bluuebunny/crossref_metadata_2025_split huggingface dataset:
SycE, a chaperone‐like protein of Yersinia enterocolitica involved in the secretion of YopE
Authors: Wattiau Pierre , Cornells Guy R. 
Published: October 2006

SummaryPathogenic yersiniae secrete a set of 11 anti‐host proteins called Yops. The yop genes, scattered around the pYV plasmid, constitute a thermoinduced regulon controlled by the product of virF gene. The secretion of the Yops also requires the presence of the products of the other vir genes and operons, namely virA, virB and virC. The large virC operon and presumably some genes of the virA region encode a new secretion system. Mutations in any of these vir genes impair the production of all the Yops. In contrast, mutations in the yerA locus, located close to yopf, specifically abolish the expression of the cyto‐toxin YopE. We describe here the counterpart of yerA in Yersinia enterocolitica W22703. We demonstrate that the gene product of yerA regulates the production of YopE at a post‐transcriptional level. It specifically binds the YopE protein. We consider that it acts as a specific chaperone and we call it SycE (for specific YopE chaperone). We hypothesize that SycE is a link between translation and the specific Yop export machinery. It is the first representative of a new family of pYV‐encoded proteins.
Sucrose‐derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis
Authors: Munro Cindy L. , Macrina Francis L. 
Published: October 2006

SummaryWe used an isogenic mutant of Streptococcus mutans V403, which differs from the wild‐type V403 in genes involved in glucan and fructan production, to examine the importance of these exopolysaccharides as factors affecting infectivity in endocarditis. Rats inoculated with V403 developed endocarditis more frequently than animals inoculated with the mutant strain which produced neither glucan nor fructan (58% versus 12%, P<0.01). In phagocytosis assays, both strains were found to be associated with the human granulocytes but a greater number of live V403 than of mutant organisms could be recovered. Colony counts recovered from fibrin plates incubated with the mutant were lower than those incubated with V403. These experiments indicate that exopolysaccharides produced by Streptococcus mutans contribute to its infectivity in endocarditis.
Dual roles of plcA in Listeria monocytogenes pathogenesis
Authors: Camilli Andrew , Tilney Lewis G. , Portnoy Daniel A. 
Published: October 2006

SummaryThe plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol‐speciftc phospholi‐pase C (PI‐PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue‐culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in‐frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the PI‐PLC in pathogenesis. PI‐PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.
Bacterial ureases: structure, regulation of expression and role in pathogenesis
Authors: Collins Carleen M. , D'Orazio Sarah E. F. 
Published: October 2006

SummaryThe nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in either the urinary tract or the gastroduodenal region, urease acts as a virulence factor. At both sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. In addition, in the urinary tract urease activity can result in the formation of urinary calculi. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen‐limiting growth conditions. For either the urea‐inducible genes or the nitrogen‐regulated genes transcription appears to be positively regulated. In the nitrogen‐regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the LysR family of transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), a member of the AraC family of transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed.
Aeromonas spp. can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway
Authors: Wong Kevin R. , Buckley J. Thomas 
Published: October 2006

SummaryAerolysin is a channel‐forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas β‐lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.
Targeted interruption of the psaA and psaB genes encoding the reaction‐centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413
Authors: Nyhus Karin J. , Thiel Teresa , Pakrasi Himadri B. 
Published: October 2006

SummaryThe two reaction‐centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA:: NmR and psaB:: NmR, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction‐centre chlorophylls of PSI) and as a consequence did not exhibit any PSI‐mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five‐ to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild‐type cells. Interestingly, the psaS:: NmR and not the psaA:: NmR mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction‐centre proteins.
Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy‐proximal secretion signals by the Escherichia coliα‐haemolysin translocator
Authors: Šebo Peter , Ladant Daniel 
Published: October 2006

SummaryThe 1706‐residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy‐proximai glycine and aspartate‐rich repeats. CyaA does not have a cleavable amino‐terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the α‐haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N–terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/ToiC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp‐1587 to lle‐1631. Interestingly, a conserved acidic' motif (Glu/Asp)‐(X)11‐Asp‐(X)3/5‐(Glu/Asp)‐(X)14‐Asp was found in the C‐terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.
Escherichia coli hha mutants, DNA supercoiling and expression of the haemolysin genes from the recombinant plasmid pANN202‐312
Authors: Carmona M. , Balsalobre C. , Muñoa F. , Mouriño M. , Jubete Y. , De la Cruz F. , Juárez A. 
Published: October 2006

