bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in
Bacillus subtilis
168
Authors: Tsuge Kenji , Itaya Mitsuhiro 
Published: July 2002

ABSTRACT

Transformation of
Bacillus subtilis
by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic
B. subtilis
recombinational repair pathway. The vector, pGETS103, a derivative of the θ-type replicating plasmid pTB19 of thermophilic
Bacillus
, had the full length of
Escherichia coli
plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of
B. subtilis
. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.
Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein
Authors: Umeyama Takashi , Horinouchi Sueharu 
Published: July 2002

ABSTRACT

A protein serine/threonine kinase, AfsK, and its target protein AfsR globally control physiological and morphological differentiation in the bacterial genus
Streptomyces
. A protein (KbpA) of 252 amino acids encoded by an open reading frame in a region upstream of
afsK
in
Streptomyces coelicolor
A3(2) was identified as an AfsK-interacting protein. The interaction site of AfsK was in the N-terminal portion containing the kinase catalytic domain. KbpA bound a nonphosphorylated form of AfsK and inhibited its autophosphorylation at serine and threonine residues. KbpA in the reaction mixture containing AfsK and AfsR also inhibited the phosphorylation of AfsR by AfsK, presumably because KbpA inhibited the conversion from the inactive, nonphosphorylated form of AfsK to the active, phosphorylated form.
kbpA
was transcribed throughout growth, and the transcription was enhanced when production of actinorhodin had already started. KbpA thus appeared to play an inhibitory role in a negative feedback system in the AfsK-AfsR regulatory pathway. Consistent with these in vitro observations,
kbpA
served as a repressor for actinorhodin production in
S. coelicolor
A3(2); disruption of
kbpA
greatly enhanced actinorhodin production, and overexpression of
kbpA
reduced the production.
Regulation of
Escherichia coli
RelA Requires Oligomerization of the C-Terminal Domain
Authors: Gropp Michal , Strausz Yael , Gross Miriam , Glaser Gad 
Published: July 2002

ABSTRACT

The
E. coli
RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in
relA
+
cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length
relA
gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.
Characterization of the Promoter of
PRS1
in
Saccharomyces cerevisiae
Identifies Three Regions Potentially Involved in Control of Expression
Authors: Hernando Yolanda , Carter Andrew T. , Sickinger Stefan , Schweizer Michael 
Published: July 2002

ABSTRACT

The transcription initiation site of the
Saccharomyces cerevisiae PRS1
gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted
PRS1
promoter linked to
lacZ
and integrated at the
ura3
locus defined three DNA regions involved in the control of
PRS1
expression. Gel shift analysis confirmed the data.
Functional Analysis of Alkane Hydroxylases from Gram-Negative and Gram-Positive Bacteria
Authors: Smits Theo H. M. , Balada Stefanie B. , Witholt Bernard , van Beilen Jan B. 
Published: July 2002

ABSTRACT

We have cloned homologs of the
Pseudomonas putida
GPo1 alkane hydroxylase from
Pseudomonas aeruginosa
PAO1,
Pseudomonas fluorescens
CHA0,
Alcanivorax borkumensis
AP1,
Mycobacterium tuberculosis
H37Rv, and
Prauserella rugosa
NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the
Pseudomonas
alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts,
Escherichia coli
GEc137 and
P. putida
GPo12, were equipped with pGEc47ΔB, which encodes all proteins necessary for growth on medium-chain-length alkanes (C
6
to C
12
), except a functional alkane hydroxylase. The third host was an
alkB
knockout derivative of
P. fluorescens
CHA0, which is no longer able to grow on C
12
to C
16
alkanes. All alkane hydroxylase homologs, except the
Acinetobacter
sp. ADP1 AlkM, allowed at least one of the three hosts to grow on
n
-alkanes.
Ralstonia paucula
(Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other
Ralstonia
Species (
R. eutropha
,
R. pickettii
,
R. gilardii
, and
R. solanacearum
)
Authors: Moissenet Didier , Bidet Philippe , Garbarg-Chenon Antoine , Arlet Guillaume , Vu-Thien Hoang 
Published: July 2002

ABSTRACT

Ralstonia paucula
(formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate
R. paucula
isolates. Eighteen strains belonging to other
Ralstonia
species (one
R. eutropha
strain, six
R. pickettii
strains, three
R. solanacearum
strains, and eight
R. gilardii
strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of
R. paucula
contains the tRNA
Ile
and tRNA
Ala
genes, which are identical to genes described for
R. pickettii
and
R. solanacearum
.
Determining Confidence Intervals When Measuring Genetic Diversity and the Discriminatory Abilities of Typing Methods for Microorganisms
Authors: Grundmann Hajo , Hori Satoshi , Tanner Gregor 
Published: July 2002

