bluuebunny/crossref_metadata_2025_split huggingface dataset:
The essential role of the promoter‐proximal subunit of CAP in pap phase variation: Lrp‐ and helical phase‐dependent activation of papBA transcription by CAP from −215
Authors: Weyand Nathan J. , Braaten Bruce A. , Van Der Woude Marjan , Tucker Julie , Low David A. 
Published: March 2003

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP–CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP‐dependent activation of papBA requires binding of the leucine‐responsive regulatory protein (Lrp) at binding sites that extend from −180 to −149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP‐dependent promoters. Also, like class I promoters, the C‐terminal domain of the α‐subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an ‘oriented heterodimer’ approach with wild‐type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter‐distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H‐NS. The results presented here show that AR1 of the promoter‐proximal CAP subunit was required for papBA transcription even in the absence of the histone‐like protein H‐NS. These results show that the promoter‐proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C‐terminal domain of the α‐subunit of RNA polymerase.
Mammalian 14‐3‐3β associates with the Chlamydia trachomatis inclusion membrane via its interaction with IncG
Authors: Scidmore M. A. , Hackstadt T. 
Published: March 2003

Chlamydiae replicate intracellularly within a vacuole that is modified early in infection to become fusogenic with a subset of exocytic vesicles. We have recently identified four chlamydial inclusion membrane proteins, IncD–G, whose expression is detected within the first 2 h after internalization. To gain a better understanding of how these Inc proteins function, a yeast two‐hybrid screen was employed to identify interacting host proteins. One protein, 14‐3‐3β, was identified that interacted specifically with IncG. The interaction between 14‐3‐3β and IncG was confirmed in infected HeLa cells by indirect immunofluorescence microscopy and interaction with a GFP‐14‐3‐3β fusion protein. 14‐3‐3 proteins are phosphoserine‐binding proteins. Immunoprecipitation studies with [32P]‐orthophosphate‐labelled cells demonstrated that IncG is phosphorylated in both chlamydia‐infected HeLa cells and in yeast cells expressing IncG. Site‐directed mutagenesis of predicted 14‐3‐3 phosphorylation sites demonstrated that IncG binds to 14‐3‐3β via a conserved 14‐3‐3‐binding motif (RS164RS166F). Finally, indirect immunofluorescence demonstrated that 14‐3‐3β interacts with Chlamydia trachomatis inclusions but not C. psittaci or C. pneumoniae inclusions. 14‐3‐3β is the first eukaryotic protein found to interact with the chlamydial inclusion; however, its unique role in C. trachomatis pathogenesis remains to be determined.
A set of ftsZ mutants blocked at different stages of cell division in Caulobacter
Authors: Wang Yan , Jones Benjamin D. , Brun Yves V. 
Published: March 2003

FtsZ is required throughout the cell division process in eubacteria and in archaea. We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus. Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding. Molecular modelling indicated that all the clustered‐charged‐to‐alanine mutations had altered amino acids at the surface of the protein. Of 13 such mutants, four were recessive‐lethal, three were dominant‐lethal, and six had no discernible phenotype. An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive‐lethal mutations and used to show that they blocked cell division at distinct stages. One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division. The effect of the recessive mutations on the subcellular localization of FtsZ was determined. Models to explain the various mutant phenotypes are discussed. This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.
The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence
Authors: Mayorga María E. , Gold Scott E. 
Published: March 2003

The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilagobypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein–protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone‐responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone‐responsive MAP kinase cascade and ubc2 is required for pheromone‐responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone‐responsive MAP kinase cascade in U. maydis.
Molecular characterization of type III secretion signals via analysis of synthetic N‐terminal amino acid sequences
Authors: Lloyd Scott A. , Sjöström Michael , Andersson Sara , Wolf‐Watz Hans 
Published: March 2003

Summary Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N‐termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2–8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N‐terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N‐terminal secretion signals. The model accurately classifies the N‐terminal sequences of native type III substrates as efficient secretion signals.
LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion
Authors: Wulff‐Strobel Christine R. , Williams Andrew W. , Straley Susan C. 
Published: March 2003

SummaryLcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ’s effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ’s effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.
Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome
Authors: Josenhans Christine , Niehus Eike , Amersbach Stefanie , Hörster Andrea , Betz Christian , Drescher Bernd , Hughes Kelly T. , Suerbaum Sebastian 
Published: March 2003

