bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of
Escherichia coli
RecA Interactions with LexA, λ CI, and UmuD by Site-Directed Mutagenesis of
recA
Authors: Mustard Julie A. , Little John W. 
Published: July 2002

ABSTRACT

An early event in the induction of the SOS system of
Escherichia coli
is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage λ CI repressor and
E. coli
UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, λ CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.
Two Types of
Bacillus subtilis tetA
(L) Deletion Strains Reveal the Physiological Importance of TetA(L) in K
+
Acquisition as well as in Na
+
, Alkali, and Tetracycline Resistance
Authors: Wang Wei , Guffanti Arthur A. , Wei Yi , Ito Masahiro , Krulwich Terry A. 
Published: July 2002

ABSTRACT

The chromosomally encoded TetA(L) protein of
Bacillus subtilis
is a multifunctional tetracycline-metal/H
+
antiporter that also exhibits monovalent cation/H
+
antiport activity and a net K
+
uptake mode. In this study,
B. subtilis
mutant strains JC112 and JC112C were found to be representative of two phenotypic types of
tetA
(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K
+
, indicating that the net K
+
uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na
+
- or K
+
-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co
+2
as well as Na
+
sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of
czcD-trkA
, a locus with a proposed involvement in K
+
uptake, occurred in both mutants. The increase was highest in the presence of Co
2+
and was higher in JC112 than in JC112C. Deletion of
czcD-trkA
resulted in diminished growth of the wild-type and both mutant strains at low [K
+
], supporting a significant role for this locus in K
+
uptake. Expression of
yheL
, which is a homologue of the Na
+
/H
+
antiporter-encoding
nhaC
gene from
Bacillus firmus
OF4, was also increased in both
tetA
(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of
yheL
were consistent with a modest role for YheL in Na
+
-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na
+
, and alkali resistance and K
+
acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.
Adenylate Kinase as a Virulence Factor ofPseudomonas aeruginosa
Authors: Markaryan Adam , Zaborina Olga , Punj Vasu , Chakrabarty A. M. 
Published: July 2002

ABSTRACTAdenylate kinase (AK; ATP:AMP phosphotransferase, EC2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg · ATP + AMP ↔ Mg · ADP + ADP. In this study we show that AK secreted by the pathogenic strains ofPseudomonas aeruginosaappears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain ofP. aeruginosa8821 and hyperproduced it as a fusion protein with glutathioneS-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection withP. aeruginosa.Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allowsP. aeruginosato exert its full virulence.
Gene Replacement Analysis of the Butyrolactone Autoregulator Receptor (FarA) Reveals that FarA Acts as a Novel Regulator in Secondary Metabolism ofStreptomyces lavendulaeFRI-5
Authors: Kitani Shigeru , Yamada Yasuhiro , Nihira Takuya 
Published: July 2002

ABSTRACTIM-2 [(2R,3R,1′R)-2-1′-hydroxybutyl-3-hydroxymethyl γ-butanolide] is a γ-butyrolactone autoregulator which, inStreptomyces lavendulaeFRI-5, switches off the production ofd-cycloserine but switches on the production of a blue pigment and several nucleoside antibiotics. To clarify the in vivo function of an IM-2-specific receptor (FarA) in the IM-2 signaling cascade ofS. lavendulaeFRI-5, afarAdeletion mutant was constructed by means of homologous recombination. On several solid media, no significant difference in morphology was observed between the wild-type strain and thefarAmutant (strain K104), which demonstrated that the IM-2–FarA system does not participate in the morphological control ofS. lavendulaeFRI-5. In liquid media, thefarAmutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the wild-type strain, suggesting that the FarA protein acts primarily as a negative regulator on the biosynthesis of these compounds in the absence of IM-2. However, contrary to the IM-2-dependent suppression ofd-cycloserine production in the wild-type strain, overproduction ofd-cycloserine was observed in thefarAmutant, indicating for the first time that the presence of both IM-2 and intact FarA are necessary for the suppression ofd-cycloserine biosynthesis.
Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of
Saccharomyces cerevisiae
Authors: Distler MacKenzie , Kulkarni Ajit , Rai Rajendra , Cooper Terrance G. 
Published: July 2002

ABSTRACT

Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in
Saccharomyces cerevisiae
. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the
S. cerevisiae
genome, raising the possibility that Dal80p might be able to “stain” DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4′,6′-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.
Regulation of Transcription of the
Bacillus subtilis pyrG
Gene, Encoding Cytidine Triphosphate Synthetase
Authors: Meng Qi , Switzer Robert L. 
Published: July 2002

