bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specific Control of Endogenous cCF10 Pheromone by a Conserved Domain of the pCF10-Encoded Regulatory Protein PrgY inEnterococcus faecalis
Authors: Chandler Josephine R. , Flynn Aron R. , Bryan Edward M. , Dunny Gary M. 
Published: July 2005

ABSTRACTConjugative transfer ofEnterococcus faecalisplasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The clonedprgYgene could complement a pCF10prgYnull mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species includingBorrelia burgdorferi,Archaeoglobus fulgidus,Arabidopsis thaliana, andHomo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.
Citrate Synthase Mutants of
Agrobacterium
Are Attenuated in Virulence and Display Reduced
vir
Gene Induction
Authors: Suksomtip Maneewan , Liu Pu , Anderson Tamara , Tungpradabkul Sumalee , Wood Derek W. , Nester Eugene W. 
Published: July 2005

ABSTRACT

A citrate synthase (CS) deletion mutant of
Agrobacterium tumefaciens
C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to
Escherichia coli
and 77% identical to
Brucella melitensis
. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in
Sinorhizobium
. The CS mutation resulted in a 10-fold reduction in
vir
gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive
virG
[
virG
(Con)] locus was introduced into this mutant, the level of
vir
gene induction was restored to nearly wild-type level. Further, the
virG
(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the
virG
(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of
A. tumefaciens
C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in
Sinorhizobium
. The function of these loci remains unknown.
Global Analysis of Cellular Factors and Responses Involved inPseudomonas aeruginosaResistance to Arsenite
Authors: Parvatiyar Kislay , Alsabbagh Eyad M. , Ochsner Urs A. , Stegemeyer Michelle A. , Smulian Alan G. , Hwang Sung Hei , Jackson Colin R. , McDermott Timothy R. , Hassett Daniel J. 
Published: July 2005

ABSTRACTThe impact of arsenite [As(III)] on several levels of cellular metabolism and gene regulation was examined inPseudomonas aeruginosa. P. aeruginosaisogenic mutants devoid of antioxidant enzymes or defective in various metabolic pathways, DNA repair systems, metal storage proteins, global regulators, or quorum sensing circuitry were examined for their sensitivity to As(III). Mutants lacking the As(III) translocator (ArsB), superoxide dismutase (SOD), catabolite repression control protein (Crc), or glutathione reductase (Gor) were more sensitive to As(III) than wild-type bacteria. The MICs of As(III) under aerobic conditions were 0.2, 0.3, 0.8, and 1.9 mM forarsB,sodA sodB,crc, andgormutants, respectively, and were 1.5- to 13-fold less than the MIC for the wild-type strain. A two-dimensional gel/matrix-assisted laser desorption ionization-time of flight analysis of As(III)-treated wild-type bacteria showed significantly (>40-fold) increased levels of a heat shock protein (IbpA) and a putative allo-threonine aldolase (GlyI). Smaller increases (up to 3.1-fold) in expression were observed for acetyl-coenzyme A acetyltransferase (AtoB), a probable aldehyde dehydrogenase (KauB), ribosomal protein L25 (RplY), and the probable DNA-binding stress protein (PA0962). In contrast, decreased levels of a heme oxygenase (HemO/PigA) were found upon As(III) treatment. Isogenic mutants were successfully constructed for six of the eight genes encoding the aforementioned proteins. When treated with sublethal concentrations of As(III), each mutant revealed a marginal to significant lag period prior to resumption of apparent normal growth compared to that observed in the wild-type strain. Our results suggest that As(III) exposure results in an oxidative stress-like response inP. aeruginosa, although activities of classic oxidative stress enzymes are not increased. Instead, relief from As(III)-based oxidative stress is accomplished from the collective activities of ArsB, glutathione reductase, and the global regulator Crc. SOD appears to be involved, but its function may be in the protection of superoxide-sensitive sulfhydryl groups.
Identification of the Iron-Responsive Genes of
Neisseria gonorrhoeae
by Microarray Analysis in Defined Medium
Authors: Ducey Thomas F. , Carson Matthew B. , Orvis Joshua , Stintzi Alain P. , Dyer David W. 
Published: July 2005

