Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors

Abstract

Bacteria utilize type VI secretion system (T6SS) to deliver antibacterial toxins to target co-habiting bacteria. Here, we report that Burkholderia gladioli strain NGJ1 deploys certain T6SS effectors (TseTBg), having both DNase and RNase activities to kill target bacteria. RNase activity is prominent on NGJ1 as well as other bacterial RNA while DNase activity is pertinent to only other bacteria. The associated immunity (TsiTBg) proteins harbor noncanonical helix-turn-helix motifs and demonstrate transcriptional repression activity, similar to the antitoxins of type II toxinantitoxin (TA) systems. Genome analysis reveals that homologs of TseTBg are either encoded as TA or T6SS effectors in diverse bacteria. Our results indicate that a new ORF (encoding a hypothetical protein) has evolved as a result of operonic fusion of TA type TseTBg homolog with certain T6SS-related genes by the action of IS3 transposable elements. This has potentially led to the conversion of a TA into T6SS effector in Burkholderia. Our study exemplifies that bacteria can recruit toxins of TA systems as T6SS weapons to diversify its arsenal to dominate during inter-bacterial competitions.

PAAR Proteins Are Versatile Clips That Enrich the Antimicrobial Weapon Arsenals of Prokaryotes

“…Few toxic effectors associated with PAAR proteins have been reported thus far. These toxins have been found to affect the pathogenicity of pathogens or directly mediate interactions among bacteria, and the ways in which they interact with PAAR proteins can be roughly divided into two types: one class of toxins is directly fused as the C-terminal extended domain of the PAAR protein, for example, Tse6 and Tse7 of Pseudomonas aeruginosa , Rhs-CT1 to CT10 of Escherichia coli , Tne2 of Pseudomonas protegens , and Rhs1 and Rhs2 of Serratia marcescens , all of which possess an N-terminal PAAR domain but a C-terminal extension containing various toxin domains ( 9 – 16 ); the other class of toxins form complexes with PAAR proteins (maybe through chaperone assistance), such as IglF of Francisella tularensis , TseT of P. aeruginosa , and TseTBg of Burkholderia gladioli ( 8 , 17 , 18 ).…”

Antibacterial T6SS effectors with a VRR-Nuc domain induce target cell death via DNA Double-Strand Breaks

“…To gain insight into the molecular function of TseV2 and TseV3 and understand their phylogenetic relationship, we used TseV1, TseV2 and TseV3 (TseV4 is 79.1% identical to TseV3 and was not used) amino acid sequences as queries in JackHMMER searches (Potter et al , 2018) for four iterations on the NCBI nr database (November 4th, 2021) to fetch a total of 2254 sequences with significant similarity (inclusion threshold ≤10 −9 and reporting threshold ≤10 −6 ). Additional JackHMMER searches were performed using selected VRR-Nuc-containing proteins as queries (Bce1019, PmgM, T1p21, KIAA1018, HP1472 and Plu1493) (Iyer et al ., 2006), and recently reported bona fide or putative T6SS effectors that also belong to the PD-(D/E)xK superfamily: TseT (Burkinshaw et al ., 2018); PoNe (Jana et al ., 2019); IdrD-CT (Sirias et al , 2020); TseTBg (Yadav et al ., 2021); Aave_0499 (Pei et al ., 2021); and TseV PA (Wang et al , 2021). A total of 39159 sequences were collected.…”

“…The fact that only one immunity protein (TsiV2.1) can neutralize the effector (TseV2) makes us wonder about the role of the additional immunity protein gene - and why such genomic context is conserved. One possibility is that the extra immunity protein could regulate the effector at the transcriptional level as has been reported for the immunity protein TsiTBg known to regulate a different PD-(D/E)xK effector (TseTBg) (Yadav et al ., 2021).…”

“…Antibacterial effectors with nuclease activity are among the most potent weapons used by an attacker to intoxicate target cells. Several T6SS effectors with nuclease activity have been reported including, Dickeya dadantii RhsA-CT and RhsB-CT (Koskiniemi et al , 2013); Agrobacterium tumefaciens Tde1 and Tde2 (Ma et al , 2014); Pseudomonas aeruginosa PA0099 (Hachani et al ., 2014), TseT (Burkinshaw et al , 2018) and Tse7 (Pissaridou, 2018); Serratia marcescens Rhs2 (Alcoforado Diniz & Coulthurst, 2015); Escherichia coli Hcp-ET1, -ET3 and -ET4 (Ma et al , 2017a), and Rhs-CT3, -CT4, -CT5, -CT6, -CT7 and -CT8 (Ma et al , 2017b); Acinetobacter baumannii Rhs2-CT (Fitzsimons et al , 2018); Vibrio parahaemolyticus PoNe (Jana et al , 2019); Aeromonas dhakensis TseI (Pei et al , 2021); and Burkholderia gladioli TseTBg (Yadav et al , 2021).…”

“…The majority of the nuclease domains mentioned above have been previously predicted by a seminal in silico study using comparative genomics (Zhang et al , 2012). Among those characterized are Ntox15 (PF15604) (Ma et al, 2014); Ntox30 (PF15532), Ntox34 (PF15606) and Ntox44 (PF15607) (Ma et al ., 2017a); Tox-REase-1 (Jana et al, 2019); Tox-REase-3 (PF15647) (Ma et al ., 2017a); Tox-REase-5 (PF15648) (Burkinshaw et al ., 2018; Yadav et al ., 2021); Tox-GHH2 (PF15635) (Hachani et al ., 2014; Pissaridou, 2018); HNH (PF01844) (Koskiniemi et al ., 2013; Alcoforado Diniz & Coulthurst, 2015; Ma et al ., 2017b); Tox-JAB-2 (Ma et al ., 2017a); AHH (PF14412) (Ma et al ., 2017a; Fitzsimons et al ., 2018); and Tox-HNH-EHHH (PF15657) (Pei et al ., 2021).…”