bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Characterization Reveals Similar Virulence Gene Content in Unrelated Clonal Groups of
Escherichia coli
of Serogroup O174 (OX3)
Authors: Tarr Cheryl L. , Nelson Adam M. , Beutin Lothar , Olsen Katharina E. P. , Whittam Thomas S. 
Published: December 2007

ABSTRACT

Most severe illnesses that are attributed to Shiga toxin-producing
Escherichia coli
are caused by isolates that also carry a pathogenicity island called the locus of enterocyte effacement (LEE). However, many cases of severe disease are associated with LEE-negative strains. We characterized the virulence gene content and the evolutionary relationships of
Escherichia coli
isolates of serogroup O174 (formerly OX3), strains of which have been implicated in cases of hemorrhagic colitis and hemolytic uremic syndrome. A total of 56 isolates from humans, farm animals, and food were subjected to multilocus virulence gene profiling (MVGP), and a subset of 16 isolates was subjected to multilocus sequence analysis (MLSA). The MLSA revealed that the O174 isolates fall into four separate evolutionary clusters within the
E. coli
phylogeny and are related to a diverse array of clonal groups, including enteropathogenic
E. coli
2 (EPEC 2), enterohemorrhagic
E. coli
2 (EHEC 2), and EHEC-O121. Of the 15 genes that we surveyed with MVGP, only 6 are common in the O174 strains. The different clonal groups within the O174 serogroup appear to have independently acquired and maintained similar sets of genes that include the Shiga toxins (
stx
1
and
stx
2
) and two adhesins (
saa
and
iha
). The absence of certain O island (OI) genes, such as those found on OI-122, is consistent with the notion that certain pathogenicity islands act cooperatively with the LEE island.
Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY
Authors: Chandler Josephine R. , Dunny Gary M. 
Published: December 2007

ABSTRACTConjugative transfer of theEnterococcus faecalisplasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.
Interaction of CheY with the C-Terminal Peptide of CheZ
Authors: Guhaniyogi Jayita , Wu Ti , Patel Smita S. , Stock Ann M. 
Published: December 2007

ABSTRACT

Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P∼CheY). The steady-state level of P∼CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P∼CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZ
C
) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol.
359:
624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZ
C
peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZ
C
interaction. In addition, we present kinetic studies of the CheZ
C
-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.
Characterization of the SOS Regulon ofCaulobacter crescentus
Authors: da Rocha Raquel Paes , de Miranda Paquola Apuã César , do Valle Marques Marilis , Menck Carlos Frederico Martins , Galhardo Rodrigo S. 
Published: December 2007

ABSTRACTThe SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs oflexAandrecA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacteriumCaulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator ofC. crescentus, which was confirmed by site-directed mutagenesis studies of theimuApromoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and thelexAstrain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.
Tn
5
Synaptic Complex Formation: Role of Transposase Residue W450
Authors: Gradman Richard J. , Reznikoff William S. 
Published: December 2007

ABSTRACT

A series of Tn
5
transposases (Tnp's) with mutations at the conserved amino acid position W450, which was structurally predicted to be important for synapsis, have been generated and characterized. This study demonstrates that W450 is involved in hydrophobic (and possibly aromatic) contacts within the Tnp monomer that negatively regulate synaptic complex formation.
Interstrain Gene Transfer inChlamydia trachomatisIn Vitro: Mechanism and Significance
Authors: DeMars Robert , Weinfurter Jason 
Published: December 2007

ABSTRACTThe high frequency of between-strain genetic recombinants ofChlamydia trachomatisamong isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by whichC. trachomatisgenerates variants that have enhanced relative fitness. A mechanism for LGT inC. trachomatishas not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describe here experiments that readily detected LGT between strains ofC. trachomatisin vitro. Host cells were simultaneously infected with an ofloxacin-resistant (Ofxr) mutant of a serovar L1 strain (L1:Ofxr-1) and a rifampin-resistant (Rifr) mutant of a serovar D strain (D:Rifr-1). Development occurred in the absence of antibiotics, and the progeny were subjected to selection for OfxrRifrrecombinants. The parental strains differed at many polymorphic nucleotide sites, and DNA sequencing was used to map genetic crossovers and to determine the parental sources of DNA segments in 14 recombinants. Depending on the assumed DNA donor, the estimated minimal length of the transferred DNA was ≥123 kb in one recombinant but was ≥336 to ≥790 kb in all other recombinants. Such trans-DNA lengths have been associated only with conjugation in known microbial LGT systems, but natural DNA transformation remains a conceivable mechanism. LGT studies can now be performed with diverse combinations ofC. trachomatisstrains, and they could have evolutionary interest and yield useful recombinants for functional analysis of allelic differences between strains.
Affinity Isolation and I-DIRT Mass Spectrometric Analysis of the
Escherichia coli
O157:H7 Sakai RNA Polymerase Complex
Authors: Lee David J. , Busby Stephen J. W. , Westblade Lars F. , Chait Brian T. 
Published: December 2007