ABSTRACT

To ensure survival, most bacteria must acquire iron, a resource that is sequestered by mammalian hosts. Pathogenic bacteria have therefore evolved intricate systems to sense iron limitation and regulate gene expression appropriately. We used a pan-
Neisseria
microarray to examine genes regulated in
Neisseria gonorrhoeae
in response to iron availability in defined medium. Overall, 203 genes varied in expression, 109 up-regulated and 94 down-regulated by iron deprivation. In iron-replete medium, genes essential to rapid bacterial growth were preferentially expressed, while iron transport functions, and predominantly genes of unknown function, were expressed in low-iron medium. Of those TonB-dependent proteins encoded in the FA1090 genome with unknown ligand specificity, expression of three was not controlled by iron availability, suggesting that these receptors may not be high-affinity transporters for iron-containing ligands. Approximately 30% of the operons regulated by iron appeared to be directly under control of Fur. Our data suggest a regulatory cascade where Fur indirectly controls gene expression by affecting the transcription of three secondary regulators. Our data also suggest that a second MerR-like regulator may be directly responding to iron availability and controlling transcription independent of the Fur protein. Comparison of our data with those recently published for
Neisseria meningitidis
revealed that only a small portion of genes were found to be similarly regulated in these closely related pathogens, while a large number of genes derepressed during iron starvation were unique to each organism.
Quorum-Sensing-Negative (
lasR
) Mutants of
Pseudomonas aeruginosa
Avoid Cell Lysis and Death
Authors: Heurlier Karin , Dénervaud Valérie , Haenni Marisa , Guy Lionel , Krishnapillai Viji , Haas Dieter 
Published: July 2005

ABSTRACT

In
Pseudomonas aeruginosa
,
N
-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of
P. aeruginosa
as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the
lasR
gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on
P. aeruginosa
under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that
lasR
mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and
lasR
mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37°C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to ≥ 9, the
lasR
mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the
lasR
mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh
−
phenotype, and five of these mutants, which were functionally complemented by
lasR

+

, had mutations in
lasR
. The observation that, in buffered nutrient yeast broth, the wild type and
lasR
mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to
lasR
mutants.
Molecular Cloning and Transcriptional and Expression Analysis of
engO
, Encoding a New Noncellulosomal Family 9 Enzyme, from
Clostridium cellulovorans
Authors: Han Sung Ok , Yukawa Hideaki , Inui Masayuki , Doi Roy H. 
Published: July 2005

ABSTRACT

Clostridium cellulovorans
produces a major noncellulosomal family 9 endoglucanase EngO. A genomic DNA fragment (40 kb) containing
engO
and neighboring genes was cloned. The nucleotide sequence contained reading frames for endoglucanase EngO, a putative response regulator, and a putative sensor histidine kinase protein. The
engO
gene consists of 2,172 bp and encodes a protein of 724 amino acids with a molecular weight of 79,474. Northern hybridizations revealed that the
engO
gene is transcribed as a monocistronic 2.6-kb mRNA. 5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR analysis indicated that the single transcriptional start site of
engO
was located 264 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the
engO
promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the σ
A
consensus promoter sequences of gram-positive bacteria. EngO contains a typical N-terminal signal peptide of 28 amino acid residues, followed by a 149-amino-acid sequence which is homologous to the family 4-9 carbohydrate-binding domain. Downstream of this domain was an immunoglobulin-like domain of 89 amino acids. The C terminus contains a family 9 catalytic domain of glycosyl hydrolase. Mass spectrometry analysis of EngO was in agreement with that deduced from the nucleotide sequence. Expression of
engO
mRNA increased from early to middle exponential phase and decreased during the early stationary phase. EngO was highly active toward carboxymethyl cellulose but showed no activity towards xylan. It was optimally active at 40 to 50°C and pH 5 to 6. The analysis of the products from the cellulose hydrolysis through thin-layer chromatography indicated its endoglucanase activity.
Hierarchical Control of Anaerobic Gene Expression in
Escherichia coli
K-12: the Nitrate-Responsive NarX-NarL Regulatory System Represses Synthesis of the Fumarate-Responsive DcuS-DcuR Regulatory System
Authors: Goh Ee-Been , Bledsoe Peggy J. , Chen Li-Ling , Gyaneshwar Prasad , Stewart Valley , Igo Michele M. 
Published: July 2005

