bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of Silent
gal
Genes in the
lac-gal
Regulon of
Streptococcus thermophilus
Authors: Vaughan Elaine E. , van den Bogaard Patrick T. C. , Catzeddu Pasquale , Kuipers Oscar P. , de Vos Willem M. 
Published: July 2002

ABSTRACT

Streptococcus thermophilus
strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with
Escherichia coli
demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order
galKTE
, which was preceded by a divergently transcribed regulator gene,
galR
, and followed by a
galM
gene and the lactose operon
lacSZ
. Results of Northern blot analysis showed that the structural
gal
genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the
galKTE
genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal
+
mutant NZ302G, the transcription of the
galR
gene was induced by growth on lactose. Disruption of
galR
indicated that it functioned as a transcriptional activator of both the
gal
and
lac
operons while negatively regulating its own expression. Sequence analysis of the
gal
promoter regions of NZ302G and nine other independently isolated Gal
+
mutants of CNRZ 302 revealed mutations at three positions in the
galK
promoter region, which included substitutions at positions −9 and −15 as well as a single-base-pair insertion at position −37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of
gusA
reporter gene fusions in strains containing the mutated promoters suggested that they were
gal
promoter-up mutations. We propose that poor expression of the
gal
genes in the galactose-negative
S. thermophilus
CNRZ 302 is caused by naturally occurring mutations in the
galK
promoter.
Structure-Function Analysis of the
Shigella
Virulence Factor IpaB
Authors: Guichon Andrea , Hersh David , Smith Mark R. , Zychlinsky Arturo 
Published: July 2002

ABSTRACT

Infection by the gram-negative bacterium
Shigella flexneri
results in dysentery, an acute inflammatory disease of the colon. Essential events in the pathogenesis of
Shigella
infections include bacterial invasion of epithelial cells, escape from the phagosome, and induction of apoptosis in macrophages. The
Shigella
virulence factor invasion plasmid antigen B (IpaB) is required for all of these processes. Induction of apoptosis is dependent on IpaB binding to the cysteine protease caspase-1 (Casp-1). The activation of this enzyme triggers both apoptosis and release of the proinflammatory cytokine interleukin-1β. Several IpaB mutants were generated to correlate function with protein subdomains. We determined that the N-terminal portion of IpaB is necessary for stable expression of IpaB. A putative amphipathic α-helical domain preserves the structure of IpaB. We found 10 consecutive residues within the amino terminus of the hydrophobic region that play a critical role in invasion, phagosomal escape, and cytotoxicity. An IpaB mutant carrying a mutation in this region binds to Casp-1 yet is not cytotoxic, even following direct delivery to the macrophage cytoplasm. These results indicate that the association between IpaB and Casp-1 is only a step in the activation of macrophage apoptosis.
Phylogenetic Evidence for Horizontal Transfer of
mutS
Alleles among Naturally Occurring
Escherichia coli
Strains
Authors: Brown Eric W. , LeClerc J. Eugene , Li Baoguang , Payne William L. , Cebula Thomas A. 
Published: July 2002

ABSTRACT

mutS
mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the
mutS
phenotype, the evolutionary structure of the
mutS
+
gene in
Escherichia coli
remains unclear. The physical proximity of
mutS
to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify
E. coli
strains in which
mutS
alleles have recombined. Comparison of
mutS
phylogeny against predicted
E. coli
“whole-chromosome” phylogenies (derived from multilocus enzyme electrophoresis and
mdh
sequences) revealed striking levels of phylogenetic discordance among
mutS
alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among
mutS
alleles. Examination of additional sites surrounding
mutS
also revealed incongruous distributions compared to
E. coli
strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min
mutS-rpoS
region is a recombinational hot spot within the
E. coli
chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by
mutS
mutators through rescue of defective
mutS
alleles with wild-type sequences.
Linear Plasmid in the Genome of
Clavibacter michiganensis
subsp.
sepedonicus
Authors: Brown Susan E. , Knudson Dennis L. , Ishimaru Carol A. 
Published: July 2002

