bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcription Profiling-Based Identification of
Staphylococcus aureus
Genes Regulated by the
agr
and/or
sarA
Loci
Authors: Dunman P. M. , Murphy E. , Haney S. , Palacios D. , Tucker-Kellogg G. , Wu S. , Brown E. L. , Zagursky R. J. , Shlaes D. , Projan S. J. 
Published: July 2002

ABSTRACT

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the
Staphylococcus aureus
genome, was used to identify open reading frames that are regulated by
agr
and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and
agr
,
sarA
, and
agr sarA
mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an
agr
- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including
hla
(alpha-toxin) and
spa
(protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by
agr
and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the
agr
and the
sarA
locus are more-global transcription regulators than previously realized.
Essential PchG-Dependent Reduction in Pyochelin Biosynthesis of
Pseudomonas aeruginosa
Authors: Reimmann Cornelia , Patel Hiten M. , Serino Laura , Barone Mario , Walsh Christopher T. , Haas Dieter 
Published: July 2002

ABSTRACT

The biosynthetic genes
pchDCBA
and
pchEF
, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the
pchR
regulatory gene on the chromosome of
Pseudomonas aeruginosa
. The 4.6-kb region located downstream of the
pchEF
genes was found to contain three additional, contiguous genes,
pchG
,
pchH
, and
pchI
, probably forming a
pchEFGHI
operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the
Yersinia pestis
and
Y. enterocolitica
proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in
pchG
abolished pyochelin formation, whereas mutations in
pchH
and
pchI
did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a
P. aeruginosa
mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed
pchDCBA
and
pchEFG
genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host
Escherichia coli
expressing
pchDCBA
and
pchEFG
together with the
E. coli entD
gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate,
l
-cysteine, ATP, NADPH, and
S
-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in
P. aeruginosa
.
Identification of a Novel Two-Component Regulatory System That Acts in Global Regulation of Virulence Factors ofStaphylococcus aureus
Authors: Yarwood Jeremy M. , McCormick John K. , Schlievert Patrick M. 
Published: July 2002

ABSTRACTWe have previously demonstrated that the presence of oxygen is necessary for the production of toxic shock syndrome toxin 1 (TSST-1) byStaphylococcus aureusin vitro. To investigate the mechanism by which oxygen might regulate toxin production, we identified homologs inS. aureusof theBacillus subtilis resDEgenes. The two-component regulatory system encoded byresDE, ResD-ResE, has been implicated in the global regulation of aerobic and anaerobic respiratory metabolism inB. subtilis. We have designated theS. aureushomologssrrAB(staphylococcal respiratory response). The effects ofsrrABexpression on expression of RNAIII (the effector molecule of theagrlocus) and on production of TSST-1 (an exotoxin) and protein A (a surface-associated virulence factor) were investigated. Expression of RNAIII was inversely related to expression ofsrrAB. Disruption ofsrrBresulted in increased levels of RNAIII, while expression ofsrrABintranson a multicopy plasmid resulted in repression of RNAIII transcription, particularly in microaerobic conditions. Disruption ofsrrBresulted in decreased production of TSST-1 under microaerobic conditions and, to a lesser extent, under aerobic conditions as well. Overexpression ofsrrABresulted in nearly complete repression of TSST-1 production in both microaerobic and aerobic conditions. Protein A production by thesrrBmutant was upregulated in microaerobic conditions and decreased in aerobic conditions. Protein A production was restored to nearly wild-type levels by complementation ofsrrABinto the null mutant. These results indicate that the putative two-component system encoded bysrrAB, SrrA-SrrB, acts in the global regulation of staphylococcal virulence factors, and may repress virulence factors under low-oxygen conditions. Furthermore,srrABmay provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of virulence factors inS. aureus.
Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745
Authors: Galli Dominique M. , Chen Jinbiao , Novak Karen F. , Leblanc Donald J. 
Published: July 2002

ABSTRACT

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from
Actinobacillus actinomycetemcomitans
, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different
A. actinomycetemcomitans
strains. However, transfer of pVT745 derivatives from
A. actinomycetemcomitans
to
Escherichia coli
JM109 by conjugation was successful only when an
E. coli
origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (
Haemophilus
-
Actinobacillus
-
Pasteurella
) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous
A. actinomycetemcomitans
strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.
N
-Acyl-
l
-Homoserine Lactone-Mediated Regulation of the Lip Secretion System in
Serratia liquefaciens
MG1
Authors: Riedel Kathrin , Ohnesorg Thomas , Krogfelt Karen A. , Hansen Thomas S. , Omori Kenji , Givskov Michael , Eberl Leo 
Published: July 2002