ABSTRACT

Hierarchical control ensures that facultative bacteria preferentially use the available respiratory electron acceptor with the most positive standard redox potential. Thus, nitrate is used before other electron acceptors such as fumarate for anaerobic respiration. Nitrate regulation is mediated by the NarX-NarL two-component system, which activates the transcription of operons encoding nitrate respiration enzymes and represses the transcription of operons for other anaerobic respiratory enzymes, including enzymes involved in fumarate respiration. These are fumarate reductase (encoded by the
frdABCD
operon), fumarase B, which generates fumarate from malate, and the DcuB permease for fumarate, malate, and aspartate. The transcription of the corresponding structural genes is activated by the DcuS-DcuR two-component system in response to fumarate or its dicarboxylate precursors. We report results from preliminary transcription microarray experiments that revealed two previously unknown members of the NarL regulon: the
aspA
gene encoding aspartate-ammonia lyase, which generates fumarate; and the
dcuSR
operon encoding the dicarboxylate-responsive regulatory system. We measured beta-galactosidase expression from monocopy
aspA-lacZ
,
frdA-lacZ
, and
dcuS-lacZ
operon fusions in response to added nitrate and fumarate and with respect to the
dcuR
and
narL
genotypes. Nitrate, acting through the NarX-NarL regulatory system, repressed the transcription of all three operons. Only
frdA-lacZ
expression, however, was responsive to added fumarate or a
dcuR

+

genotype. Phospho-NarL protein protected operator sites in the
aspA
and
dcuS
promoter regions from DNase I cleavage in vitro. The overall results are consistent with the hypothesis that nitrate represses
frdA
operon transcription not only directly, by repressing
frdA
promoter activity, but also indirectly, by repressing
dcuS
promoter activity.
Rapid Detection of Colicin E9-Induced DNA Damage Using
Escherichia coli
Cells Carrying SOS Promoter-
lux
Fusions
Authors: Vankemmelbeke Mireille , Healy Bryan , Moore Geoffrey R. , Kleanthous Colin , Penfold Christopher N. , James Richard 
Published: July 2005

ABSTRACT

ColE9 is a plasmid-encoded protein antibiotic produced by
Escherichia coli
and closely related species that kills
E. coli
cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of
E. coli
cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-
lux
fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.
A Cystic Fibrosis Epidemic Strain of
Pseudomonas aeruginosa
Displays Enhanced Virulence and Antimicrobial Resistance
Authors: Salunkhe Prabhakar , Smart Catherine H. M. , Morgan J. Alun W. , Panagea Stavroula , Walshaw Martin J. , Hart C. Anthony , Geffers Robert , Tümmler Burkhard , Winstanley Craig 
Published: July 2005

ABSTRACT

The Liverpool epidemic strain (LES) of
Pseudomonas aeruginosa
is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC β-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the
ampC
gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in
lasR
. When fed to
Drosophila melanogaster
, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.
An Atypical KdpD Homologue from the CyanobacteriumAnabaenasp. Strain L-31: Cloning, In Vivo Expression, and Interaction withEscherichia coliKdpD-CTD
Authors: Ballal Anand , Bramkamp Marc , Rajaram Hema , Zimmann Petra , Apte Shree Kumar , Altendorf Karlheinz 
Published: July 2005

