bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stringent Response Activates Quorum Sensing and Modulates Cell Density-Dependent Gene Expression in
Pseudomonas aeruginosa
Authors: van Delden Christian , Comte Rachel , Bally And Marc 
Published: July 2002

ABSTRACT

During nutrient starvation,
Escherichia coli
elicits a stringent response involving the ribosome-associated protein RelA. Activation of RelA results in a global change in the cellular metabolism including enhanced expression of the stationary-phase sigma factor RpoS. In the human pathogen
Pseudomonas aeruginosa
, a complex quorum-sensing circuitry, linked to RpoS expression, is required for cell density-dependent production of many secreted virulence factors, including LasB elastase. Quorum sensing relies on the activation of specific transcriptional regulators (LasR and RhlR) by their corresponding autoinducers (3-oxo-C
12
-homoserine lactone [HSL] and C
4
-HSL), which function as intercellular signals. We found that overexpression of
relA
activated the expression of
rpoS
in
P. aeruginosa
and led to premature, cell density-independent LasB elastase production. We therefore investigated the effects of the stringent response on quorum sensing. Both
lasR
and
rhlR
gene expression and autoinducer synthesis were prematurely activated during the stringent response induced by overexpression of
relA
. Premature expression of
lasR
and
rhlR
was also observed when
relA
was overexpressed in a PAO1
rpoS
mutant. The stringent response induced by the amino acid analogue serine hydroxamate (SHX) also led to premature production of the 3-oxo-C
12
-HSL autoinducer. This response to SHX was absent in a PAO1
relA
mutant. These findings suggest that the stringent response can activate the two quorum-sensing systems of
P. aeruginosa
independently of cell density.
Infrequent Genetic Exchange and Recombination in the Mitochondrial Genome of
Candida albicans
Authors: Anderson James B. , Wickens Claire , Khan Mustafa , Cowen Leah E. , Federspiel Nancy , Jones Ted , Kohn Linda M. 
Published: July 2002

ABSTRACT

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast
Candida albicans
. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of
C. albicans
from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of
C. albicans
, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.
Conserved Gene Cluster at Replication Origins of the α-Proteobacteria
Caulobacter crescentus
and
Rickettsia prowazekii
Authors: Brassinga Ann Karen C. , Siam Rania , Marczynski Gregory T. 
Published: July 2002

ABSTRACT

A 30-kb region surrounding the replication origin in
Caulobacter crescentus
was analyzed. Comparison to the genome sequence of another α-proteobacterium,
Rickettsia prowazekii
, revealed a conserved cluster of genes (RP001,
hemE, hemH
, and RP883) that overlaps the established origin of replication in
C. crescentus
and the putative origin of replication in
R. prowazekii
. The genes flanking this cluster differ between these two organisms. We therefore propose that this conserved gene cluster can be used to identify the origin of replication in other α-proteobacteria.
Gene Expression in
Pseudomonas aeruginosa
: Evidence of Iron Override Effects on Quorum Sensing and Biofilm-Specific Gene Regulation
Authors: Bollinger Nikki , Hassett Daniel J. , Iglewski Barbara H. , Costerton J. William , McDermott Timothy R. 
Published: July 2002

ABSTRACT

Prior studies established that the
Pseudomonas aeruginosa
oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study,
sodA
(encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a
lasI
or
lasR
mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that
sodA
transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of
sodA
was greatest in the wild-type strain and least in the
lasR
mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased
lasI
::
lacZ
transcription in response to iron limitation, whereas the expression level in the
las
mutants was minimal and iron starvation induction of
lasI
::
lacZ
did not occur. Studies comparing Mn-SOD activity in
P. aeruginosa
biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the
lasI
and
lasR
mutant biofilms but could be suppressed if the growth medium was amended with 25 μM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.
Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic
Escherichia coli
Strain
Authors: Wagner Patrick L. , Neely Melody N. , Zhang Xiaoping , Acheson David W. K. , Waldor Matthew K. , Friedman David I. 
Published: July 2002

ABSTRACT

Shiga toxins (Stxs), encoded by the
stxA
and
stxB
genes, are important contributors to the virulence of
Escherichia coli
O157:H7 and other Stx-producing
E. coli
(STEC) strains. The
stxA
and
stxB
genes in STEC strains are located on the genomes of resident prophages of the λ family immediately downstream of the phage late promoters (
p
R′
). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate
p
R′
promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of
p
R′
as well as terminator kilobases distal to
p
R′
. To test if this Q-directed processive transcription plays a role in
stx
2
AB
expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which
p
R′
and part of Q were deleted but which has an intact
p
Stx, the previously described
stx
2
AB
-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the
p
R′
promoter. Since transcription initiating at
p
R′
ultimately requires activation of the phage lytic cascade, expression of
stx
2
AB
in STEC depends primarily on prophage induction. By showing this central role for the prophage in
stx
2
AB
expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.
glnD
and
mviN
Are Genes of an Essential Operon in
Sinorhizobium meliloti
Authors: Rudnick Paul A. , Arcondéguy Tania , Kennedy Christina K. , Kahn Daniel 
Published: July 2002