ABSTRACT
We describe here a method for determining confidence intervals for a commonly used index of diversity. This approach facilitates the comparison of the genetic population structure of microorganisms isolated from different environments and improves the objective assessment of the discriminatory power of typing techniques.
Application of the Sherlock Mycobacteria Identification System Using High-Performance Liquid Chromatography in a Clinical Laboratory
Authors: Kellogg James A. , Bankert David A. , Withers Gisela S. , Sweimler William , Kiehn Timothy E. , Pfyffer Gaby E. 
Published: July 2002

ABSTRACT

There is a growing need for a more accurate, rapid, and cost-effective alternative to conventional tests for identification of clinical isolates of
Mycobacterium
species. Therefore, the ability of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.) using computerized software and a Hewlett-Packard series 1100 high-performance liquid chromatograph to identify mycobacteria was compared to identification using phenotypic characteristics, biochemical tests, probes (Gen-Probe, Inc.), gas-liquid chromatography, and, when necessary, PCR-restriction enzyme analysis of the 65-kDa heat shock protein gene and 16S rRNA gene sequencing. Culture, harvesting, saponification, extraction, derivatization, and chromatography were performed following MIDI's instructions. Of 370 isolates and stock cultures tested, 327 (88%) were given species names by the SMIS. SMIS software correctly identified 279 of the isolates (75% of the total number of isolates and 85% of the named isolates). The overall predictive value of accuracy (correct calls divided by total calls of a species) for SMIS species identification was 85%, ranging from only 27% (3 of 11) for
M. asiaticum
to 100% for species or groups including
M. malmoense
(8 of 8),
M. nonchromogenicum
(11 of 11), and the
M. chelonae-abscessus
complex (21 of 21). By determining relative peak height ratios (RPHRs) and relative retention times (RRTs) of selected mycolic acid peaks, as well as phenotypic properties, all 48 SMIS-misidentified isolates and 39 (91%) of the 43 unidentified isolates could be correctly identified. Material and labor costs per isolate were $10.94 for SMIS, $26.58 for probes, and $42.31 for biochemical identification. The SMIS, combined with knowledge of RPHRs, RRTs, and phenotypic characteristics, offers a rapid, reasonably accurate, cost-effective alternative to more traditional methods of mycobacterial species identification.
Nucleic Acid Sequence-Based Amplification of
Aspergillus
RNA in Blood Samples
Authors: Loeffler Juergen , Hebart Holger , Cox Philipp , Flues Nicole , Schumacher Ulrike , Einsele Hermann 
Published: July 2002

ABSTRACT

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of
Aspergillus
RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the
Aspergillus
18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant
Aspergillus
species, including
Aspergillus fumigatus
. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of
Aspergillus
RNA with potential for routine diagnosis, including the possibility to test the viability of cells.
Specificities and Functions of the
recA
and
pps1
Intein Genes of
Mycobacterium tuberculosis
and Application for Diagnosis of Tuberculosis
Authors: Saves Isabelle , Lewis Lee-Ann , Westrelin Fabrice , Warren Robin , Daffé Mamadou , Masson Jean-Michel 
Published: July 2002

ABSTRACT

The worldwide recrudescence of tuberculosis and the widespread appearance of antibiotic resistance have strengthened the need for rapid and specific diagnostic tools. The prevailing microbiological identification of
Mycobacterium tuberculosis
, the causative agent of tuberculosis, which implies the use of in vitro cultures and acid-fast staining microscopy, is time-consuming. Detection of
M. tuberculosis
directly in clinical samples through PCR amplification of mycobacterium-specific genes, designed to shorten diagnostic delay, demonstrated reliability and high sensitivity. However, the quality of the diagnosis depends on the specificity of the target sequence for
M. tuberculosis
complex strains. In the present study, we demonstrated the specificity of
recA
and
pps1
inteins for this complex and thus the feasibility of using intein-coding sequences as a new target for PCR diagnosis. Indeed, the
recA
and
pps1
genes of 36 clinical isolates of
M. tuberculosis
and 10 field strains of
M. bovis
were found to be interrupted by an intein sequence at the RecA-a and Pps1-b sites, respectively, while a large number of nontuberculous mycobacterial species failed to demonstrate these insertions. Besides, the MtuPps1, which was cloned and expressed in
Escherichia coli
, was shown to possess an endonuclease activity. The intein cleaves the 40-bp sequence spanning the intein insertion site Pps1-b in the inteinless
pps1
gene. In addition to the PCR amplification of
recA
and
pps1
intein genes as a tool for diagnosis, the specific endonuclease activity could represent a new molecular approach to identify
M. tuberculosis
.
Real-Time LightCycler PCR for Detection and Discrimination of
Bordetella pertussis
and
Bordetella parapertussis
Authors: Kösters Katrin , Reischl Udo , Schmetz Johanna , Riffelmann Marion , Wirsing von König Carl Heinz 
Published: July 2002

