AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements.

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.

Characterization of the bacteriocins and the PrtR regulator in a plant-associated Pseudomonas strain.

The emergence of antibiotic resistant bacterial strains demands the development of new antimicrobial agents. In the last decades, bacteriocins have gained significant interest due to their potential application as biopreservatives in the food industry and as therapeutic agents in medicine. Recent studies project the use of these antimicrobials in agriculture as biocontrol agents. The characterization of bacteriocins and their genetic regulation, however, have been scarcely studied in plant-associated bacteria. In this report, an in-silico and proteomic analysis was performed to identify the bacteriocins produced by Pseudomonas fluorescens SF4c. More than one functional bacteriocin was detected in this strain (S-type bacteriocins and phage-tail-like bacteriocins [tailocins]). It is known that the regulator PrtR represses bacteriocin production in P. aeruginosa under normal condition. However, the mechanism for tailocin regulation remains unknown in plant-associated pseudomonads. In this work, an orthologue of the prtR of P. aeruginosa was identified in the SF4c-tailocin cluster and a prtR null mutant constructed. The expression and production of tailocins was abolished in this mutant; thus evidencing that, unlike P. aeruginosa, PrtR is a positive regulator of tailocins expression in P. fluorescens.

Fengycins From Bacillus amyloliquefaciens MEP218 Exhibit Antibacterial Activity by Producing Alterations on the Cell Surface of the Pathogens Xanthomonas axonopodis pv. vesicatoria and Pseudomonas aeruginosa PA01.

Bacillus amyloliquefaciens MEP218 is an autochthonous bacterial isolate with antibacterial and antifungal activities against a wide range of phytopathogenic microorganisms. Cyclic lipopeptides (CLP), particularly fengycins, produced by this bacterium; are the main antimicrobial compounds responsible for the growth inhibition of phytopathogens. In this work, the CLP fraction containing fengycins with antibacterial activity was characterized by LC-ESI-MS/MS. In addition, the antibacterial activity of these fengycins was evaluated on the pathogens Xanthomonas axonopodis pv. vesicatoria (Xav), a plant pathogen causing the bacterial spot disease, and Pseudomonas aeruginosa PA01, an opportunistic human pathogen. In vitro inhibition assays showed bactericidal effects on Xav and PA01. Atomic force microscopy images revealed dramatic alterations in the bacterial surface topography in response to fengycins exposure. Cell damage was evidenced by a decrease in bacterial cell heights and the loss of intracellular content measured by potassium efflux assays. Furthermore, the viability of MRC-5 human normal lung fibroblasts was not affected by the treatment with fengycins. This study shows in vivo evidence on the less-known properties of fengycins as antibacterial molecules and leaves open the possibility of using this CLP as a novel antibiotic.

Effectiveness of tailocins produced by Pseudomonas fluorescens SF4c in controlling the bacterial-spot disease in tomatoes caused by Xanthomonas vesicatoria.

The development of alternatives for the use of chemical pesticides for plant disease control is the present-day and ongoing challenge for achieving sustainable agriculture. Pseudomonas fluorescens SF4c, native strain from wheat, produces tailocins (phage-tail-like bacteriocins) with antimicrobial activity against several phytopathogenic strains. We thus investigated the efficacy of foliar application of these bacteriocins to control the bacterial-spot disease in tomato caused by Xanthomonas vesicatoria Xcv Bv5-4a. The disease severity and incidence index were reduced by 44 and 36%, respectively; while the number of viable cells of X. vesicatoria Xcv Bv5-4a decreased after bacteriocin treatment. Furthermore, bacteriocin was effective in reducing bacterial-spot-disease symptoms on tomato fruits even when applied 12 h after infection. Tailocin activity was not affected by abiotic influences such as adjuvant, light and temperature and, biotic factors such as apoplastic-fluids. In contrast, no antibacterial activity of these tailocins was observed when the bacteriocin was exposed to extremely dry conditions. Finally, that no cytotoxic effects on mammalian cells were observed with this representative tailocins is highly significant and demonstrates the safety of such compounds in humans. All these findings indicate that the SF4c tailocins represent an attractive alternative to copper-containing bactericides for use in the control of bacterial spot.

Effect of a Pseudomonas fluorescens tailocin against phytopathogenic Xanthomonas observed by atomic force microscopy.

Phage tail-like bacteriocins, called tailocins, represent a class of protein complexes produced by a multitude of bacteria. Pseudomonas fluorescens SF4c, a strain isolated from wheat rhizosphere, produces a bacteriocin similar to phage tail-like pyocins of Pseudomonas aeruginosa. This tailocin has antimicrobial activity against several phytopathogenic strains of the genus Xanthomonas and Pseudomonas. In this work, the effect of the SF4c tailocin on the phytopathogenic strain X. axonopodis pv vesicatoria Xcv Bv5-4a was analyzed through Atomic Force Microscopy (AFM). We demonstrated that tailocins adhere and cause damage to the cell envelope of strain Xcv Bv5-4a. This results in a rapid leakage of intracellular materials, with the subsequent decrease of cell volume. Finally, lysis of sensitive bacteria occurs. This study provides, to our knowledge, the first evidence about the effect of a tailocin analyzed by AFM. Further studies are in progress to evaluate the use of SF4c tailocin in the biocontrol of bacterial spot on tomato.