ABSTRACT

To evaluate the role of uridylyl-transferase, the
Sinorhizobium meliloti glnD
gene was isolated by heterologous complementation in
Azotobacter vinelandii
. The
glnD
gene is cotranscribed with a gene homologous to
Salmonella mviN
.
glnD1
::Ω or
mviN1
::Ω mutants could not be isolated by a powerful sucrose counterselection procedure unless a complementing cosmid was provided, indicating that
glnD
and
mviN
are members of an indispensable operon in
S. meliloti
.
PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other
Erwinia chrysanthemi
Polygalacturonases
Authors: Hugouvieux-Cotte-Pattat Nicole , Shevchik Vladimir E. , Nasser William 
Published: July 2002

ABSTRACT

Erwinia chrysanthemi
3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by
pel
genes, which play a major role in the soft-rot disease caused by this bacterium on various plants.
E. chrysanthemi
also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes,
pehV
,
pehW
, and
pehX,
encoding exo-poly-α-
d
-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named
pehN,
encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the
pehN
gene does not significantly alter the virulence of
E. chrysanthemi
. Regulation of
pehN
transcription was analyzed by using gene fusions. Like other pectinase genes,
pehN
transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of
pehN
is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the
pel
genes but activates that of
pehV
,
pehW
, and
pehX
, also activates transcription of
pehN
. The three regulators KdgR, PecS, and CRP act by direct interaction with the
pehN
promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent
pehN
transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in
peh
regulation.
New Type of Osmoregulated Solute Transporter Identified in Halophilic Members of theBacteriaDomain: TRAP Transporter TeaABC Mediates Uptake of Ectoine and Hydroxyectoine inHalomonas elongataDSM 2581T
Authors: Grammann Katrin , Volke Angela , Kunte Hans Jörg 
Published: July 2002

ABSTRACTThe halophilic bacteriumHalomonas elongatasynthesizes as its main compatible solute the aspartate derivative ectoine. We constructed a deletion mutant ofH. elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes. The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters. One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine. Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC). The nucleotide sequence upstream ofteaCwas determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified. Deletion ofteaAandteaBproved that all three genes are mandatory for ectoine uptake. Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T). The affinity of the cells for ectoines was determined (Ks= 21.7 μM), suggesting that the transporter TeaABC exhibits high affinity for ectoines. An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated. Deletion ofteaCandteaBCin the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane.
Differential Regulation of Multiple Proteins ofEscherichia coliandSalmonella entericaSerovar Typhimurium by the Transcriptional Regulator SlyA
Authors: Spory Andrea , Bosserhoff Armin , von Rhein Christine , Goebel Werner , Ludwig Albrecht 
Published: July 2002

ABSTRACTSlyA is a transcriptional regulator ofEscherichia coli,Salmonella enterica, and other bacteria belonging to theEnterobacteriaceae. The SlyA protein has been shown to be involved in the virulence ofS. entericaserovar Typhimurium, but its role inE. coliis unclear. In this study, we employed the proteome technology to analyze the SlyA regulons of enteroinvasiveE. coli(EIEC) andSalmonellaserovar Typhimurium. In both cases, comparative analysis of the two-dimensional protein maps of a wild-type strain, a SlyA-overproducing derivative, and a correspondingslyAmutant revealed numerous proteins whose expression appeared to be either positively or negatively controlled by SlyA. Twenty of the putative SlyA-induced proteins and 13 of the putative SlyA-repressed proteins of the tested EIEC strain were identified by mass spectrometry. The former proteins included several molecular chaperones (GroEL, GroES, DnaK, GrpE, and CbpA), proteins involved in acid resistance (HdeA, HdeB, and GadA), the “starvation lipoprotein” (Slp), cytolysin ClyA (HlyE or SheA), and several enzymes involved in metabolic pathways, whereas most of the latter proteins proved to be biosynthetic enzymes. Consistently, the resistance of the EIECslyAmutant to heat and acid stress was impaired compared to that of the wild-type strain. Furthermore, the implication of SlyA in the regulation of several of the identifiedE. coliproteins was confirmed at the level of transcription withlacZfusions. Twenty-three of theSalmonellaserovar Typhimurium proteins found to be affected by SlyA were also identified by mass spectrometry. With the exception of GroEL these differed from those identified in the EIEC strain and included proteins involved in various processes. The data suggest that gene regulation by SlyA might be crucial for intracellular survival and/or replication of both EIEC andSalmonellaserovar Typhimurium in phagocytic host cells.
Characterization of
comQ
and
comX
, Two Genes Required for Production of ComX Pheromone in
Bacillus subtilis
Authors: Schneider Katherine Bacon , Palmer Tanya M. , Grossman Alan D. 
Published: July 2002

