bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Characterization ofBurkholderia cenocepaciaMutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability
Authors: Farmer Kate L. , Thomas Mark S. 
Published: December 2003

ABSTRACTThe opportunistic pathogenBurkholderia cenocepaciaproduces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenizedB. cenocepaciawith the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. ThecysWgene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.
Osmoregulatory Systems ofEscherichia coli:Identification of Betaine-Carnitine-Choline Transporter Family MemberBetU and Distributions ofbetUandtrkGamongPathogenic and NonpathogenicIsolates
Authors: Ly Anh , Henderson James , Lu Annie , Culham Doreen E. , Wood Janet M. 
Published: December 2003

ABSTRACTMultiple transporters mediate osmoregulatory solute accumulation inEscherichia coliK-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K+(micromolar concentrations) and retained locustrkH, which encodes an osmoregulatory K+transporter. Both lacked thetrkHhomologuetrkG. Transporters ProP and ProU account for all glycine-betaine uptake activity inE. coliK-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. GenebetUwas isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence oftrkGandbetUwithin the ECOR collection, representatives of theE. colipathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. GenetrkGwas present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. GenebetUwas more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). GenestrkGandbetUmay have been acquired by lateral gene transfer, sincetrkGis part of theracprophage andbetUis flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the speciesE. coli, but they are not characteristic of a particular phylogenetic group or pathotype.
The BH1999 Protein of
Bacillus halodurans
C-125 Is Gentisyl-Coenzyme A Thioesterase
Authors: Zhuang Zhihao , Song Feng , Takami Hideto , Dunaway-Mariano Debra 
Published: December 2003

ABSTRACT

In this study, we have shown that recombinant BH1999 from
Bacillus halodurans
catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a
k
cat
/

K
m

of 1.6 × 10
6
M
−1
s
−1
and the hydrolysis of 3-hydroxybenzoyl-CoA with a
k
cat
/

K
m

of 3.0 × 10
5
M
−1
s
−1
. All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.
The Genome-Sequenced Variant of
Campylobacter jejuni
NCTC 11168 and the Original Clonal Clinical Isolate Differ Markedly in Colonization, Gene Expression, and Virulence-Associated Phenotypes
Authors: Gaynor Erin C. , Cawthraw Shaun , Manning Georgina , MacKichan Joanna K. , Falkow Stanley , Newell Diane G. 
Published: December 2003

ABSTRACT

The genome sequence of the enteric bacterial pathogen
Campylobacter jejuni
NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of
C. jejuni
-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods. These observations highlight the capacity of
C. jejuni
to adapt to multiple environmental niches, the likelihood that this adaptation involves genetic evolution, and provides the first whole-genome molecular exploration of the effect of laboratory culture and storage on colonization and virulence properties of this pathogen.
Sequence Analysis of the Mobile Genome Island pKLC102 of
Pseudomonas aeruginosa
C
Authors: Klockgether Jens , Reva Oleg , Larbig Karen , Tümmler Burkhard 
Published: December 2003

ABSTRACT

The
Pseudomonas aeruginosa
plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with
P. aeruginosa
clone K chromosomes at one of the two tRNA
Lys
genes, pKLC102 is incorporated into the tRNA
Lys
gene only close to the
pilA
locus. Targeting of the other tRNA
Lys
copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred
oriV
and genes for replication, partitioning, and conjugation, including a
pil
cluster encoding type IV thin sex pili and an 8,524-bp
chvB
glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase,
att
, and a syntenic set of conserved hypothetical genes also observed in the tRNA
Gly
-associated genome islands of
P. aeruginosa
clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS
6100
elements. Intramolecular transposition of a copy of IS
6100
led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in
P. aeruginosa
clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.
Pseudomonas syringae
Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter
Authors: Schechter Lisa M. , Roberts Kathy A. , Jamir Yashitola , Alfano James R. , Collmer Alan 
Published: December 2003

ABSTRACT

Pseudomonas syringae
pv. tomato strain DC3000 is a pathogen of tomato and
Arabidopsis
. The
hrp
-
hrc
-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most
avr
or
hop
genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of
P. syringae
TTSS effectors into plant cells. This system includes a cloned
P. syringae hrp
gene cluster and the model plant
Nicotiana benthamiana
. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the
P. syringae
Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes
hopPtoS4
::
tnpA
and
hopPtoAG
::
tnpA
. We also confirmed that HopPtoK, HopPtoC, and AvrPphE
Pto
are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in
P. syringae.
Surface Layer Protein EA1 Is Not a Component of
Bacillus anthracis
Spores but Is a Persistent Contaminant in Spore Preparations
Authors: Williams David D. , Turnbough Charles L. 
Published: December 2003

ABSTRACT

EA1 is an abundant, highly antigenic, surface layer protein of
Bacillus anthracis
vegetative cells. Recent studies indicate that EA1 is also a component of
B. anthracis
spores and a potential marker for spore detection. We show here that EA1 is not a spore component but a persistent contaminant in spore preparations.
Expression and Regulation of a Silent Operon,
hyf
, Coding for Hydrogenase 4 Isoenzyme in
Escherichia coli
Authors: Self William T. , Hasona Adnan , Shanmugam K. T. 
Published: December 2003

