Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments.

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells.IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

Induction of pre-chorismate, jasmonate and salicylate pathways by Burkholderia sp. RR18 in peanut seedlings.

AIMS: To characterize the mechanisms by which bacteria in the peanut rhizosphere promote plant growth and suppress Aspergillus niger, the fungus that causes collar rot of peanut. METHODS AND RESULTS: In all, 131 isolates cultured from the peanut rhizosphere were assayed for growth promotion in a seedling germination assay. The most effective isolate, RR18, was identified as Burkholderia sp. by 16S sequencing analysis. RR18 reduced collar rot disease incidence and increased the germination rate and biomass of peanut seeds, and had broad-spectrum antifungal activity. Quantitative analyses showed that RR18 induced long-lasting accumulation of jasmonic acid, salicylic acid and phenols, and triggered the activity of six defence enzymes related to these changes. Comparative proteomic analysis of treated and untreated seedlings revealed a clear induction of four abundant proteins, including a member of the pre-chorismate pathway, a regulator of clathrin-coated vesicles, a transcription factor and a hypothetical protein. CONCLUSION: Burkholderia sp. RR18 promotes peanut growth and disease resistance, and stably induces two distinct defence pathways associated with systemic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a strain of the Burkholderia cepacia complex can elicit both salicylic- and jasmonic-acid-mediated defences, in addition to having numerous other beneficial properties.

Genome Mining Shows Ubiquitous Presence and Extensive Diversity of Toxin-Antitoxin Systems in Pseudomonas syringae.

Bacterial toxin-antitoxin (TA) systems consist of two or more adjacent genes, encoding a toxin and an antitoxin. TA systems are implicated in evolutionary and physiological functions including genome maintenance, antibiotics persistence, phage defense, and virulence. Eight classes of TA systems have been described, based on the mechanism of toxin neutralization by the antitoxin. Although studied well in model species of clinical significance, little is known about the TA system abundance and diversity, and their potential roles in stress tolerance and virulence of plant pathogens. In this study, we screened the genomes of 339 strains representing the genetic and lifestyle diversity of the Pseudomonas syringae species complex for TA systems. Using bioinformatic search and prediction tools, including SLING, BLAST, HMMER, TADB2.0, and T1TAdb, we show that P. syringae strains encode 26 different families of TA systems targeting diverse cellular functions. TA systems in this species are almost exclusively type II. We predicted a median of 15 TA systems per genome, and we identified six type II TA families that are found in more than 80% of strains, while others are more sporadic. The majority of predicted TA genes are chromosomally encoded. Further functional characterization of the predicted TA systems could reveal how these widely prevalent gene modules potentially impact P. syringae ecology, virulence, and disease management practices.

Development and evaluation of in situ novel intragastric controlled-release formulation of hydrochlorothiazide.

In situ forming intragastric controlled-release formulation is a new technology in the field of oral controlled-release delivery systems. The objective of this study was to develop formulations that can control drug release up to 24 hours. In addition, a combination of appropriate polymers and solvents was selected that could form a drug loaded gel at the process temperature of 60-70 °C, which gel could turn into a rigid mass upon exposure to dissolution fluid at body temperature. The drug release mechanism from this rigid mass was controlled by different formulation factors such as different polymer grades, polymer concentrations, hydrophobicity or hydrophilicity of solvents, different drug loadings, and physicochemical properties of additional excipients. After evaluating different formulation factors, Ethocel 10 FP and triethyl citrate were selected for further studies using hydrochlorothiazide as a model drug. Polynomial correlation between viscosity of the blank gel and drug release profile was also obtained.