ABSTRACT

Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing. ComX pheromone is a modified 10-amino-acid peptide used by
Bacillus subtilis
to modulate changes in gene expression in response to crowding.
comQ
and
comX
are required for production of ComX pheromone. We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone. We overexpressed
comQ
and
comX
separately and together and found that overexpression of
comX
alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter. These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of
comX
is limiting for production of ComX pheromone. We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone. Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone. We have also identified and mutated a putative isoprenoid binding domain of ComQ. Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid.
Comparison of In Vitro Activities of Voriconazole and Five Established Antifungal Agents against Different Species of Dermatophytes Using a Broth Macrodilution Method
Authors: Perea Sofia , Fothergill Annette W. , Sutton Deanna A. , Rinaldi Michael G. 
Published: July 2002

ABSTRACTThe in vitro activities of voriconazole against 19 different species of dermatophytes were compared with those of terbinafine, itraconazole, ketoconazole, griseofulvin, and fluconazole. MICs were determined according to a National Committee for Clinical Laboratory Standards broth macrodilution method. Voriconazole appeared more active than ketoconazole, griseofulvin, and fluconazole and less active than itraconazole and terbinafine. Based on these results, voriconazole merits further investigation as a potentially useful agent for the treatment of dermatophytosis.
Heterogeneity of
Pseudomonas aeruginosa
in Brazilian Cystic Fibrosis Patients
Authors: Silbert Suzane , Barth Afonso Luis , Sader Hélio S. 
Published: July 2002

ABSTRACT

The aim of this study was to assess the diversity and genomic variability of
Pseudomonas aeruginosa
isolates from cystic fibrosis (CF) patients being treated at a university hospital in Brazil. Ninety-seven isolates of
P. aeruginosa
from 43 CF patients were characterized by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested for susceptibility to 20 antimicrobial agents by broth microdilution. It was possible to evaluate single isolates from 20 patients and multiple isolates (two to seven) from 23 patients collected during a 22-month period. Among all of the unrelated patients, we detected only one pair of patients sharing a common strain. Among the 77 isolates from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were recognized. The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display differences in their susceptibilities. Mucoid isolates were more resistant (
P
< 0.001) than nonmucoid isolates to five antibiotics. Our results indicate that CF patients remain colonized by more than one strain of
P. aeruginosa
for long periods of time. In addition, the finding of several different genotypes in the same patient suggests that the colonizing strain may occasionally be replaced.
Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the
sodA
Gene as a Target
Authors: Poyart Claire , Quesne Gilles , Boumaila Claire , Trieu-Cuot Patrick 
Published: July 2002

ABSTRACT

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (
sodA
int
) to the
sodA
gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or
hsp60
gene sequences. Sequence analysis revealed that the staphylococcal
sodA
genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the
sodA
gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The
sodA
sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
Surveillance of Invasive
Streptococcus pneumoniae
Infection in the Province of Quebec, Canada, from 1996 to 1998: Serotype Distribution, Antimicrobial Susceptibility, and Clinical Characteristics
Authors: Jetté L. P. , Delage G. , Ringuette L. , Allard R. , De Wals P. , Lamothe F. , Loo V. ,   the Pneumococcus Study Group
Published: July 2002

ABSTRACT

In the province of Quebec, Canada, from 1996 to 1998, 3,650 invasive
Streptococcus pneumoniae
infections were reported. A total of 1,354 isolates were serotyped and tested for antimicrobial susceptibility. The distribution of serotypes remained stable over the 3 years, with serotypes 14, 6B, 4, 9V, 23F, and 19F accounting for 61% of the isolates. Overall, 90% of isolates were included in the current 23-valent vaccine and 67% were included in the 7-valent conjugate vaccine. We were able to determine that resistance to penicillin and to other antibiotics is increasing.
Clinical Evaluation of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Rapid Detection of
Mycobacterium tuberculosis
in Select Nonrespiratory Specimens
Authors: Woods Gail L. , Bergmann John S. , Williams-Bouyer Natalie 
Published: July 2002

ABSTRACT

The performance of the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of extrapulmonary tuberculosis was evaluated by testing 178 nonrespiratory specimens from 158 patients. Criteria for specimen inclusion were (i) a positive smear for acid-fast bacilli (
n
= 54) and (ii) the source if the smear was negative (tissue biopsies and aspirates and abscess material were tested;
n
= 124). Results were compared to those of mycobacterial culture; clinical history was reviewed when MTD and culture results disagreed. Forty-eight specimens (27.0%) were positive for mycobacteria, including 23
Mycobacterium tuberculosis
complex specimens; of which 21 were smear positive. Twenty-five specimens were MTD positive; 20 of these grew
M. tuberculosis
complex. All of the five MTD-positive,
M. tuberculosis
complex culture-negative specimens were considered truly positive, based on review of the medical record. Of the three MTD-negative,
M. tuberculosis
complex culture-positive specimens, two contained inhibitory substances; one of the two was smear positive. Excluding the latter specimen from analysis, after chart review, the sensitivity, specificity, and positive and negative predictive values of the MTD were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient. Given the few smear-negative samples from patients with extrapulmonary tuberculosis in our study, additional similar studies that include more smear-negative,
M. tuberculosis
complex culture-positive specimens to confirm our data are desirable.