ABSTRACT

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen
Clavibacter michiganensis
subsp.
sepedonicus
revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.
Characterization of the
Bacillus subtilis ywsC
Gene, Involved in γ-Polyglutamic Acid Production
Authors: Urushibata Yuji , Tokuyama Shinji , Tahara Yasutaka 
Published: July 2002

ABSTRACT

The genes required for γ-polyglutamic acid (PGA) production were cloned from
Bacillus subtilis
IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the
ywsC
and
ywtABC
genes of
B. subtlis
168. Northern blot analysis showed that the four genes constitute an operon. Three genes,
ywsC
,
ywtA,
and
ywtB
, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Δ
ywsC
and Δ
ywtA
strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Δ
ywsC
strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the
ywsC
gene.
14
C-labeled PGA was synthesized by the purified proteins from
l
-[
14
C]-glutamate in the presence of ATP and MnCl
2
, through an acylphosphate intermediate, indicating that the
ywsC
gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.
raiIR
Genes Are Part of a Quorum-Sensing Network Controlled by
cinI
and
cinR
in
Rhizobium leguminosarum
Authors: Wisniewski-Dyé F. , Jones J. , Chhabra S. R. , Downie J. A. 
Published: July 2002

ABSTRACT

Analysis of
N
-acyl-
l
-homoserine lactones (AHLs) produced by
Rhizobium leguminosarum
bv. viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species. Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (
raiI
and
raiR
) that are required for production of AHLs. The
raiIR
genes are located adjacent to
dad
genes (involved in
d
-alanine catabolism) on a large indigenous plasmid. RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for
raiI
expression. The
raiR
gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream
raiI
promoter. Using gene fusions and analysis of AHLs produced, we showed that expression of
raiI
is strongly reduced in strains carrying mutations in
cinI
or
cinR
, genes which determine a higher-level quorum-sensing system that is required for normal expression of
raiIR
. The product of CinI,
N
-(3-hydroxy-7-
cis
tetradecenoyl) homoserine lactone, can induce
raiR
-dependent
raiI
expression, although higher levels of expression are induced by other AHLs. Expression of
raiI
in a strain of
Agrobacterium
that makes no AHLs resulted in the identification of
N
-(3-hydroxyoctanoyl)-
l
-homoserine lactone (3OH,C
8
-HSL) as the major product of RaiI, although other AHLs that comigrate with
N
-hexanoyl-,
N
-heptanoyl-, and
N
-octanoyl-homoserine lactones were also made at low levels. The
raiI
gene was strongly induced by 3OH,C
8
-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the
raiI
promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid. Thus, the
raiIR
and
cinIR
genes are part of a complex regulatory network that influences AHL biosynthesis in
R. leguminosarum
.
Analysis for a Limited Number of Gene Codons Can Predict Drug Resistance of
Mycobacterium tuberculosis
in a High-Incidence Community
Authors: Van Rie Annelies , Warren Robin , Mshanga Idris , Jordaan Annemarie M , van der Spuy Gian D. , Richardson Madalene , Simpson John , Gie Robert P. , Enarson Donald A. , Beyers Nulda , van Helden Paul D. , Victor Thomas C. 
Published: July 2002

ABSTRACT

Correct and rapid diagnosis is essential in the management of multidrug-resistant tuberculosis (MDR-TB). In this population-based study of 61 patients with drug-resistant tuberculosis, we evaluated the frequency of mutations and compared the performance of genotypic (mutation analysis by dot blot hybridization) and phenotypic (indirect proportion method) drug resistance tests. Three selected codons (
rpoB531, rpoB526
, and
katG315
) allowed identification of 90% of MDR-TB cases. Ninety percent of rifampin, streptomycin, and ethambutol resistance and 75% of isoniazid resistance were detected by screening for six codons:
rpoB531, rpoB526, rrs-513, rpsL43, embB306
, and
katG315
. The performance (reproducibility, sensitivity, and specificity) of the genotypic method was superior to that of the routine phenotypic method, with the exception of sensitivity for isoniazid resistance. A commercialized molecular genetic test for a limited number of target loci might be a good alternative for a drug resistance screening test in the context of an MDR “DOTS-plus” strategy.
Rapid Detection of Mutations in the 23S rRNA Gene of
Helicobacter pylori
That Confers Resistance to Clarithromycin Treatment to the Bacterium
Authors: Matsumura Masayuki , Hikiba Yoko , Ogura Keiji , Togo Goichi , Tsukuda Izumi , Ushikawa Kenji , Shiratori Yasushi , Omata Masao 
Published: July 2002

