
. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of
E. faecalis
and
L. lactis
revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on
L. lactis
also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the
nisA
promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.
Mu and IS
1
Transpositions Exhibit Strong Orientation Bias at the
Escherichia coli bgl
Locus
Authors: Manna Dipankar , Wang Xiuhua , Higgins N. Patrick 
Published: July 2002

ABSTRACT

The region upstream of the
Escherichia coli bgl
operon is an insertion hot spot for several transposons. Elements as distantly related as Tn
1
, Tn
5
, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the
bgl
promoter. In vivo, Mu insertions were focused in two narrow zones of DNA near
bgl
, and both zones exhibited a striking orientation bias. Five hot spots upstream of the
bgl
cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of
bgl
transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS
1
insertions that activate the
bgl
operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.
Identification of Some DNA Damage-Inducible Genes of
Mycobacterium tuberculosis
: Apparent Lack of Correlation with LexA Binding
Authors: Brooks Patricia C. , Movahedzadeh Farahnaz , Davis Elaine O. 
Published: July 2002

ABSTRACT

The repair of DNA damage is expected to be particularly important to intracellular pathogens such as
Mycobacterium tuberculosis
, and so it is of interest to examine the response of
M. tuberculosis
to DNA damage. The expression of
recA
, a key component in DNA repair and recombination, is induced by DNA damage in
M. tuberculosis
. In this study, we have analyzed the expression following DNA damage in
M. tuberculosis
of a number of other genes which are DNA damage inducible in
Escherichia coli
. While many of these genes were also induced by DNA damage in
M. tuberculosis
, some were not. In addition, one gene (
ruvC
) which is not induced by DNA damage in
E. coli
was induced in
M. tuberculosis
, a result likely linked to its different transcriptional arrangement in
M. tuberculosis
. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in
E. coli
. However, two of the genes which were DNA damage inducible in
M. tuberculosis
did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in
M. tuberculosis
.
Identification of the
varR
Gene as a Transcriptional Regulator of Virginiamycin S Resistance in
Streptomyces virginiae
Authors: Namwat Wises , Lee Chang-Kwon , Kinoshita Hiroshi , Yamada Yasuhiro , Nihira Takuya 
Published: July 2002

ABSTRACT

A gene designated
varR
(for
virginiae
antibiotic resistance regulator) was identified in
Streptomyces virginiae
89 bp downstream of a
varS
gene encoding a virginiamycin S (VS)-specific transporter. The deduced
varR
product showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the
varS
promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of
varS
in a VS-dependent manner. Northern blot analysis revealed that
varR
was cotranscribed with upstream
varS
as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of the
varS
promoter region clarified two adjacent VarR binding sites in the
varS
promoter.
Roles of therecJandrecNGenes in Homologous Recombination and DNA Repair Pathways ofNeisseria gonorrhoeae
Authors: Skaar Eric P. , Lazio Matthew P. , Seifert H. Steven 
Published: July 2002

ABSTRACTThe paradigm of homologous recombination comes fromEscherichia coli, where the genes involved have been segregated into pathways. In the human pathogenNeisseria gonorrhoeae(the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcalrecNandrecJgenes in the recombination-based processes of the gonococcus, these genes were inactivated in theN. gonorrhoeaestrain FA1090. We report that bothrecNandrecJloss-of-function mutants show decreased DNA repair ability. In addition, therecJmutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while therecNmutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of eitherrecJorrecNat an irrelevant locus. These results describe the role ofrecJandrecNin the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.
Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of
Riemerella anatipestifer
Authors: Crasta Karen C. , Chua Kim-Lee , Subramaniam Sumathi , Frey Joachim , Loh Hilda , Tan Hai-Meng 
Published: July 2002

ABSTRACT

Riemerella anatipestifer
is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin,
cam
, from a strain of
R. anatipestifer
was cloned and expressed in
Escherichia coli.
Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in
E. coli
XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with
Staphylococcus aureus
. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp
Bam
HI-
Bst
XI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying
cam
obtained by PCR cloning into
E. coli
M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of
O
-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.
Nosocomial Outbreak Due to a Multiresistant Strain of
Pseudomonas aeruginosa
P12: Efficacy of Cefepime-Amikacin Therapy and Analysis of β-Lactam Resistance
Authors: Dubois Véronique , Arpin Corinne , Melon Monique , Melon Bernard , Andre Catherine , Frigo Cécile , Quentin Claudine 
Published: July 2002

ABSTRACT

Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains of
Pseudomonas aeruginosa
P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepime-amikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49- and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant
P. aeruginosa
P12 exhibiting at least four mechanisms of β-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime.
Molecular Epidemiology of
Mycobacterium malmoense
Infections in Scotland
Authors: Doig C. , Muckersie L. , Watt B. , Forbes K. J. 
Published: July 2002

