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Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter. Lisa M. Schechter, 2004.Pseudomonas syringae pv . tomato strain DC3000 is a pathogen of tomato and Arabidopsis . The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity . In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues . Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P . syringae TTSS effectors into plant cells . This system includes a cloned P . syringae hrp gene cluster and the model plant Nicotiana benthamiana . Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation . AvrB, tested because it is poorly secreted in cultures by the P . syringae Hrp system, was translocated into plant cells as effectively as AvrPto . The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA . We also confirmed that HopPtoK, HopPtoC, and AvrPphEPto are translocated into plant cells . These results increased the number of Hrp system-secreted proteins in DC3000 to 40 . Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics . Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P . syringae. Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins. Charléric Bornet, 2004.In Enterobacter aerogenes, ß-lactam resistance often involves a decrease in outer membrane permeability induced by modifications of porin synthesis . In ATCC 15038 strain, we observed a different pattern of porin production associated with a variable antibiotic susceptibility . We purified Omp35, which is expressed under conditions of low osmolality and analyzed its pore-forming properties in artificial membranes . This porin was found to be an OmpF-like protein with high conductance values . It showed a noticeably higher conductance compared to Omp36 and a specific location of WNYT residues in the L3 loop . The importance of the constriction region in the porin function suggests that this organization is involved in the level of susceptibility to negative large cephalosporins such as ceftriaxone by bacteria producing the Omp35 porin subfamily . Monomeric NarB Is a Dual-Affinity Nitrate Reductase, and Its Activity Is Regulated Differently from That of Nitrate Uptake in the Unicellular Diazotrophic Cyanobacterium Synechococcus sp . Strain RF-1. Tung-Hei Wang, 2003.Synechococcus sp . strain RF-1 is a unicellular freshwater cyanobacterium that fixes N2 aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen . Synechococcus sp . strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth . Under the diazotrophic growth, the nitrate uptake in Synechococcus sp . strain RF-1 was induced by nitrate or nitrite but repressed by ammonium . In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay . The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate . The different expression patterns of nitrate uptake and NR in Synechococcus sp . strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR . Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate . The recombinant NR encoded by the narB gene of Synechococcus sp . strain RF-1, expressed in E . coli, also showed the biphasic kinetics with similar pH and temperature profiles . By in-gel NR activity assay, the recombinant NarB was found to exist as a single form . Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability . When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single Km for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics . Identification and Characterization of Two Novel Clostridial Bacteriocins, Circularin A and Closticin 574. Robèr Kemperman, 2003.Two novel antibacterial peptides of clostridial species were purified, N-terminally sequenced, and characterized . Moreover, their structural genes were identified . Closticin 574 is an 82-amino-acid bacteriocin produced by Clostridium tyrobutyricum ADRIAT 932 . The supernatant of the producing strain showed a high level of activity against the indicator strain C . tyrobutyricum . The protein is synthesized as a preproprotein that is possibly secreted via the general secretion pathway, after which it is hydrolyzed at an Asp-Pro site . Circularin A is produced by Clostridium beijerinckii ATCC 25752 as a prepeptide of 72 amino acids . Cleavage of the prepeptide between the third leucine and fourth valine residues followed by a head-to-tail ligation between the N and C termini creates a circular antimicrobial peptide of 69 amino acids . The unusually small circularin A leader peptide of three amino acids is cleaved off in this process . The supernatant of C . beijerinckii ATCC 25752 showed a broad antibacterial activity range . Differences in Susceptibility of Listeria monocytogenes Strains to Sakacin P, Sakacin A, Pediocin PA-1, and Nisin. T. Katla, 2003.Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin . The individual 50% inhibitory concentrations (IC50) were determined in a microtiter assay and expressed in nanograms per milliliter . The IC50 of sakacin P ranged from 0.01 to 0.61 ng ml-1 . The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml-1, respectively . The use of a large number of strains and the accuracy of the IC50 determination revealed patterns not previously described, and for the first time it was shown that the IC50 of sakacin P divided the L . monocytogenes strains into two distinct groups . Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism . The results from these studies essentially confirmed the grouping based on the IC50 of sakacin P . A high correlation was found between the IC50 of sakacin P and that of pediocin PA-1 for the 200 strains . Surprisingly, the correlation between the IC50 of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC50 of sakacin A and the class I bacteriocin nisin .
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