ABSTRACT

The analysis of
Serratia liquefaciens
MG1 ′
luxAB
insertion mutants that are responsive to
N
-butanoyl-
l
-homoserine lactone revealed that expression of
lipB
is controlled by the
swr
quorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.
Dissimilatory Fe(III) and Mn(IV) Reduction by
Shewanella putrefaciens
Requires
ferE
, a Homolog of the
pulE
(
gspE
) Type II Protein Secretion Gene
Authors: DiChristina Thomas J. , Moore Charles M. , Haller Carolyn A. 
Published: July 2002

ABSTRACT

Shewanella putrefaciens
strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb
S. putrefaciens
wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468–1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (
ferE
) whose translated product displayed 87% sequence similarity to
Aeromonas hydrophila
ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type
ferE
with an insertionally inactivated
ferE
construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking
ferE
revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (
pulC
to -
N
homologs) was found in the unannotated genome sequence of
Shewanella oneidensis
(formerly
S. putrefaciens
) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer
+
) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer
−
) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of
S. putrefaciens
proteins involved in anaerobic respiration on Fe(III) and Mn(IV).
Intramolecular Regulation of the Opposing (p)ppGpp Catalytic Activities of Rel

Seq

, the Rel/Spo Enzyme from
Streptococcus equisimilis
Authors: Mechold Undine , Murphy Helen , Brown Larissa , Cashel Michael 
Published: July 2002

ABSTRACT

Catalytic and regulatory domains of the Rel/Spo homolog of
Streptococcus equisimilis
affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel

Seq

protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3′-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn
2+
-dependent 3′-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel

Seq

to mediate (p)ppGpp accumulation during amino acid starvation in
S. equisimilis
is absent when the protein is expressed ectopically in
Escherichia coli.
Fusing the N-terminal half of Rel

Seq

with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in
E. coli
by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.
Plasmid-Encoded
asp
Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction
Authors: Abe Keietsu , Ohnishi Fumito , Yagi Kyoko , Nakajima Tasuku , Higuchi Takeshi , Sano Motoaki , Machida Masayuki , Sarker Rafiquel I. , Maloney Peter C. 
Published: July 2002

ABSTRACT

Tetragenococcus halophila
D10 catalyzes the decarboxylation of
l
-aspartate with nearly stoichiometric release of
l
-alanine and CO
2
. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative
asp
operon consisting of two genes, which we designated
aspD
and
aspT
, encoding an
l
-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the
asp
operon in pD1 were in the following order: promoter →
aspD
→
aspT
. The deduced amino acid sequence of AspD showed similarity to the sequences of two known
l
-aspartate-β-decarboxylases from
Pseudomonas dacunhae
and
Alcaligenes faecalis
. Hydropathy analyses suggested that the
aspT
gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the
Escherichia coli
expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the
asp
operon in
E. coli
coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in
E. coli
and purified from cell extracts. The purified AspDHis clearly exhibited activity of
l
-aspartate-β-decarboxylase. Recombinant AspT was solubilized from
E. coli
membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the
asp
operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.
Agilent 2100 Bioanalyzer for Restriction Fragment Length Polymorphism Analysis of the
Campylobacter jejuni
Flagellin Gene
Authors: Nachamkin Irving , Panaro Nicholas J. , Li Ming , Ung Huong , Yuen Po Ki , Kricka Larry J. , Wilding Peter 
Published: July 2002

ABSTRACT

The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing
Campylobacter jejuni
. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.
Chlamydial Serology: Comparative Diagnostic Value of Immunoblotting, Microimmunofluorescence Test, and Immunoassays Using Different Recombinant Proteins as Antigens
Authors: Bas S. , Muzzin P. , Ninet B. , Bornand J. E. , Scieux C. , Vischer T. L. 
Published: July 2002