ABSTRACT

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of
Bordetella pertussis
and
Bordetella parapertussis
in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for
B
.
pertussis
and
B
.
parapertussis
, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for
B
.
pertussis
and/or
B
.
parapertussis
. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (
P
= 0.08) to 2.3-fold (simplex PCR) (
P
= 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.
Maternal SDF1 3′A Polymorphism Is Associated with Increased Perinatal Human Immunodeficiency Virus Type 1 Transmission
Authors: John Grace C. , Rousseau Christine , Dong Tao , Rowland-Jones Sarah , Nduati Ruth , Mbori-Ngacha Dorothy , Rostron Tim , Kreiss Joan K. , Richardson Barbra A. , Overbaugh Julie 
Published: July 2002

ABSTRACT
Genetic polymorphisms in chemokine and chemokine receptor genes influence susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression, but little is known regarding the association between these allelic variations and the ability of the host to transmit virus. In this study, we show that the maternal heterozygous SDF1 genotype (SDF1 3′A/wt) is associated with perinatal transmission of HIV-1 (risk ratio [RR], 1.8; 95% confidence interval [CI], 1.0 to 3.3) and particularly postnatal breastmilk transmission (RR, 3.1; 95% CI, 1.1 to 8.6). In contrast, the infant SDF1 genotype had no effect on mother-to-infant transmission. These data suggest that SDF1, which is a ligand for the T-tropic HIV-1 coreceptor CXCR4, may affect the ability of a mother to transmit the virus to her infant. This suggests that a genetic polymorphism in a gene encoding a chemokine receptor ligand may be associated with increased infectivity of the index case and highlights the importance of considering transmission as well as clinical outcome in designing chemokine-based therapies for HIV-1.
Immune Responses and Viral Replication in Long-Term Inapparent Carrier Ponies Inoculated with Equine Infectious Anemia Virus
Authors: Hammond Scott A. , Li Feng , McKeon Brian M. , Cook Sheila J. , Issel Charles J. , Montelaro Ronald C. 
Published: July 2002

ABSTRACT

Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627–9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840–3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10
3
-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.
Internalization of Adenovirus by Alveolar Macrophages Initiates Early Proinflammatory Signaling during Acute Respiratory Tract Infection
Authors: Zsengellér Zsuzsanna , Otake Kazuhisa , Hossain Shaikh-Abu , Berclaz Pierre-Yves , Trapnell Bruce C. 
Published: July 2002

ABSTRACT

Adenovirus is a common respiratory pathogen which causes a broad range of distinct clinical syndromes and has recently received attention for its potential for in vivo gene delivery. Although adenovirus respiratory tract infection (ARTI) results in dose-dependent, local inflammation, the pathogenesis of this remains unclear. We hypothesized that alveolar macrophages (AMφ) rapidly internalize adenovirus following in vivo pulmonary administration and then initiate inflammatory signaling within the lung. To evaluate the role of AMφ in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AMφ and correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF-
α
], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and MIP-1α) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-PCR]) and protein (by enzyme-linked immunosorbent assay) and by identification of cells expressing TNF-α and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labeled with the fluorescent dye (Cy3), was rapidly and widely distributed on epithelial surfaces of airways and alveoli and was very rapidly (∼1 min) localized within AMφ. At 30 min after infection AMφ but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-α and IL-6, thus identifying AMφ as the cell source of initial cytokine signaling. IL-6, TNF-α, MIP-2, and MIP-1α levels progressively increased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h (
P
< 0.05). To begin to define the molecular mechanism(s) by which adenovirus initiates the inflammatory signaling in macrophages, TNF-α expression from adenovirus-infected RAW264.7 macrophages was evaluated in vitro. TNF-α expression was readily detected in adenovirus-infected RAW cell supernatant with kinetics similar to AMφ during in vivo infection. Blockage of virus uptake at specific cellular sites, including internalization (by wortmannin), endosome acidification and/or lysis (by chloroquine) or by Ca
2+
chelation (by BAPTA) completely blocked TNF-α expression. In conclusion, results showed that during ARTI, (i) AMφ rapidly internalized adenovirus, (ii) expression of inflammatory mediators was initiated within AMφ and not airway epithelial or other cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidification. These results have implications for our understanding of the role of the AMφ in the initiation of inflammation following adenovirus infection and adenovirus-mediated gene transfer to the lung.
Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon
Authors: Wang Xiuyan , Li Ming , Zheng Hongyong , Muster Thomas , Palese Peter , Beg Amer A. , Garcı́a-Sastre Adolfo 
Published: July 2002

ABSTRACT
The alpha/beta interferon (IFN-α/β) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-α/β-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-α/β genes, including IFN-β. It is known that transactivation of the IFN-β promoter depends on NF-κB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-κB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-κB pathway during delNS1 virus infection prevented the transactivation of the IFN-β promoter, demonstrating a functional link between NF-κB activation and IFN-α/β synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-κB pathway and of IFN-β synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-κB pathway may thus play a key role in the pathogenesis of influenza A virus.