Summary Helicobacter pylori is thought to regulate gene ex‐pression with a very small set of regulatory genes. We identified a previously unannotated open reading frame (ORF) in the H. pylori 26695 genome (HP1122) as a putative H. pylori flgM gene (σ28 factor antagonist) by a motif‐based bioinformatic approach. Deletion of HP1122 resulted in a fourfold increase in transcription of the σ28‐dependent major flagellin gene flaA, supporting the function of HP1122 as H. pylori FlgM. Helicobacter pylori FlgM lacks a conserved 20‐amino‐acid N‐terminal domain of enterobacterial FlgM proteins, but was able to interact with the Salmonella typhimuriumσ28 (FliA) and inhibit the expression of FliA‐dependent genes in Salmonella. Helicobacter pylori FlgM inhibited FliA to the same extent in a Salmonella strain with an intact flagellar export system and in an export‐deficient strain. Helicobacter pylori FliA was able to drive transcription of FliA‐dependent genes in Salmonella. The effects of mutations in the H. pylori flgM and fliA genes on the H. pylori transcriptome were analysed using whole genome DNA microarrays. The antagonistic roles of FlgM and FliA in controlling the transcription of the major flagellin gene flaA were confirmed, and two additional FliA/FlgM dependent operons (HP472 and HP1051/HP1052) were identified. None of the three genes contained in these operons has a known function in flagellar biogenesis in other bacteria. Like other motile bacteria, H. pylori has a FliA/FlgM pair of sigma and anti‐sigma factors, but the genes controlled by these differ markedly from the Salmonella/Escherichia coli paradigm.
A tyrosine‐phosphorylated 12‐amino‐acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals
Authors: Campellone Kenneth G. , Giese Ndrew , Tipper Onald J. , Leong John M. 
Published: March 2003

SummaryEnteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12‐residue sequence of EPEC Tir containing Y474 that confers actin‐signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation‐dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.
Examination of Salmonella gene expression in an infected mammalian host using the green fluorescent protein and two‐colour flow cytometry
Authors: Bumann Dirk 
Published: March 2003

SummaryQuantitative data on Salmonella gene expression in infected hosts are largely

lacking because of technical problems. One attractive reporter, the green fluorescent

protein (GFP), is widely used in vitro but is difficult to quantify in infected

tissues because of the preponderance of background particles with similar fluorescence.

Here, bacterial GFP emission was spectrally distinguished from host autofluorescence

by two‐colour flow cytometry. Using this technique, the in vivo activity of

three well‐characterized promoters (PsicA, PssaH

and PpagC) was determined. Their spatial and temporal activity

patterns are in close agreement with predictions based on previous data and the colonization

defects of corresponding deletion strains. To identify additional Salmonella

promoters that are induced in infected animals, a genomic library was sorted by flow

cytometry yielding four independent promoters. Genes expressed from PpibB

and PsifA contribute to virulence, and chorismate mutase expressed

from ParoQ might participate in aromatic acid biosynthesis, which

is also required for virulence. Promoter P3g appears to be part of a mobile genetic element that is lacking in the completely sequenced strain LT2.
Identification of genes that are associated with DNA repeats in prokaryotes
Authors: Jansen Ruud. , Embden Jan. D. A. van , Gaastra Wim. , Schouls Leo. M. 
Published: March 2003

SummaryUsing in silico analysis we studied a novel family of repetitive DNA sequences

that is present among both domains of the prokaryotes (Archaea and Bacteria), but

absent from eukaryotes or viruses. This family is characterized by direct repeats,

varying in size from 21 to 37 bp, interspaced by similarly sized non‐repetitive sequences.

To appreciate their characteri‐stic structure, we will refer to this family as the

clustered regularly interspaced short palindromic repeats (CRISPR). In most species

with two or more CRISPR loci, these loci were flanked on one side by a common leader

sequence of 300–500 b. The direct repeats and the leader sequences were conserved

within a species, but dissimilar between species. The presence of multiple chromosomal

CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR‐associated (cas)

genes were identified in CRISPR‐containing prokaryotes that were absent from CRISPR‐negative

prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus,

indicating that the cas genes and CRISPR loci have a functional relationship.