ABSTRACT

To improve the reliability of the serodiagnosis of
Chlamydia trachomatis
infections, an immunoblot analysis, a microimmunofluorescence titration, and different immunoassays using synthetic peptides derived from species-specific epitopes in variable domain IV of the major outer membrane protein or recombinant antigens (heat shock protein 70 [hsp70], hsp60, hsp10, polypeptide encoded by open reading frame 3 of the plasmid [pgp3], macrophage infectivity potentiator, and a fragment of the total lipopolysaccharide) were evaluated. Because cross-reactions between chlamydial species have been reported, the microimmunofluorescence tests were also performed with
Chlamydia pneumoniae
and
Chlamydia psittaci
used as antigens, and
C. pneumoniae
-specific antibodies were also determined by immunoassays. Since the presence of antimicrobial antibodies must be interpreted in light of their prevalence in the general population, responses obtained with serum samples from patients with well-defined infection (i.e., with positive urethral or endocervical
C. trachomatis
DNA amplification) were compared to those obtained with samples from healthy blood donors. The best sensitivity (86%) with a specificity of 81% was obtained for immunoblotting results, when the number of individuals with ≥10 immunoglobulin G (IgG) and/or ≥2 IgM responses to the different
C. trachomatis
antigens was considered. A 13-kDa antigen was recognized by most of the samples (86% for IgG) from patients with acute urogenital infection but rarely (3%) by those from healthy blood donors (
P
< 0.0001). The sensitivity and specificity results obtained for serum antibodies to peptides or recombinant antigens were slightly lower than those results obtained for the number of responses to whole
C. trachomatis
antigens, which were 76 and 77%, respectively, when IgG responses to both recombinant hsp60 and pgp3 were considered.
Human Babesiosis in Japan: Isolation of
Babesia microti
-Like Parasites from an Asymptomatic Transfusion Donor and from a Rodent from an Area Where Babesiosis Is Endemic
Authors: Wei Qiang , Tsuji Masayoshi , Zamoto Aya , Kohsaki Masatoshi , Matsui Toshimitsu , Shiota Tsunezo , Telford Sam R. , Ishihara Chiaki 
Published: July 2002

ABSTRACT

To determine the source of infection for the Japanese index case of human babesiosis, we analyzed blood samples from an asymptomatic individual whose blood had been transfused into the patient. In addition, we surveyed rodents collected from near the donor's residence. Examination by microscopy and PCR failed to detect the parasite in the donor's blood obtained 8 months after the donation of the blood that was transfused. However, we were able to isolate
Babesia
parasites by inoculating the blood sample into SCID mice whose circulating red blood cells (RBCs) had been replaced with human RBCs. A
Babesia
parasite capable of propagating in human RBCs was also isolated from a field mouse (
Apodemus speciosus
) captured near the donor's residential area. Follow-up surveys over a 1-year period revealed that the donor continued to be asymptomatic but had consistently high immunoglobulin G (IgG) titers in serum and low levels of parasitemia which were microscopically undetectable yet which were repeatedly demonstrable by inoculation into animals. The index case patient's sera contained high titers of IgM and, subsequently, rising titers of IgG antibodies, both of which gradually diminished with the disappearance of the parasitemia. Analysis of the parasite's rRNA gene (rDNA) sequence and immunodominant antigens revealed the similarity between donor and patient isolates. The rodent isolate also had an rDNA sequence that was identical to that of the human isolates but that differed slightly from that of the human isolates by Western blot analysis. We conclude that the index case patient acquired infection by transfusion from a donor who became infected in Japan, that parasitemia in an asymptomatic carrier can persist for more than a year, and that
A. speciosus
serves as a reservoir of an agent of human babesiosis in Japan.
Identification of
Mycobacterium
spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries
Authors: Cloud J. L. , Neal H. , Rosenberry R. , Turenne C. Y. , Jama M. , Hillyard D. R. , Carroll K. C. 
Published: July 2002

ABSTRACT

Current methods for identification of
Mycobacterium
spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify
Mycobacterium
isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of
Mycobacterium
spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.
High Variability between Results of Different In-House Tests for Cytomegalovirus (CMV) Monitoring and a Standardized Quantitative Plasma CMV PCR Assay
Authors: von Müller Lutz , Hampl Walter , Hinz Joachim , Meisel Helga , Reip Angela , Engelmann Elisabeth , Heilbronn Regine , Gärtner Barbara , Krämer Oliver , Einsele Hermann , Hebart Holger , Ljubicic Tatjana , Löffler Jürgen , Mertens Thomas 
Published: July 2002

ABSTRACT
A total of 2,718 blood samples were analyzed in five virological laboratories for the presence of cytomegalovirus (CMV) by in-house tests and one standardized plasma PCR assay. Results from in-house tests showed remarkable variability. Detection of CMV pp65 antigen or DNA from cells was more sensitive than that by plasma CMV PCR assay.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50/Rta Protein Activates the Entire Viral Lytic Cycle in the HH-B2 Primary Effusion Lymphoma Cell Line
Authors: Gradoville Lyndle , Gerlach Jennifer , Grogan Elizabeth , Shedd Duane , Nikiforow Sarah , Metroka Craig , Miller George 
Published: July 2002

ABSTRACT
Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.