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 non-canonical helix-turn-helix motifs and demonstrate transcriptional repression activity, similar to the antitoxins of type II toxin-antitoxin (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.

Keywords: DNA adenine methylase; LysR proteins; effector neutralization; protein-DNA interaction; restriction modification system.

Figure 1. Burkholderia gladioli strain NGJ1 utilizes two different type VI secretion system (T6SS) apparatus for antibacterial activity

1. A

   Antibacterial activity of NGJ1, T6SS‐1⁻, and T6SS‐2⁻ strains against target bacteria.

2. B

   Schematic representation of two different T6SS apparatus‐encoding gene clusters (named as T6SS‐1 and T6SS‐2) of NGJ1. The locus id (as per Burkholderia genome database) of upstream and downstream genes of the T6SS locus is provided. The gene disrupted in the respective T6SS mutants is marked by triangle (△).

3. C, D

   Immunoblots showing secretion profile of Hcp‐1 (associated with T6SS‐1) and Hcp‐2 (associated with T6SS‐2) proteins, using protein‐specific peptide antibody in different NGJ1 strains. The T6SS‐1⁻ mutant was defective in Hcp‐1 secretion (due to polar effect of plasmid integration, synthesis was also prevented) but proficient in Hcp‐2 secretion. On the other hand, the T6SS‐2⁻ mutant was defective in secretion of Hcp‐2 (although the protein was synthesized) but proficient in secretion of Hcp‐1.

Data information: In (A), graphs show mean values ± SD of three biological replicates. Asterisk * indicates significant difference at P < 0.05 and “ns” indicates non‐significant difference (estimated using one‐way ANOVA). In (C, D), P: total protein from bacterial pellet and S: total protein from cell‐free supernatant. Similar results were obtained in at least three independent experiments. Source data are available online for this figure.

Figure 2. The TseTBg effectors of NGJ1 exhibit potent antibacterial activity

1. Growth curve of recombinant BL21 (DE3) cells upon IPTG‐mediated induction of proteins, represented as colored line graphs.

2. Bacterial two‐hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons. Interaction between T25‐zip and T18‐zip was used as positive control while pKNT25 and pUT18C (empty vectors) were used as negative control. Appearance of blue color suggests positive interaction while the absence of color suggests lack of interaction.

3. Yeast two‐hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR (PAAR^TseTBg) proteins encoded in their operon. Interaction between P53 and SV40 large T‐antigen (T) proteins was used as positive control. The pGBKT7 and pGADT7 (empty vectors) were used as negative control.

Data information: In (A), graphs represent mean ± SD of three biological replicates. Asterisk ** indicates significant difference at P < 0.01, and “ns” indicates non‐significant difference (estimated using one‐way ANOVA) at the given time point. In (B, C), similar results were obtained in at least three independent experiments.

Figure EV1. TseTBg effector proteins elicit nuclear degradation/ cell death in Escherichia coli

Fluorescence microscopic images of DAPI (nuclear staining dye)‐stained E. coli cells that express TseTBg, TseTBg + TsiTBg, TseTBg:TsiTBg, and TseTBg:TsiTBg^ΔImm52 proteins upon IPTG (1 mM) induction. Lack of DAPI staining suggested nuclear degradation/ cell death while proper staining reflected intact nuclei. DIC: differential interference contrast microscopy image. Data information: scale bar = 10µm. Similar results were obtained in at least three independent experiments. Source data are available online for this figure.

Figure EV2. RT–PCR analysis reflects genes of TseTBg operon of NGJ1 being expressed as single transcriptional unit

1. Strategy used for validating the expression of different genes of TseTBg operon as single transcriptional unit.

2. PCR amplification of PAAR^TseTBg1 and TseTBg1 genes from cDNA synthesized using TsiTBg1‐specific primer.

3. PCR amplification of PAAR^TseTBg2 and TseTBg2 genes from cDNA synthesized using TsiTBg2‐specific primer.

Data information: In (B, C), lanes 1 and 3 represent gene expression during NGJ1 interaction with Escherichia coli, while lanes 2 and 4 represent gene expression during NGJ1 interaction with Agrobacterium tumefaciens. Similar results were obtained in at least three independent experiments. M: DNA size marker. Source data are available online for this figure.