SummaryThe hha gene of Escherichia coli was identified as modulating the expression of the haemolysin (hly) genes encoded by the recombinant plasmid pANN202‐312. hha mutants harbouring plasmid pANN202‐312 showed increased haemolysin production. The product of the hha gene, the Hha protein, shows strong homology to the YmoA protein of Yersinia enterocolitica, which plays a role in the thermoregulation of various V. enterocolitica virulence genes. We show in this study that the Hha protein modulates the expression of haemolysin at the transcriptional level in ceils harbouring plasmid pANN202‐312. In addition, hha mutants show alterations in the level of plasmid DNA supercolting. This suggests that the hha mutation increases haemolysin expression through changes in the DNA topology. This hypothesis is supported by our finding that gyr mutations, inhibitors of DNA gyrase such as novo‐biocin, or growth in conditions reported to reduce levels of negative supercoiling, such as low osmolarity medium, increase haemolysin production.
Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal‐clostridial cell wall lytic enzyme
Authors: Croux C. , Ronda C. , Lopez R. , Garcia J. L. 
Published: October 2006

SummaryBacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline‐binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline‐binding protein (CLC) that has been purified using affinity chromatography on DEAE‐cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline‐dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin‐defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.
The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion
Authors: Poquet Isabelle , Kornacker Michael G. , Pugsley Anthony P. 
Published: October 2006

SummaryThe analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino‐proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino‐terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cyto‐plasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase‐specific secretion genes. A non‐secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightty lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient, the pullulanase activity remained at the bottom while the NADH oxidase floated to the top. Thus, there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane. Instead, the results support our previous proposal that lipoproteins with an aspartate +2 residue are specifically enriched in a distinct domain of the cell envelope that contains material from both the cytoplasmic and the outer membranes. Possible explanations for the requirement for the aspartate residue in pullulanase secretion are discussed.
Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin
Authors: Smith J. M. , Davies J. E. , Holden D. W. 
Published: October 2006

Summary Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxjn called restrictocin. To investigate the role of restrictocin in (PA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one drsruptant, using an anti‐restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infectediung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclearleucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.
Translational initiation at the coat‐protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure
Authors: de Smit Maarten H. , van Duin Jan 
Published: October 2006

SummaryMaximal translation of the coat‐protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30‐fold reduction of translational efficiency. By site‐directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20‐fold to almost the level of the full‐length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full‐length MS2 RNA, but there it does not Impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat‐protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.
ColE1 multimer formation triggers inhibition of Escherichia coli cell division
Authors: Patient Michaela E. , Summers David K. 
Published: October 2006

SummaryMultimer formation and consequent copy number depression are acknowledged causes of multicopy plasmid instability. Multimer resolution sites (among which ColE1 cer is best‐characterized) have been identified in a variety of plasmids. They participate in the conversion of multimers to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. We show that multimer resolution alone is insufficient to ensure stable maintenance of ColE1‐like plasmids in a recombination‐proficient host. The expression of Red, a transcript encoded within cer and expressed in multimer‐containing cells, is also required. The appearance of Red correlates with the inhibition of division of multimer‐containing cells, presumably allowing time for the conversion of multimers to monomers by site‐specific recombination.
Pore‐forming and haemolytic properties of the Gardnerella vaginalis cytoiysin
Authors: Cauci Sabina , Monte Rossella , Ropele Monica , Missero Caterina , Not Tarcisio , Quadrifoglio Franco , Menestrina Gianfranco 
Published: October 2006

SummaryThe pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 × 106 HU mg−1 being obtained. The toxin‐induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose‐response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol−1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh‐haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens theta‐toxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram‐positive and Gram‐negative bacteria, respectively. The toxin shares several features with the family of the so‐called ‘sulphydryl‐activated’ cytolysins produced by Gram‐positive bacteria, although Gvh does not truly belong to this family, being deactivated by β‐mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.
An unusual promoter controls cell‐cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon
Authors: Stephens Craig M. , Shapiro Lucille 
Published: October 2006

SummaryTranscription of flagellar genes in Caulobacter crescentus is programmed to occur during the predivisional stage of the cell cycle. The mechanism of activation of Class II flagellar genes, the highest identified genes in the Caulobacter flagellar hierarchy, is unknown. As a step toward understanding this process, we have defined cis‐acting sequences necessary for expression of a Class II flagellar operon, fliLM. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Transcription from this promoter‐containing fragment was severely reduced when chromosomal DNA replication was inhibited. Extensive mutational analysis of the promoter region from ‐42 to ‐5 identified functionally important nucleotides at ‐36 and ‐35, between ‐29 and ‐22 and at ‐12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel Sigma subunit interacts with an activation factor bound to the central region of the promoter.