Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome

Abstract

The genome of Pseudomonas fluorescens F113, a model rhizobacterium and a plant growth-promoting agent, encodes three putative type VI secretion systems (T6SSs); F1-, F2- and F3-T6SS. Bioinformatic analysis of the F113 T6SSs has revealed that they belong to group 3, group 1.1, and group 4a, respectively, similar to those previously described in Pseudomonas aeruginosa. In addition, in silico analyses allowed us to identify genes encoding a total of five orphan VgrG proteins and eight putative effectors (Tfe), some with their cognate immunity protein (Tfi) pairs. Genes encoding Tfe and Tfi are found in the proximity of P. fluorescens F113 vgrG, hcp, eagR and tap genes. RNA-Seq analyses in liquid culture and rhizosphere have revealed that F1- and F3-T6SS are expressed under all conditions, indicating that they are active systems, while F2-T6SS did not show any relevant expression under the tested conditions. The analysis of structural mutants in the three T6SSs has shown that the active F1- and F3-T6SSs are involved in interbacterial killing while F2 is not active in these conditions and its role is still unknown.. A rhizosphere colonization analysis of the double mutant affected in the F1- and F3-T6SS clusters showed that the double mutant was severely impaired in persistence in the rhizosphere microbiome, revealing the importance of these two systems for rhizosphere adaption.

Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

“…RNA was isolated from F113 and derivatives cultures grown in SA medium [37] at exponential (Optical density (OD) 600 = 0.6) and stationary phase (OD 600 =1.2) [38]. All the steps of the RNA isolation were done at 4 °C except otherwise specified.…”

“…Strains were grown overnight in SA medium and diluted in FP to inoculate 1 ml of 1·10 8 Colony forming Units (CFUs) at the base of each alfalfa 7 day-old seedling. RNA isolation was carried out 7 days post-inoculation [38]. After removing shoots of alfalfa plants, roots and vermiculite were placed on 10 ml of 60% (v/v) RNAlater [40] in pH 7.4 phosphate-buffered saline and vortexed for 2 min in order to resuspend bacterial cells.…”

“…After RNA sequencing, reads were trimmed and quality-filtered using Trimmomatic v 0.38 [45], specifying a four nts sliding window with an average phred quality of 15 and 50 nts as minimum read length as described in [38]. Then, trimmed reads were randomly subsampled into two technical replicates with 10000000 reads each using an own designed Python script.…”

The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

“…It should be noted that methyltransferase motifs have been found in other proteins within T6SS clusters of other bacteria although they are structurally diverse and different from Tsb1. Some examples are PF00487 from Rhizobium etli Mim1 [26]; Azc_2606 and Azc_2704; from A. caulinodans [30] and Tfe7 homologous to the ribosomal RNA large subunit methyltransferase D required for the fully methylation of 23S ribosomal RNA [47]. Interestingly, no gene encoding an immunity pair was found linked to these genes, suggesting a distinct activity of the antibacterial effectors as is the case of antifungal effectors described in the opportunistic pathogen Serratia marcescens [48].…”

The Azospirillum brasilense type VI secretion system promotes cell aggregation, biocontrol protection against phytopathogens and attachment to the microalgae Chlorella sorokiniana

“…The A. brasilense T6SS provides antibacterial activity against competitors, including phytopathogens A number of reports have demonstrated that the T6SS is involved in regulating bacterial communities by targeting effector toxins into competing bacteria sharing the same ecological niche (Hood et al, 2010;Sana et al, 2016;Wexler et al, 2016;Anderson et al, 2017;Chassaing and Cascales, 2018;Allsopp et al, 2020;Wood et al, 2020;Dur an et al, 2021). We therefore asked whether the A. brasilense T6SS might confer protection to the microalgae or plants by delivering antibacterial toxins into phytopathogens.…”