ABSTRACT

We developed a new method capable of detecting point mutations in the 23S rRNA gene of
Helicobacter pylori
using a LightCycler. Our method can detect a mutation in this gene in less than 1 h and can process many samples at once, thereby contributing to the selection of patients suitable for clarithromycin-based therapy.
Comparison of Three Nucleic Acid Amplification Assays of Cerebrospinal Fluid for Diagnosis of Cytomegalovirus Encephalitis
Authors: Bestetti Arabella , Pierotti Chiara , Terreni Mariarosa , Zappa Alessandra , Vago Luca , Lazzarin Adriano , Cinque Paola 
Published: July 2002

ABSTRACT

The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (
n
= 15) or other central nervous system conditions (
n
= 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions.
Serological, Epidemiological, and Molecular Differences between Human T-Cell Lymphotropic Virus Type 1 (HTLV-1)-Seropositive Healthy Carriers and Persons with HTLV-I Gag Indeterminate Western Blot Patterns from the Caribbean
Authors: Rouet François , Meertens Laurent , Courouble Géry , Herrmann-Storck Cécile , Pabingui Raymond , Chancerel Bruno , Abid Adda , Strobel Michel , Mauclere Philippe , Gessain Antoine 
Published: July 2002

ABSTRACT

To investigate the significance of serological human T-cell lymphotropic virus type 1 (HLTV-1) Gag indeterminate Western blot (WB) patterns in the Caribbean, a 6-year (1993 to 1998) cross-sectional study was conducted with 37,724 blood donors from Guadeloupe (French West Indies), whose sera were routinely screened by enzyme immunoassay (EIA) for the presence of HTLV-1 and -2 antibodies. By using stringent WB criteria, 77 donors (0.20%) were confirmed HTLV-1 seropositive, whereas 150 (0.40%;
P
< 0.001) were considered HTLV seroindeterminate. Among them, 41.3% (62) exhibited a typical HTLV-1 Gag indeterminate profile (HGIP). Furthermore 76 (50.7%) out of the 150 HTLV-seroindeterminate subjects were sequentially retested, with a mean duration of follow-up of 18.3 months (range, 1 to 70 months). Of these, 55 (72.4%) were still EIA positive and maintained the same WB profile whereas the others became EIA negative. This follow-up survey included 33 persons with an HGIP. Twenty-three of them (69.7%) had profiles that did not evolve over time. Moreover, no case of HTLV-1 seroconversion could be documented over time by studying such sequential samples. HTLV-1 seroprevalence was characterized by an age-dependent curve, a uniform excess in females, a significant relation with hepatitis B core (HBc) antibodies, and a microcluster distribution along the Atlantic coast of Guadeloupe. In contrast, the persons with an HGIP were significantly younger, had a 1:1 sex ratio, did not present any association with HBc antibodies, and were not clustered along the Atlantic façade. These divergent epidemiological features, together with discordant serological screening test results for subjects with HGIP and with the lack of HTLV-1 proviral sequences detected by PCR in their peripheral blood mononuclear cell DNA, strongly suggest that an HGIP does not reflect true HTLV-1 infection. In regard to these data, healthy blood donors with HGIP should be reassured that they are unlikely to be infected with HTLV-1 or HTLV-2.
Assessment of
Helicobacter pylori vacA
and
cagA
Genotypes and Host Serological Response
Authors: Figueiredo Céu , Quint Wim , Nouhan Nathalie , van den Munckhof Henk , Herbrink Paul , Scherpenisse Joost , de Boer Wink , Schneeberger Peter , Perez-Perez Guillermo , Blaser Martin J. , van Doorn Leen-Jan 
Published: July 2002