ABSTRACTThekdpFABCoperon ofEscherichia coli, coding for the high-affinity K+transport system KdpFABC, is transcriptionally regulated by the products of the adjacently locatedkdpDEgenes. The KdpD protein is a membrane-bound sensor kinase consisting of a large N-terminal domain and a C-terminal transmitter domain interconnected by four transmembrane segments (the transmembrane segments together with the C-terminal transmitter domain of KdpD are referred to as CTD), while KdpE is a cytosolic response regulator. We have cloned and sequenced thekdpoperon from a nitrogen-fixing, filamentous cyanobacterium,Anabaenasp. strain L-31 (GenBank accession. numberAF213466). ThekdpABCgenes are similar in size to those ofE. coli, but thekdpDgene is short (coding only for 365 amino acids), showing homology only to the N-terminal domain ofE. coliKdpD. AkdpE-like gene is absent in the vicinity of this operon.AnabaenaKdpD with six C-terminal histidines was overproduced inE. coliand purified by Ni2+-nitrilotriacetic acid affinity chromatography. With antisera raised against the purifiedAnabaenaKdpD, the protein was detected inAnabaenasp. strain L-31 membranes. The membrane-associated or soluble form of theAnabaenaKdpD(6His) could be photoaffinity labeled with the ATP analog 8-azido-ATP, indicating the presence of an ATP binding site. The coproduction ofAnabaenaKdpD withE. coliKdpD-CTD decreasedE. coli kdpFABCexpression in response to K+limitation in vivo relative to the wild-type KdpD-CTD protein. In vitro experiments revealed that the kinase activity of theE. coliKdpD-CTD was unaffected, but its phosphatase activity increased in the presence ofAnabaenaKdpD(6His). To our knowledge this is the first report where a heterologous N-terminal domain (AnabaenaKdpD) is shown to affect intransKdpD-CTD (E. coli) activity, which is just opposite to that observed for the KdpD-N-terminal domain ofE. coli.
Genome-Wide Detection and Analysis of Cell Wall-Bound Proteins with LPxTG-Like Sorting Motifs
Authors: Boekhorst Jos , de Been Mark W. H. J. , Kleerebezem Michiel , Siezen Roland J. 
Published: July 2005

ABSTRACT

Surface proteins of gram-positive bacteria often play a role in adherence of the bacteria to host tissue and are frequently required for virulence. A specific subgroup of extracellular proteins contains the cell wall-sorting motif LPxTG, which is the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases. A comprehensive set of putative sortase substrates was identified by in silico analysis of 199 completely sequenced prokaryote genomes. A combination of detection methods was used, including secondary structure prediction, pattern recognition, sequence homology, and genome context information. With the hframe algorithm, putative substrates were identified that could not be detected by other methods due to errors in open reading frame calling, frameshifts, or sequencing errors. In total, 732 putative sortase substrates encoded in 49 prokaryote genomes were identified. We found striking species-specific variation for the LPxTG motif. A hidden Markov model (HMM) based on putative sortase substrates was created, which was subsequently used for the automatic detection of sortase substrates in recently completed genomes. A database was constructed, LPxTG-DB (
http://bamics3.cmbi.kun.nl/sortase_substrates
), containing for each genome a list of putative sortase substrates, sequence information of these substrates, the organism-specific HMMs based on the consensus sequence of the sortase recognition motif, and a graphic representation of this consensus.
Gene Transfer and Genome Plasticity inThermotoga maritima, a Model Hyperthermophilic Species
Authors: Mongodin Emmanuel F. , Hance Ioana R. , DeBoy Robert T. , Gill Steven R. , Daugherty Sean , Huber Robert , Fraser Claire M. , Stetter Karl , Nelson Karen E. 
Published: July 2005

ABSTRACTThe genome sequence of the hyperthermophilic bacteriumThermotoga maritimaMSB8 presents evidence for lateral gene transfer events between bacterial and archaeal species. To estimate the extent of genomic diversity across the orderThermotogales, a comparative genomic hybridization study was initiated to compare nineThermotogastrains to the sequencedT. maritimaMSB8. Many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environmental niche that these individual species occupy. A detailed analysis of some of the predicted variable regions demonstrates many examples of the deletion/insertion of complete cassettes of genes and of gene rearrangements and insertions of DNA within genes, with the C or N terminus being retained. Although the mechanism for gene transfer in this lineage remains to be elucidated, this analysis suggests possible associations with repetitive elements and highlights the possible benefits of rampant genetic exchange to these species.