Figure EV3. NGJ1 utilizes TseTBg2 effector to kill prey bacteria in a T6SS dependent manner

1. Western blot analysis reflecting the T6SS‐2‐dependent secretion of TseTBg2 protein in NGJ1. The total proteins from cell‐free supernatant (S) as well as pellet (P) of different strains were immunoblotted using TseTBg2‐specific peptide antibody.

2. Antibacterial activity of the TseTBg2 mutants (NGJ203 and NGJ204; two independent mutants) and wild‐type NGJ1 bacteria against Agrobacterium tumefaciens and Escherichia coli.

Data information: In (A), M: Protein size marker. Similar results were obtained in at least three independent experiments. In (B), asterisks ** and * indicate significant difference at P < 0.01 and P < 0.05, respectively, while “ns” indicates non‐significant difference (estimated using paired t‐test). Graphs show mean values ± SD of three biological replicates. Source data are available online for this figure.

Figure 3. The TseTBg effector proteins of NGJ1 harbor potent DNase and RNase activity

1. A, B

   Effect of purified TseTBg (TseTBg1/TseTBg2) and one of the variants (TseTBg2^D116A,K129A) proteins on degradation of bacterial genomic DNA.

2. C, D

   RNase activity of TseTBg effector proteins, in the presence or absence of cognate TsiTBg immunity proteins.

3. E

   Effect of TseTBg1, TseTBg2, TseTBg2^D116A,K129A proteins on degradation of pHM1, a broad host‐range plasmid isolated from Escherichia coli and NGJ1.

4. F

   Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1, reintroduced into E. coli from NGJ1.

5. G

   Effect of ectopic expression of DNA adenine methylase (Dam) protein on TseTBg proteins mediated degradation of plasmid DNA.

Data information: In (A–G), similar results were obtained in at least three independent experiments. M; DNA marker (1 kb); −; negative control.

Figure EV4. Effect of ectopic expression of TseTBg2 variant protein (TseTBg2^D116A,K129A) on the growth of recombinant Escherichia coli cells

1. Growth of recombinant BL21 (DE3) cells with or without induction of TseTBg2 and its variant (TseTBg2^D116A,K129A, DNase⁻RNase⁺).

2. Growth curve of recombinant BL21 (DE3) cells with or without IPTG‐mediated induction of TseTBg2 or TseTBg2^D116A,K129A proteins.

Data information: In (A, B), VC, vector control. In (A), similar results were obtained in at least three independent experiments. +/− indicates presence/absence of 1 mM IPTG. In (B), graph represents mean ± SD of three biological replicates. Asterisk * indicates significant difference at P < 0.05 and “ns” indicates non‐significant difference (estimated using one‐way ANOVA) at the given time point.

Figure 4. The TsiTBg immunity proteins of NGJ1 demonstrate transcriptional repressor activity

1. A, B

   Schematics reflecting the presence of a non‐canonical HTH domain and an Imm52 domain in TsiTBg1 and TsiTBg2 proteins.

2. C, D

   Representation of predicted external (pExt^TseTBg1/pExt^TseTBg2) and internal (pInt^TseTBg1/pInt^TseTBg2) promoter regions in TseTBg1 and TseTBg2 operons. Strategy for obtaining promoter:Gus reporter constructs in pBI101 is also provided.

3. E, F

   Graph showing the effect of native TsiTBg and HTH or Imm52 domain‐deleted variant of TsiTBg proteins on Gus reporter gene expression (quantified using 4‐methylumbelliferyl ß‐d‐glucuronide, MUG, as substrate) under the external (pExt^TseTBg1/pExt^TseTBg2) and internal (pInt^TseTBg1/pInt^TseTBg2) promoters in Escherichia coli.