ABSTRACT

On the basis of
hyf-lacZ
fusion studies, the
hyf
operon of
Escherichia coli
, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain. However, mutant FhlA proteins (constitutive activators of the
hyc
-encoded hydrogenase 3 isoenzyme) activated
hyf-lacZ
. HyfR, an FhlA homolog encoded by the
hyfR
gene present at the end of the
hyf
operon, also activated transcription of
hyf-lacZ
but did so only when
hyfR
was expressed from a heterologous promoter. The HYD4 isoenzyme did not substitute for HYD3 in H
2
production. Optimum expression of
hyf-lacZ
required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator.
Increased G→A Transition Frequencies Displayed by Primer Grip Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Cases-González Clara E. , Menéndez-Arias Luis 
Published: December 2003

ABSTRACT
A genetic screen based on the blue-white β-galactosidase complementation assay designed to detect G→A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.
Impacts of Epitope Expression Kinetics and Class I Downregulation on the Antiviral Activity of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes
Authors: Ali Ayub , Lubong Rachel , Ng Hwee , Brooks David G. , Zack Jerome A. , Yang Otto O. 
Published: December 2003

ABSTRACT

The determinants of CD8
+
cytotoxic T-lymphocyte (CTL) antiviral activity against human immunodeficiency virus type 1 (HIV-1) remain poorly defined. Although recent technological advances have markedly enhanced the ability to detect HIV-1-specific T cells, commonly used assays do not reveal their direct interaction with virus. We investigated two determinants of CTL antiviral efficiency by manipulating HIV-1 and measuring the effects on CTL suppression of viral replication in acutely infected cells. Translocation of a Gag epitope into the early protein Nef markedly increased the activity of CTL recognizing that epitope, in comparison to HIV-1 expressing the epitope normally in the late protein Gag. Because this epitope translocation resulted not only in earlier expression but also in loss of major histocompatibility complex class I downregulation by Nef, the activities of CTL against a panel of viral constructs differing in kinetics of epitope expression and class I downmodulation were compared. The results indicated that both the timing of epitope expression and the reduction of class I have profound effects on the ability of CTL to suppress HIV-1 replication in acutely infected cells. The epitope targeting of CTL and viral control of class I therefore likely play important roles in the ability of CTL to exert pressure on HIV-1.
Identification of an Extracellular Domain within the Human PiT2 Receptor That Is Required for Amphotropic Murine Leukemia Virus Binding
Authors: Feldman Steven A , Farrell Karen B. , Murthy Ravi K. , Russ Jill L. , Eiden Maribeth V. 
Published: December 2003

ABSTRACT
Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.
Competitive Fitness of Nevirapine-Resistant Human Immunodeficiency Virus Type 1 Mutants
Authors: Collins Jennifer A. , Thompson M. Gregory , Paintsil Elijah , Ricketts Melisa , Gedzior Joanna , Alexander Louis 
Published: December 2003

ABSTRACT
Determining the fitness of drug-resistant human immunodeficiency virus type 1 (HIV-1) strains is necessary for the development of population-based studies of resistance patterns. For this purpose, we have developed a reproducible, systematic assay to determine the competitive fitness of HIV-1 drug-resistant mutants. To demonstrate the applicability of this assay, we tested the fitness of the five most common nevirapine-resistant mutants (103N, 106A, 181C, 188C, and 190A), with mutations in HIV-1 reverse transcriptase (RT), singly and in combination (for a total of 31 variants) in a defined HIV-1 background. For these experiments, the 27 RT variants that produced viable virus were cocultured with wild-type virus without nevirapine. The ratios of the viral species were determined over time by utilization of a quantitative real-time RT-PCR-based assay. These experiments revealed that all of the viable variants were less fit than the wild type and demonstrated that the order of relative fitness of the single mutants tested was as follows: 103N > 181C > 190A > 188C > 106A. This order correlated with the commonality of these mutants as a result of nevirapine monotherapy. These investigations also revealed that, on average, the double mutants were less fit than the single mutants and the triple mutants were less fit than the double mutants. However, the fitness of the single and double mutants was often not predictive of the fitness of the derivative triple mutants, suggesting the presence of complex interactions between the closely aligned residues that confer nevirapine resistance. This complexity was also evident from the observation that all three of the replication-competent quadruple mutants were fitter than most of the triple mutants, and in some cases, even the double mutants. Our data suggest that, in many cases, viral fitness is the determining factor in the evolution of nevirapine-resistant mutants in vivo, that interactions between the residues that confer nevirapine resistance are complex, and that these interactions substantially affect reverse transcriptase structure and/or function.
Genetic and Biochemical Analysis of
cis
Regulatory Elements within the Keratinocyte Enhancer Region of the Human Papillomavirus Type 31 Upstream Regulatory Region during Different Stages of the Viral Life Cycle
Authors: Sen Ellora , Alam Samina , Meyers Craig 
Published: December 2003

ABSTRACT

Using linker scanning mutational analysis, we recently identified potential
cis
regulatory elements contained within the 5′ upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the viral life cycle. For the present study, we extended the linker scanning mutational analysis to identify potential
cis
elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the viral life cycle. The linker scanning mutational analysis identified viral
cis
elements located in the KE region that regulate transcription in the presence and absence of any viral gene products or viral DNA replication and determine the role of host tissue differentiation on viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same
cis
regulatory elements at different stages of the HPV differentiation-dependent life cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify
cis
regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the viral life cycle. The variable recruitment of transcription factors to the same
cis
element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.
cis
-Acting Sequences That Contribute to the Synthesis of Relaxed-Circular DNA of Human Hepatitis B Virus
Authors: Liu Ning , Ji Lin , Maguire Megan L. , Loeb Daniel D. 
Published: December 2003

ABSTRACT

Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of
cis
-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about
cis
-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for
cis
-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5′r, and 3′r were not analyzed in the study. Results from Southern blotting showed that multiple
cis
-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of
cis
-acting sequences that contribute to the synthesis of HBV RC DNA.