ABSTRACT

Clinical isolates of
Mycobacterium malmoense
collected over 5 years from patients across Scotland with a variety of diseases have been characterized by pulsed-field gel electrophoresis (PFGE), ribotyping, and 16S ribosomal DNA gene sequencing. Results indicate that this species harbors little genetic diversity and that the different strain types that were identified by PFGE showed no correlation with geographical origin or date of isolation.
Detection of
Mycobacterium avium
in Blood Samples of Patients with AIDS by Using PCR
Authors: Rodrigues Vívian de F. Sumnienski , Queiroz Mello Fernanda C. , Ribeiro Marta Osório , Fonseca Leila , Kritski Afrânio Lineu , Rossetti Maria Lucia , Zaha Arnaldo 
Published: July 2002

ABSTRACT

Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect
Mycobacterium avium
. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease.
Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein
Authors: Boyko Vitaly , van der Laak Jessica , Ferralli Jacqueline , Suslova Elena , Kwon Myoung-Ok , Heinlein Manfred 
Published: July 2002

ABSTRACT
Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.
Retroviral Vector Targeting to Human Immunodeficiency Virus Type 1-Infected Cells by Receptor Pseudotyping
Authors: Somia Nikunj V. , Miyoshi Hiroyuki , Schmitt Mark J. , Verma Inder M. 
Published: July 2002

ABSTRACT
We report the generation of retroviral vectors based on Moloney murine leukemia virus that specifically transduce cells infected with T-cell-tropic human immunodeficiency virus type 1 (HIV-1). This vector was pseudotyped with T-cell-tropic HIV-1 receptors CD4 and CXCR4. We demonstrate that transduction is contingent upon HIV-1 gp120 and gp41 expression.
A Single Amino Acid Alteration in the Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Glycoprotein Confers Resistance to the Inhibitory Effects of Zanamivir on Receptor Binding and Neuraminidase Activity
Authors: Murrell Matthew T. , Porotto Matteo , Greengard Olga , Poltoratskaia Natalia , Moscona Anne 
Published: July 2002

ABSTRACT

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-
N
-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.
Cellular Membrane-Binding Ability of the C-Terminal Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope Transmembrane Protein gp41
Authors: Chen Steve S.-L. , Lee Sheau-Fen , Wang Chin-Tien 
Published: July 2002

ABSTRACTThe amphipathic α-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and anEscherichia coliβ-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the β-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated β-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.
The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95
Authors: Takemoto Masaya , Shimamoto Takuya , Isegawa Yuji , Yamanishi Koichi 
Published: July 2002

ABSTRACTAn immediate-early (IE) gene of human herpesvirus 6 (HHV-6), U95, has similarity at the amino acid level to the murine cytomegalovirus (MCMV) IE2 gene and is related to the human cytomegalovirus (HCMV) US22 gene family. Sequence analyses of U95 cDNA clones revealed that the transcription start site was located about 1.6 kbp upstream of the putative initiating ATG and that the transcript consisted of two exons. A single intron extended from nucleotides 142589 to 144229, which contained ORF U94. A protein with a molecular mass of about 120 kDa was translated from this cDNA clone in an in vitro transcription-translation assay. The transcription start site was found to be 220 bp downstream of the R3 region by primer extension analysis. HHV-6 has three repetitive elements, R1, R2, and R3, in or near the IE-A locus. R3 is composed of 24 copies of a 104- to 107-bp sequence element, which contains multiple putative binding sites for cellular transcription factors such as AP2 and NF-κB, and its biological significance has yet to be elucidated. The region between −710 and +46 relative to the transcription start site of U95 was analyzed in this study. Deletion from −710 to −396, corresponding to three copies of an R3 unit, decreased the promoter activity by 15-fold, and coexpression of IκBα(S32A/S36A) repressed it to almost the same level. Electrophoretic mobility shift assays showed that NF-κB family members p50 and c-Rel bound to NF-κB sites derived from the R3 region. These results demonstrate that R3 strongly enhances the U95 promoter activity and that NF-κB and binding sites for NF-κB in the R3 region play an important role in its activation. Because U95 promoter activity correlated with the number of R3 units, which each contained an NF-κB site, the repetitive organization of R3 is important for regulating U95 transcription.
Functional and Antigenic Characterization of Human, Rhesus Macaque, Pigtailed Macaque, and Murine DC-SIGN
Authors: Baribaud Frédéric , Pöhlmann Stefan , Sparwasser Tim , Kimata Monica T. Yu , Choi Yang-Kyu , Haggarty Beth S. , Ahmad Navid , Macfarlan Todd , Edwards Terri G. , Leslie George J. , Arnason Jon , Reinhart Todd A. , Kimata Jason T. , Littman Dan R. , Hoxie James A. , Doms Robert W. 
Published: July 2002

ABSTRACT
DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.