Data information: In (C, D), TSS represents putative transcription start site. In (E, F), VC: vector control; ‐: absence of TsiTBg protein. Graphs show mean values ± SD of three independent biological replicates. Asterisk * indicates significant difference at P < 0.05 and “ns” indicates non‐significant difference (estimated using paired t‐test).

Figure 5. Electromobility shift assay (EMSA) reflecting promoter DNA‐binding ability of TseTBg1 protein

1. Interaction of external (pExt^TseTBg1) and internal (pInt^TseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein.

2. Interaction of pExt^TseTBg1 and pInt^TseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain‐deleted variant of TseTBg1 protein (TsiTBg1^ΔHTH).

3. Interaction of pExt^TseTBg2 and pInt^TseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein.

4. Interaction of control (random) DNA with the TsiTBg1 protein.

Data information: In (A–D), lower panel represents visualization of the protein on the gel. Equimolar concentration of promoter DNA probe (5 pmol) was titrated with increasing concentration of protein (2–80 pmol). Similar results were obtained in at least three independent experiments. Source data are available online for this figure.

Figure 6. Phylogenetic analysis indicates homologs of TseTBg proteins to be encoded as TA or T6SS effectors in different bacteria

Phylogenetic tree of different TseTBg homologs (n = 121) constructed by RAxML tool using Maximum Likelihood algorithm. The tree is drawn to scale wherein branch lengths indicate number of substitutions per site. For each of the TseTBg homologs, gene neighborhood conservation (depicted by color‐coded box) by webFlaGs tool is also presented. The full output of webFlaGs analysis is provided in Dataset EV2. Bacterial classification is denoted in the outermost bar line. Data information: The TseTBg homologs when encoded together with TsiTBg homologs are considered as TA whereas, when TseTBg and TsiTBg homologs are encoded along with certain T6SS related (PAAR, DUF4123, and hypothetical protein [H.P]) proteins, they are considered as T6SS effectors. *VgrG is encoded (instead of PAAR) as upstream ORF in these operons.

Figure 7. Potential conversion of TseTBg homologs of TA system into T6SS effectors in Burkholderia pseudomallei

1. The genomic loci containing TseTBg (toxin) and TsiTBg (antitoxin) homologs in different B. pseudomallei strains.

2. In B. pseudomallei 3000015237 isolate MX2014, two IS3 family transposable elements that carry two of the T6SS‐related genes (PAAR and DUF4123) are present upstream to the TA genes. However, in B. pseudomallei Nau24B‐3 and vgh16R strains, the IS3 element which flank the TA and T6SS‐related genes have been excised and a new ORF encoding hypothetical protein (H.P) has evolved at its place.

3. The excision of IS3 family transposase has created operonic fusion of TA and T6SS related genes. Moreover, a new ORF of 474 bp that encode a H.P has evolved at the locus, through adoption of 3′ end of the DUF4123 and 5′ end of the toxin gene.

Data information: In (A–C), H.P, hypothetical protein, PRT, Phosphoribosyl transferase, DPP, Decaprenyl diphosphatase, MT, MerR transcriptional regulator, AT, Aspartate aminotransferase. In (A, B), genomic location of each of the locus on the contig/chromosome is provided. In (B), asterisks indicate presence of multiple IS3 family transposase.

Figure EV5. Representation of genomic loci harboring TseTBg homologs as TA or T6SS effectors in different Burkholderia pseudomallei strains

The TseTBg homologs (toxins) and TsiTBg homologs (antitoxins) were either encoded alone (considered as TA) or encoded along with certain T6SS‐related genes (PAAR, DUF4123, and H.P; considered as T6SS effector). The conservation of flanking genes suggests that TA or T6SS effector type TseTBg homologs are encoded at the same genomic locus in different B. pseudomallei strains. Data information: H.P—hypothetical protein, PRT—Phosphoribosyltransferase. Genomic location of each of the locus on the contig/chromosome is provided.