ABSTRACT

Helicobacter pylori
strains can be distinguished by genotyping of virulence-associated genes, such as
vacA
and
cagA
. Because serological discrimination between strain types would reduce the need for endoscopy, 61 patients carrying
H. pylori
were studied by
vacA
and
cagA
genotyping of
H. pylori
in gastric biopsy specimens and by detection of specific serum antibodies. Serological responses to
H. pylori
were determined by Helicoblot (versions 2.0 and 2.1). Antibodies to CagA also were determined by a rapid anti-CagA assay (Pyloriset screen CagA) as well as by two noncommercially developed enzyme immunoassays, each using a recombinant CagA protein. Assessment of performance of the Helicoblot assays indicated substantial interobserver variation, with kappa values between 0.20 and 0.93. There was no relationship between the serological profiles on the Helicoblot and the genotypes from the same patients, except for strong associations between the presence of anti-CagA and the
cagA
-positive and
vacA
s1
H. pylori
genotypes. Detection of anti-CagA by the five different assays varied considerably, with kappa values ranging from 0.21 to 0.78. Using the
cagA
genotype as the “gold standard,” the sensitivity and specificity of the anti-CagA assays varied from 71.4 to 85.7% and from 54.2 to 100%, respectively. Thus, serological profiles of antibodies to
H. pylori
are heterogeneous and, with the exception of anti-CagA antibodies, show no relation to the
H. pylori vacA
and
cagA
genotypes. Detection of anti-CagA antibodies is strongly dependent on the test used.
Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome
Authors: Adler Heiko , Messerle Martin , Wagner Markus , Koszinowski Ulrich H. 
Published: July 2002

ABSTRACT

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in
Escherichia coli
. During propagation in
E. coli
, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by
loxP
sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.
The Human Cytomegalovirus IE86 Protein Can Block Cell Cycle Progression after Inducing Transition into the S Phase of Permissive Cells
Authors: Murphy Eain A. , Streblow Daniel N. , Nelson Jay A. , Stinski Mark F. 
Published: July 2002

ABSTRACT
Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.
Identification of an Export Control Sequence and a Requirement for the KH Domains in ICP27 from Herpes Simplex Virus Type 1
Authors: Soliman Tarik M. , Silverstein Saul J. 
Published: July 2002

ABSTRACT
The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an RNA-binding protein that performs multiple functions required for the expression of HSV-1 genes during a productive infection. One essential function involves shuttling between the nucleus and the cytoplasm. Some of the domains identified in ICP27 include a leucine-rich nuclear export sequence (NES), a nuclear localization signal, three KH-like RNA-binding domains, and an RGG-box type RNA-binding motif. To study the contribution of two of the essential domains in ICP27 to HSV gene expression, we generated recombinant herpesviruses carrying deleterious mutations in the NES and KH domains of ICP27. To accomplish this, we fused the green fluorescent protein (GFP) to ICP27 and utilized fluorescence as a marker to isolate recombinant herpesviruses. Fusion of GFP to wild-type ICP27 did not disturb its localization or function or significantly reduce virus yield. Analysis of HSV gene expression in cells infected with a recombinant virus carrying a point mutation in the first KH-like RNA-binding domain revealed that nuclear export of ICP27 was not blocked, and the expression of only a subset of ICP27-dependent late genes was affected. These findings suggest that individual KH-like RNA-binding motifs in ICP27 may be involved in binding distinct RNAs. Analysis of recombinant viruses carrying a lethal mutation in the NES of ICP27 was not accomplished because this mutation results in a strong dominant-negative phenotype. Finally, we demonstrate that shuttling by ICP27 is regulated by an export control sequence adjacent to its NES that functions like the inhibitory sequence element found adjacent to the NES of NS1 from influenza virus.