The cas3 gene showed motifs characteristic for helicases of the superfamily

2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting

that these genes are involved in DNA metabolism or gene expression. The spatial coherence

of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.
NAD+‐glycohydrolase acts as an intracellular toxin to enhance the extracellular survival of group A streptococci
Authors: Bricker Angela L. , Cywes Colette , Ashbaugh Cameron D. , Wessels Michael R. 
Published: March 2003

SummaryGroup A streptococci (GAS) produce several secreted products that are thought to enhance pathogenicity by facilitating spread of the organisms through host tissues. Two such products, streptolysin O (SLO) and NAD+‐glycohydrolase,
appear to be functionally linked, in that SLO is required for transfer of NAD+‐glycohydrolase
into epithelial cells. However, the effects of NAD+‐glycohydrolase
on host cells are largely unexplored. We now report that SLO‐mediated delivery of
NAD+‐glycohydrolase to the cytoplasm of human keratinocytes results
in major changes in host cell biology that enhance GAS pathogenicity. We derived
isogenic mutant strains deficient in the expression of SLO, NAD+‐glycohydrolase
or both proteins in the background of a virulent, M‐type 3 strain of GAS. All three
mutant strains were internalized by human keratinocytes more rapidly and in higher
numbers than were organisms from the wild‐type strain. Association of the mutant
strains with keratinocytes also resulted in reduced cytotoxicity and reduced keratinocyte
apoptosis compared with wild‐type GAS. These results support a model in which NAD+‐glycohydrolase
contributes to GAS pathogenesis by modulating host cell signalling pathways to inhibit
GAS internalization, to augment SLO‐mediated cytotoxicity and to induce keratinocyte
apoptosis. We conclude that NAD+‐glycohydrolase is a novel type of bacterial toxin that acts intracellularly in the infected host to enhance the survival and proliferation of an extracellular pathogen.
Diversity in the serine recombinases
Authors: Smith Margaret C. M. , Thorpe Helena M. 
Published: March 2003

SummaryMost site‐specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases orλintegrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N‐terminal catalytic domain and a C‐terminal DNA binding domain. Phage‐encoded serine recombinases (e.g.φC31 integrase) cause integration and excision with strictly controlled directionality, and have an N‐terminal catalytic domain but much longer C‐terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N‐terminal to the catalytic domain (e.g. IS607transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.
ParE toxin encoded by the broad‐host‐range plasmid RK2 is an inhibitor of Escherichia coli gyrase
Authors: Jiang Yong , Pogliano Joe , Helinski Donald R. , Konieczny Igor 
Published: March 2003

SummaryBroad‐host‐range plasmid RK2 encodes a post‐segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication‐proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase‐ and gyrase‐dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.
An accessory sec locus of Streptococcus gordonii is required for export of the surface protein GspB and for normal levels of binding to human platelets
Authors: Bensing Barbara A. , Sullam Paul M. 
Published: March 2003

SummaryThe translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are
not required for viability or for the translocation of most exported proteins. The
genes (secA2 and secY2) reside in a region of the chromosome required
for the export of GspB, a 286 kDa cell wall‐anchored protein. Loss of GspB surface
expression is associated with a significant reduction in the binding of M99 to human
platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99
has a second, canonical SecA homologue that is essential for viability. At least
two other Gram‐positive species, Streptococcus pneumoniae and Staphylococcus
aureus, encode two sets of SecA and SecY homologues. One set is more similar
to SecA and SecY of Escherichia coli, whereas the other set is more similar
to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2–secA2 loci suggests that, in each of these Gram‐positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine‐rich repeat protein.
Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans
Authors: Weissman Ziva , Shemer Revital , Kornitzer Daniel 
Published: March 2003

SummaryEfficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron‐poor host environment. In Saccharomyces cerevisiae,

high‐affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis

in the trans‐Golgi compartment requires a copper‐transporting P‐type ATPase,

ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue

from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct

from a previously described C. albicans copper‐transporting P‐type ATPase,

CaCrp1, which appears to be specifically involved in copper detoxification. Regulation

of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate

that it is required for high‐affinity iron import, making it the bona fide CCC2

homologue of C. albicans. Remarkably, in a mouse model of systemic infection,

the Caccc2Δ strain displayed robust proliferation and no significant

reduction in pathogenicity, suggesting the existence of alternative mechanisms of

iron uptake from host tissues. We identify haemin and haemoglobin as potential iron

sources that can be used by C. albicans in a CaCcc2‐independent manner.