ABSTRACT

The
B. subtilis pyrG
gene, which encodes CTP synthetase, is located far from the pyrimidine biosynthetic operon on the chromosome and is independently regulated. The
pyrG
promoter and 5′ leader were fused to
lacZ
and integrated into the chromosomes of several
B. subtilis
strains having mutations in genes of pyrimidine biosynthesis and salvage. These mutations allowed the intracellular pools of cytidine and uridine nucleotides to be manipulated by the composition of the growth medium. These experiments indicated that
pyrG
expression is repressed by cytidine nucleotides but is largely independent of uridine nucleotides. The start of
pyrG
transcription was mapped by primer extension to a position 178 nucleotides upstream of the translation initiation codon. A factor-independent termination hairpin lying between the
pyrG
promoter and its coding region is essential for regulation of
pyrG
expression. Primer-extended transcripts were equally abundant in repressed and derepressed cells when the primer bound upstream of the terminator, but they were much less abundant in repressed cells when the primer bound downstream of the terminator. Furthermore, deletion of the terminator from
pyrG-lacZ
fusions integrated into the chromosome yielded elevated levels of expression that was not repressible by cytidine. We suggest that cytidine repression of
pyrG
expression is mediated by an antitermination mechanism in which antitermination by a putative
trans
-acting protein is reduced by elevated levels of cytidine nucleotides. Conservation of sequences and secondary structural elements in the
pyrG
5′ leaders of several other gram-positive bacteria indicates that their
pyrG
genes are regulated by a similar mechanism.
Role of
Agrobacterium
VirB11 ATPase in T-Pilus Assembly and Substrate Selection
Authors: Sagulenko Evgeniy , Sagulenko Vitaliya , Chen Jun , Christie Peter J. 
Published: July 2002

ABSTRACT

The VirB11 ATPase is a subunit of the
Agrobacterium tumefaciens
transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of
virB11
mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing
virB11
dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of
virB11
mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i)
virB11/virB11
∗ merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.
The PPP-Family Protein Phosphatases PrpA and PrpB of
Salmonella enterica
Serovar Typhimurium Possess Distinct Biochemical Properties
Authors: Shi Liang , Kehres David G. , Maguire Michael E. 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Typhimurium requires Mn
2+
, but only a few Mn
2+
-dependent enzymes have been identified from bacteria. To characterize Mn
2+
-dependent enzymes from serovar Typhimurium, two putative PPP-family protein phosphatase genes were cloned from serovar Typhimurium and named
prpA
and
prpB
. Their DNA-derived amino acid sequences showed 61% identity to the corresponding
Escherichia coli
proteins and 41% identity to each other. Each phosphatase was expressed in
E. coli
and purified to near electrophoretic homogeneity. Both PrpA and PrpB absolutely required a divalent metal for activity. As with other phosphatases of this class, Mn
2+
had the highest affinity and stimulated the greatest activity. The apparent

K
a

of PrpA for Mn
2+
of 65 μM was comparable to that for other bacterial phosphatases, but PrpB had a much higher affinity for Mn
2+
(1.3 μM). The pH optima were pH 6.5 for PrpA and pH 8 for PrpB, while the optimal temperatures were 45 to 55°C for PrpA and 30 to 37°C for PrpB. Each phosphatase could hydrolyze phosphorylated serine, threonine, or tyrosine residues, but their relative specific activities varied with the specific substrate tested. These differences suggest that each phosphatase is used by serovar Typhimurium under different growth or environmental conditions such as temperature or acidity.
Molecular and Phenotypic Analysis ofCaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter
Authors: Nishikawa Akiko , Poster Jay B. , Jigami Yoshifumi , Dean Neta 
Published: July 2002

ABSTRACTCell surface mannan is implicated in almost every aspect of pathogenicity ofCandida albicans. InSaccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation inC. albicans,we isolated theCaVRG4gene and analyzed the effects of loss of its function.CaVRG4encodes a functional homologue of theS. cerevisiaeGDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm ofC. albicansin a pattern reminiscent of localization of Vrg4p in the Golgi apparatus inS. cerevisiae. Like partial loss ofScVRG4function, partial loss ofCaVRG4function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy ofCaVRG4under control of the methionine-repressibleMET3promoter, did not grow in the presence of methionine and cysteine, demonstrating thatCaVRG4is essential for viability. MutantCandida vrg4strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because theVRG4gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.
Multistate Outbreak of
Salmonella
Serovar Muenchen Infections Associated with Alfalfa Sprouts Grown from Seeds Pretreated with Calcium Hypochlorite
Authors: Proctor Mary E. , Hamacher Marge , Tortorello Mary Lou , Archer John R. , Davis Jeffrey P. 
Published: July 2002

ABSTRACT

During September 1999, a multistate outbreak of
Salmonella
serovar Muenchen infection associated with eating raw alfalfa sprouts was identified in Wisconsin. Despite use of a calcium hypochlorite sanitizing procedure to pretreat seeds before sprouting, at least 157 outbreak-related illnesses were identified in seven states having sprouters who received alfalfa seed from a specific lot. The continued occurrence of sprout-related outbreaks despite presprouting disinfection supports the concern that no available treatment will eliminate pathogens from seeds before sprouting and reinforces the need for additional safeguards to protect the public. A lack of consumer knowledge regarding exposure to sprouts documented in this investigation suggests that more-targeted outreach to high-risk individuals may be needed to reduce their risk.
Performance of Eight Methods, Including Two New Rapid Methods, for Detection of Oxacillin Resistance in a Challenge Set of
Staphylococcus aureu
s Organisms
Authors: Swenson Jana M. , Williams Portia P. , Killgore George , O'Hara Caroline Mohr , Tenover Fred C. 
Published: July 2002

ABSTRACT

Using a set of 55
Staphylococcus aureus
challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of
S. aureus
to oxacillin. The methods were evaluated by using the presence of the
mecA
gene, as detected by PCR, as the “gold standard.” The strains included 19
mecA
-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 μg/ml) and 36
mecA
-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 μg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the
mecA-
negative strains increased to 4 to 8 μg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.
Clustering of South African
Helicobacter pylori
Isolates from Peptic Ulcer Disease Patients Is Demonstrated by Repetitive Extragenic Palindromic-PCR Fingerprinting
Authors: Kidd M. , Atherton J. C. , Lastovica A. J. , Louw J. A. 
Published: July 2002

ABSTRACT

The present report assesses the association between clonal groupings, disease, and the virulence fingerprint of 76 South African

Helicobacter pylori cagA
+

strains isolated from 57 Cape-colored subjects. Two methods, repetitive extragenic palindromic (REP)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to generate DNA fingerprints, and computer-assisted analysis was used to derive clusters. The two PCR techniques were only partially complementary (48%). REP-PCR fingerprints identified a distinct pathological cluster consisting of strains from 63% of the patients and was strongly associated with both disease (
P
< 0.00001) and the vacuolating cytotoxin A (
vacA
) signal sequence type (
P
< 0.003). RAPD-PCR fingerprinting was not associated with disease and was less strongly associated with
vacA
(
P
< 0.05) than REP-PCR was. Hierarchical analysis indicated that isolates from patients with peptic ulcer disease tended to cluster differently than isolates from patients with gastritis alone or gastric adenocarcinoma. These relationships are consistent with a loosely clonal population structure associated with disease for
H. pylori
in the Cape-colored population in South Africa.
Possible Approach for Serodiagnosis of Ascariasis by Evaluation of Immunoglobulin G4 Response UsingAscaris lumbricoidesSomatic Antigen
Authors: Bhattacharyya Tanusree , Santra Amal , Majumder Debendra N. Guha , Chatterjee Bishnu P. 
Published: July 2002

ABSTRACTSomatic antigen ofAscaris lumbricoideswas purified to homogeneity (molecular mass, 34 kDa) by ammonium sulfate fractionation and successive chromatographic procedures, namely, gel permeation, ion exchange, and high-performance gel permeation liquid chromatographies. The antigen showed strong binding with immunoglobulin G (IgG) inAscaris-infested patients and was cross-reactive with IgE and IgG in patients infested with other nematodes. It reacted specifically with IgG4 (P< 0.001) in 63Ascaris-infested patients, which represented 65% of the total IgG response, though cross-reactivity with IgG1, IgG2, and IgG3 subclasses was observed, indicating the unique specificity of this test system and its potential utility in the serodiagnosis of ascariasis.
Disseminated Toxoplasmosis, Resulting from Infection of Allograft, after Orthotopic Liver Transplantation: Usefulness of Quantitative PCR
Authors: Botterel F. , Ichai P. , Feray C. , Bouree P. , Saliba F. , Tur Raspa R. , Samuel D. , Romand S. 
Published: July 2002

ABSTRACT

Disseminated toxoplasmosis is a life-threatening disease in liver transplant recipients that can result from an organ-transmitted infection. We report here a case of fatal disseminated toxoplasmosis after orthotopic liver transplantation from a seropositive donor (immunoglobulin G [IgG]
+
and IgM
−
) in a patient who was nonimmune for toxoplasmosis prior to transplantation. Quantitative PCR analyses of various clinical specimens, including serum samples, appeared retrospectively to be a valuable diagnostic tool that might guide therapeutic attitudes.