ABSTRACT

Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the
Escherichia coli
K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic
Escherichia coli
strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although
E. coli
O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the “core”
E. coli
genome.
Genetics for
Pseudoalteromonas
Provides Tools To Manipulate Marine Bacterial Virus PM2
Authors: Kivelä Hanna M. , Madonna Stefania , Krupovìč Mart , Tutino M. Luisa , Bamford Jaana K. H. 
Published: December 2007

ABSTRACT

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (
Corticoviridae
) has been limited so far. The isolation of an autonomously replicating DNA element of
Pseudoalteromonas haloplanktis
TAC125 and construction of a shuttle vector replicating in both
Escherichia coli
and
Pseudoalteromonas
enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2
sus2
mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative
Pseudoalteromonas
species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.
Role of the Multidrug Resistance Regulator MarA in Global Regulation of the
hdeAB
Acid Resistance Operon in
Escherichia coli
Authors: Ruiz Cristian , McMurry Laura M. , Levy Stuart B. 
Published: December 2007

ABSTRACT

MarA, a transcriptional regulator in
Escherichia coli
, affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of
hdeAB
and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of
hdeAB
by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of
hdeAB
. Moreover, repression of
hdeAB
by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of
hdeAB
by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on
hdeAB
under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of
hdeAB
expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of
hdeAB
by MarA depends on pH, growth phase, and other regulators of
hdeAB
and is associated with reduced resistance to acid conditions.
Clostridium perfringens
Spore Germination: Characterization of Germinants and Their Receptors
Authors: Paredes-Sabja Daniel , Torres J. Antonio , Setlow Peter , Sarker Mahfuzur R. 
Published: December 2007

ABSTRACT

Clostridium perfringens
food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (
cpe
) gene (C-
cpe
), while
C. perfringens
-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne
cpe
gene (P-
cpe
).
C. perfringens
spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-
cpe
and P-
cpe
isolates in that (i) while a mixture of
l
-asparagine and KCl was a good germinant for spores of C-
cpe
and P-
cpe
isolates, KCl and, to a lesser extent,
l
-asparagine triggered spore germination in C-
cpe
isolates only; and (ii)
l
-alanine or
l
-valine induced significant germination of spores of P-
cpe
but not C-
cpe
isolates. Spores of a
gerK
mutant of a C-
cpe
isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with
l
-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca
2+
and dipicolinic acid. In contrast, spores of a
gerAA
mutant of a C-
cpe
isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in
C. perfringens
spore germination. In sum, this study identified nutrient germinants for spores of both C-
cpe
and P-
cpe
isolates of
C. perfringens
and provided evidence that proteins encoded by the
gerK
operon are required for both nutrient-induced and non-nutrient-induced spore germination.
Differential Regulation of High-Affinity Phosphate Transport Systems of
Mycobacterium smegmatis
: Identification of PhnF, a Repressor of the
phnDCE
Operon
Authors: Gebhard Susanne , Cook Gregory M. 
Published: December 2007

ABSTRACT

The uptake of phosphate into the cell via high-affinity, phosphate-specific transport systems has been studied with several species of mycobacteria. All of these species have been shown to contain several copies of such transport systems, which are synthesized in response to phosphate limitation. However, the mechanisms leading to the expression of the genes encoding these transporters have not been studied. This study reports on the investigation of the regulation of the
pstSCAB
and the
phnDCE
operons of
Mycobacterium smegmatis
. The
phn
locus contains an additional gene,
phnF
, encoding a GntR-like transcriptional regulator. Expression analyses of a
phnF
deletion mutant demonstrated that PhnF acts as a repressor of the
phnDCE
operon but does not affect the expression of
pstSCAB
. The deletion of
pstS
, which is thought to cause the constitutive expression of genes regulated by the two-component system SenX3-RegX3, led to the constitutive expression of the transcriptional fusions
pstS
-
lacZ, phnD
-
lacZ
, and
phnF
-
lacZ
, suggesting that
phnDCE
and
phnF
are conceivably new members of the SenX3-RegX3 regulon of
M. smegmatis
. Two presumptive binding sites for PhnF in the intergenic region between
phnD
and
phnF
were identified and shown to be required for the repression of
phnD
and
phnF
, respectively. We propose a model in which the transcription of
pstSCAB
is controlled by the two-component SenX3-RegX3 system, while
phnDCE
and
phnF
are subject to dual control by SenX3-RegX3 and PhnF.
Genetic and Biochemical Analysis of CodY-Binding Sites inBacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L. 
Published: December 2007

ABSTRACTCodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons inBacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of theB. subtilis ylmAandyurPgenes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of theylmA, yurP, dppA, andilvBgenes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo oflacZfusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified forLactococcus lactisCodY, with up to five mismatches play an important role in the interaction ofB. subtilisCodY with DNA.
A Combination ofsbmAandtolCMutations inEscherichia coliK-12 Tn10-Carrying Strains Results in Hypersusceptibility to Tetracycline
Authors: de Cristóbal Ricardo E. , Vincent Paula A. , Salomón Raúl A. 
Published: December 2007

ABSTRACTPreviously, we demonstrated thatEscherichia coli tolCmutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 μg/ml to resistance at 40 μg/ml. In this study, we found that the addition of ansbmAmutation to atolC::Tn10mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 μg/ml. Inactivation ofsbmAalone partially inhibited high-level tetracycline resistance, from resistance at 200 μg/ml to resistance at 120 μg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10tetracycline resistance.
Thromboprophylaxis in cancer patients with central venous catheters
Authors: Nautiyal Amit , Radhakrishnan Sangeetha , Singh Sonal , Navaneethan Sankar D , Chaukiyal Pooja 
Published: December 2007

SummaryIt was the aim of the review to determine the risks and benefits of primary thromboprophylaxis with anticoagulants in cancer patients with central venous devices. Medline, Central and Google Scholar databases were searched for randomized controlled trials (RCTs) in June 2006.Two reviewers extracted data and appraised the quality of RCTs. Results were expressed as relative risk (RR) with 95% confidence intervals (CI) using random effects model for the outcomes of catheter-related thrombosis, bleeding and thrombocytopenia. Eight RCTs (1,428 patients) were included. There was no statistically significant difference in the risk of catheter-related thrombosis for the use of warfarin versus placebo (3 trials, 425 patients, RR 0.75, 95% CI 0.24–2.35,p=0.63),heparin versus placebo (4 trials,886 patients, RR 0.46 95% CI 0.18–1.20, p=0.06) or warfarin, unfractionated heparin or low-molecular-weight heparin versus placebo (7 trials, 1,311 patients, RR 0.59, 95% CI 0.31–1.13, p=0.11). Substantial statistical heterogeneity was noted among these trials (I2>50%).The use of anticoagulants showed no statistically significant difference in the risk of overall bleeding (5 trials, 1,193 patients, RR 1.24, 95% CI 0.84–1.82, p=0.28), and thrombocytopenia for heparin versus placebo (4 trials, 958 patients, RR 0.85, 95% CI 0.49, 1.46, p=0.55) without any statistical heterogeneity (I2=0%). In cancer patients with central venous devices, thromboprophylaxis has no significant effect on the risk of catheter related thrombosis or bleeding. The use of primary thromboprophylaxis in cancer patients with central venous catheters while not causing any harm provides no benefit.
Cathepsin G, a leukocyte protease, activates coagulation factor VIII
Authors: Rozenshteyn Diana , Gale Andrew J 
Published: December 2007

SummaryNeutrophils and monocytes express cathepsin G and can also bind to activated platelets, thus they can be localized to the site of active coagulation. Previous studies have suggested that cathepsin G inactivated coagulation factorVIII (FVIII) and was thus anticoagulant. But other studies have indicated procoagulant functions for cathepsin G in activation of coagulation factorV or activation of platelets among other possible mechanisms. Therefore, it remains unclear if cathepsin G is anticoagulant or procoagulant. We investigated the effects of human neutrophil cathepsin G on FVIII/VIIIa. Cathepsin G activates FVIII to a partially active form while having only a minor inactivating effect on thrombin- activated FVIIIa. This inactivation is mostly due to decreased stability of FVIIIa since a disulfide bond that prevents A2 subunit dissociation from FVIIIa prevents any loss of activity due to cathepsin G proteolysis. FVIII that has been cleaved by cathepsin G can still be activated by thrombin if A2 subunit dissociation is prevented. Cathepsin G cleavages of FVIII are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. Therefore, it is possible that cathepsin G from neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII.