bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein secretion by heterologous bacterial ABC‐transporters: the C‐terminus secretion signal of the secreted protein confers high recognition specificity
Authors: Duong F. , Lazdunski A. , Murgier M. 
Published: October 2006

Summary Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) is released by its own specific secretion machinery which is an ABC‐transporter. Despite sequence similarities between components of ABC‐transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC‐transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C‐terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated a‐haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the trans‐locator which confers specificity to the secretion process. Secretion was size‐dependent. However inclusion of glycine‐rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20kDa larger than the normal maximal size.
Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae
Authors: Alioing Geneviève , Granadel Chantal , Morrison Donald A. , Claverys Jean‐Pierre 
Published: October 2006

Summary An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide‐binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild‐type strain and we investigated the response of the various mutants to treatment with synthetic competence‐stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide‐binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide‐binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two‐component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid‐insertion mutation abolished competence completely. In addition, the triple AmiA‐AliA‐AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.
The Bacillus subtilis soj‐spo0J locus is required for a centromere‐like function involved in prespore chromosome partitioning
Authors: Sharpe Michaela E. , Errington Jeffery 
Published: October 2006

SummaryDuring sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell division. Pre‐spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere‐like apparatus that anchors the oriC region of the prespore chromosome in the pole of the cell.
CheC and CheD interact to regulate methylation of Bacillus subtilis methyl‐accepting chemotaxis proteins
Authors: Rosario Mia Mae L. , Ordal George W. 
Published: October 2006

SummaryIn this study, we have demonstrated that two unique proteins inBacillus subtilischemotaxis, CheC and CheD, interact. We have shown this interaction both by using the yeast two‐hybrid system and by precipitation ofin vitrotranslated products using glutathione‐S‐transferase fusions and glutathione agarose beads. We have also shown that CheC inhibitsB. subtilisCheR‐mediated methylation ofB. subtilismethyl‐accepting chemotaxis proteins (MCPs) but not ofEscherichia coliMCPs. It was previously reported thatcheCmutants tend to swim smoothly and do not adapt to addition of attractant;cheDmutants have very poorly methylated MCPs and are very tumbiy, similar tocheAmutants. We hypothesize that CheC exerts its effect on MCP methylation inB. subtilisby controlling the binding of CheD to the MCPs. In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA. The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis inB. subtilis, whereby high levels of CheY‐P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY‐P.
Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli
Authors: Sakellaris Harry , Balding Donna P. , Scott June R. 
Published: October 2006

Summary Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1. For the assembly of the major‐pilin subunit, CooA, into pili, each of four linked genes, cooB,A,C, and D, is required. In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera. CooD appears to be an integral pilus protein because it co‐purifies with, and is strongly associated with, CS1 pili. In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1‐piliated strains) was detected in periplasmic inter‐molecular complexes with the major‐pilin subunit CooA. CooB is an assembly protein found exclusively in the periplasm of CS1‐piliated strains. CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure. Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1‐piliated E. coli. Based on this information, we have proposed a model for CS1 ‐pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E. coli. As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution.
Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells
Authors: Lebrun Maryse , Mengaud Jérôme , Ohayon Hélène , Nato Farida , Cossart Pascale 
Published: October 2006

SummaryEntry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram‐positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C‐terminal end. By immunofluorescence and immunogold labelling, we show that in wild‐type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C‐terminal region of internalin is necessary for cell‐surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain‐swapping’strategy ‐ replacement of the cell wall anchor of InIA by the membrane anchor of ActA ‐ we show that the reduced ability to adhere and enter cells of strains expressing InIA‐ActA correlates with a lower amount of surface‐exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.
Activation of transcription at divergent urea‐dependent promoters by the urease gene regulator UreR
Authors: D'Orazio Sarah E. F. , Thomas Venetta , Collins Carleen M. 
Published: October 2006

Summary The Proteus mirabilis and plasmid‐encoded urease loci contain seven contiguous structural and accessory genes (ureDABCEFG) and the divergently transcribed ureR, which codes for an AraC‐like transcriptional activator. Previously, it was shown that the plasmid‐encoded ureR to ureD intergenic region contained divergent promoters (ureRp and ureDp). Transcription from these promoters required both the effector molecule urea and the activator protein UreR. In this report, we demonstrate that the P. mirabilis urease gene cluster contains similar divergent urea‐ and UreR‐dependent promoters. The ureR gene products from either urease locus were able to activate transcription at both the plasmid‐encoded and P. mirabilis promoters. The minimal concentration of urea required to activate transcription at ureRp or ureDp from either gene cluster was approximately 4 mM. The transcriptional start sites for the plasmid‐encoded and P. mirabilis divergent promoters were similar in an Escherichia coli DH5α background, as determined by primer‐extension analysis. However, in P. mirabilis HI4320, transcription of ureR initiated predominately at an alternative site. Physical mapping and inhibition studies were used to localize the UreR‐binding sites within the plasmid‐encoded ureRp and ureDp intergenic sequences to regions of 68 bp and 86 bp, respectively. Gel shift analysis demonstrated that UreR bound to a 135 bp fragment in the approximate centre of the plasmid‐encoded ureR to ureD intergenic region. The results presented here suggest that the P. mirabilis and plasmid‐encoded urease gene clusters utilize similar mechanisms of transcriptional activation in response to urea.
Customized secretion chaperones in pathogenic bacteria
Authors: Wattiau Pierre , Woestyn Sophie , Cornelis Guy R. 
Published: October 2006

SummaryPathogenic yersiniae secrete about a dozen anti‐host proteins, the Yops, by a pathway which does not involve cleavage of a classical signal peptide. The Yop secretory apparatus, called Ysc, for Yop secretion, is the archetype of type III secretion systems (which serve for the secretion of virulence proteins by several animal and plant pathogens) and is related to the flagellar assembly apparatus. The Yop secretion signal is N‐terminal but has not been defined to date. Apart from the Ysc machinery, secretion of at least four Yops requires cytoplasmic proteins called Syc (for specific Yop chaperone). Each Syc protein binds to its cognate Yop. Unlike most cytoplasmic chaperones, these proteins do not have an ATP‐binding domain, and are presumably devoid of ATPase activity. They share a few common properties: an acidic pl, a size in the range of 15–20 kDa, and a putative amphipathic α‐helix in the C‐terminal portion. They were recently shown to have counterparts in other pathogenic bacteria, where they appear to have a similar function.
Optimized BlaM‐transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence
Authors: Odenbreit Stefan , Till Markus , Haas Rainer 
Published: October 2006

Summary Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini‐blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β‐lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild‐type strain.
Purification and functional characterization of PecS, a regulator of virulence‐factor synthesis in Erwinia chrysanthemi
Authors: Praillet Thierry , Nasser William , Robert‐Baudouy Janine , Reverchon Sylvie 
Published: October 2006

SummaryThe Erwinia chrysanthemi pecS gene encodes a repressor that negatively regulates the expression of virulence factors such as pectinases or cellulases. The cloned pecS gene was overexpressed using a phage T7 system. The purification of PecS involved DEAE‐anion exchange and TSK‐heparin columns and delivered the PecS protein that was purified to homogeneity. The purified repressor displayed an 18 kDa apparent molecular mass and an isoelectric point near to neutrality (PI = 6.5). Gel‐filtration experiments revealed that the PecS protein is a dimer. Bandshift assays demonstrated that the PecS protein could specifically bind in vitro to the regulatory sites of the in vivo PecS‐regulated genes. The interaction between the PecS protein and its DNA‐binding site was characterized by a relatively low affinity (about 10−8 M). DNase I footprintings revealed short protected sequences only with the most in vivo PecS‐regulated genes. Alignment of these PecS‐binding sites did not show a well‐conserved consensus sequence. lmmunoblotting demonstrated that the copy number of the PecS protein was approximately 50 dimers per cell. The low affinity of the PecS repressor for its DNA targets and the low cellular PecS content suggest the existence of E. chrysanthemi‐specific factors able to potentiate PecS protein activity in vivo.
Reciprocal regulation of the differentiation of Myxococcus xanthus by Pkn5 and Pkn6, eukaryotic‐like Ser/Thr protein kinases
Authors: Zhang Wandong , Lnouye Masayori , Inouye Sumiko 
Published: October 2006

Summary Myxococcus xanthus contains a large family of genes encoding eukaryotic‐like serinehhreonine kinases. Among them, two genes, pkn5 and pkn6, are divergently located on the chromosome and share a 46 bp promoter region between their transcription initiation sites, as determined by RNA protection. Pkn5, consisting of 380 amino acid residues, is a soluble protein in the cytoplasm, while Pkn6, consisting of 710 amino acid residues, is a transmembrane protein. Its membrane topology was determined using the Pkn6‐PhoA fusion protein in Escherichia coli, which has a single transmembrane domain with the N‐terminal domain in the cytoplasm and the C‐terminal domain outside the cytoplasmic membrane. Both proteins, when expressed in E. coli, were autophosphorylated: Pkn5 only at Ser, and Pkn6 at both Ser and Thr. In M. xanthus, both genes are expressed constitutively throughout the life cycle, with slight increases at an early stage of development. Most strikingly, a pkn5‐deletion strain forms fruiting bodies much faster than the wild‐type strain, while a pknb‐deletion strain develops slower than the wild‐type strain. These results, together with the fact that the pkn5‐deletion strain is able to form fruiting bodies on semi‐rich media, suggest that Pkn5 and Pkn6 have reciprocal roles in M. xanthus growth and development. Furthermore, Pkn6 may be a transmembrane sensor of external signals for development, while Pkn5 is a kinase that negatively regulates M. xanthus development.
Construction of a ferritin‐deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress
Authors: Wai Sun Nyunt , Nakayama Koji , Umene Kenichi , Moriya Tetsuhiro , Amako Kazunobu 
Published: October 2006

SummaryThe ferritin‐encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin‐deficient mutant was constructed. The growth of ferritin‐deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin‐deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron‐mediated oxidative stress.
Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase
Authors: Neece Sue H. , Carles‐Kinch Kelly , Tomso Daniel J. , Kreuzer Kenneth N. 
Published: October 2006

SummaryThe bacteriophage T4‐encoded type II DNA topoisomerase is the major target for the antitumour agent m‐AMSA (4‐(9‐acridinylamino)methanesulphon‐m‐anisidide) in phage‐infected bacterial cells. Inhibition of the purified enzyme by m‐AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double‐strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase‐deficient mutants were shown to be resistant to m‐AMSA, indicating that m‐AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m‐AMSA, strongly suggesting that recombination participates in the repair of topoisomerase‐mediated damage. Third, m‐AMSA stimulated recombination in phage‐infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m‐AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage‐infected cells.
The activation of PhoB by acetylphosphate
Authors: McCleary W. R. 
Published: October 2006

Summary PhoB is a response‐regulator protein from Escherichia coli that controls an adaptive response to limiting phosphate. It is activated by autophosphorylation of a conserved aspartate residue within its regulatory domain. Its primary phospho‐donor is its cognate histidine kinase PhoR; however, it also becomes phos‐phorylated when incubated with acetylphosphate. To further characterize its activation, PhoB was considered to be an acetylphosphatase whose enzymatic mechanism involves a phospho‐enzyme intermediate. The kinetic constants for autophosphorylation were determined using 32P‐ and fluorescence‐based assays and indicated that PhoB has a Km for acetylphosphate of between 7 and 8 mM. These constants are not consistent with an in vivo role for acetylphosphate in the normal control of the Pho regulon. In addition, when PhoB was phosphorylated by acetylphosphate it eluted from a high‐performance liquid chromatography (HPLC) size‐exclusion column in two peaks. The larger form of PhoB eluted from the column in a similar manner to a chemically cross‐linked dimer of PhoB. The smaller form of PhoB is a monomer. Phosphorylated PhoB bound pho‐box DNA approximately 10 times tighter than PhoB. These observations show that PhoB forms a dimer when phosphorylated and suggest that the characteristics of activated PhoB result from its dimer ization.
The F plasmid traY gene product binds DNA as a monomer or a dimer: structural and functional implications
Authors: Nelson William C. , Matson Steven W. 
Published: October 2006

Summary The F factor traY gene product (TraYp) is a site‐specific DNA‐binding protein involved in initiation of DNA transfer during bacterial conjugation. The sequence of TraYp exhibits a unique direct‐repeat structure predicted to have a ribbon‐helix‐helix DNA‐binding motif in each repeat unit. The stoichiometry of TraYp binding to DNA was determined to further support the hypothesis that TraYp is a member of the ribbon‐helix‐helix family of DNA‐binding proteins. A gluta‐thione‐S‐transferase‐traY fusion protein was purified and shown to possess almost wild‐type DNA‐binding activity. DNA‐binding experiments were performed in which the DNA ligand was incubated with either the fusion protein, the wild‐type protein, or both. The results indicate that TraYp can bind DNA as a monomer or a dimer. Thus a TraYp monomer folds into a stable three‐dimensional structure similar to that of a dimer of the ribbon‐helix‐helix proteins Arc or Mnt. A homology model of a TraYp monomer has been constructed using the co‐crystal structure of Arc bound to DNA as a template to provide additional support for this conclusion. In addition, we have shown that an origin of the transfer‐deletion mutant lacking approximately half of the TraYp‐binding site can only be bound by a monomer of TraYp. The functional implications of this result are discussed.
Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter
Authors: Polissi Alessandra , Georgopoulos Costa 
Published: October 2006

Summary The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low‐copy‐number plasmid vector it was able to suppress the temperature‐sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP‐binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP‐binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single‐amino‐acid substitutions in non‐conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single‐amino‐acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP‐binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W‐acetyl [3H]‐glucosamine, a precursor of lipopolysaccharides, accumulates at the non‐permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild‐type msbA gene. A possible role for MsbA