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Cited by 5Bacterial Genetics and BiotechnologyPlant Pathogenic Bacteria StudiesCRISPR and Genetic Engineering

The DOI matches its Crossref record; 45 of the 45 references it cites were found in a registry or an index.

Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors

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Santos, Mary Nia M., Pintor, Katherine L., Hsieh, Pei-Yu, Cheung, Yee-Wai, Sung, Li-Kang, Shih, Yu-Ling, Lai, Erh-Min·2024·Frontiers in Microbiology
DOI 10.3389/fmicb.2024.1351590PMC PMC10902643

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Abstract

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d ), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa- associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc- associated gene 2 ( v2c ) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro . Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii .

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Figures

Figure 1 V2c is a PAAR-linked specialized effector containing a C-terminal Tox-SHH motif. (A) A schematic diagram of V2c protein showing the N-terminal DUF4150 PAAR region and the catalytic region (amino acids 380–409), which covers the Tox-SHH motif. (B) Partial sequence alignment of orthologs of V2c from BLASTP. The strain name and accession number are indicated on the left. SHH motif is highlighted in yellow, with H383 (indicated in red) as the metal ion coordinating residue, and H384 and H408 (indicated in green) of the HNH motif for catalysis and metal ion binding, respectively, are targeted for mutagenesis. The star corresponds to the specific domains shown in panel (C): *DUF4150/PAAR-like, **DUF4150/PAAR-like + FIX-like, ***FIX-like superfamily, ****PAAR + Rhs core domain. (C) Domain architectures of V2c orthologs and Tox-SHH-containing proteins found in this study. Full-length sequences were analyzed, and the domains were from the Pfam database with E value threshold set at 10−5. (D) A cartoon model of V2c (residues 380–409). The structure of V2c predicted by AlphaFold indicates V2c that consists of a His-Me finger domain with two antiparallel β-strands (yellow) connected with an Ω loop (light blue), a histidine (H384) at the C-terminus of the first β-strand and is followed by an α-helix (magenta). The confidence score of this region is higher than 90, indicating high structure accuracy.
Figure 1 V2c is a PAAR-linked specialized effector containing a C-terminal Tox-SHH motif. (A) A schematic diagram of V2c protein showing the N-terminal DUF4150 PAAR region and the catalytic region (amino acids 380–409), which covers the Tox-SHH motif. (B) Partial sequence alignment of orthologs of V2c from BLASTP. The strain name and accession number are indicated on the left. SHH motif is highlighted in yellow, with H383 (indicated in red) as the metal ion coordinating residue, and H384 and H408 (indicated in green) of the HNH motif for catalysis and metal ion binding, respectively, are targeted for mutagenesis. The star corresponds to the specific domains shown in panel (C): *DUF4150/PAAR-like, **DUF4150/PAAR-like + FIX-like, ***FIX-like superfamily, ****PAAR + Rhs core domain. (C) Domain architectures of V2c orthologs and Tox-SHH-containing proteins found in this study. Full-length sequences were analyzed, and the domains were from the Pfam database with E value threshold set at 10−5. (D) A cartoon model of V2c (residues 380–409). The structure of V2c predicted by AlphaFold indicates V2c that consists of a His-Me finger domain with two antiparallel β-strands (yellow) connected with an Ω loop (light blue), a histidine (H384) at the C-terminus of the first β-strand and is followed by an α-helix (magenta). The confidence score of this region is higher than 90, indicating high structure accuracy.CC BY · PubMed Central
Figure 2 V2c exhibits growth inhibition and DNase activity in vivo and in vitro. (A) Growth inhibition analysis of expression of v2c and its variants with or without v3c in E. coli DH10B. E. coli cultures were induced with 1 mM IPTG at 0 h for v3c expressed from the pTrc200 plasmid, followed by L-arabinose (Ara) induction at 1 h to induce v2c from the pJN105 plasmid. Cell growth was recorded every hour at OD600. Strain expressing empty vectors (vectors) were used as a control. (B) Number of colony-forming units (log10 CFU/mL) quantified after growth inhibition assay at 8 h. Data in panel (A,B) represent mean ± SEM of four independent experiments (n = 4), each averaged with three technical repeats. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test. (C) Plasmid degradation assay performed by E. coli DH10B cells harboring pTrc200 and pJN105 plasmids (vector) or the derivatives induced with (+) or without (−) Ara for 2 h. Plasmid DNA was extracted, and the degradation pattern was observed in agarose gel. (D) In vitro DNase activity assay performed by using pTrc200HA plasmid DNA as DNA substrate and incubated with purified V2c or its variants. Nuclease-free water (ddH2O), protein elution buffer (elution buffer), and eluate from empty vector expression (pET28a) served as negative controls, while Benzonase nuclease served as a positive control. The digestion of the DNA substrate was determined by agarose gel electrophoresis. The reaction samples of V2c or its variants were detected by Western blotting using an anti-V2c antibody (α-V2c). The results of panel (C,D) are representative of three independent experiments.
Figure 2 V2c exhibits growth inhibition and DNase activity in vivo and in vitro. (A) Growth inhibition analysis of expression of v2c and its variants with or without v3c in E. coli DH10B. E. coli cultures were induced with 1 mM IPTG at 0 h for v3c expressed from the pTrc200 plasmid, followed by L-arabinose (Ara) induction at 1 h to induce v2c from the pJN105 plasmid. Cell growth was recorded every hour at OD600. Strain expressing empty vectors (vectors) were used as a control. (B) Number of colony-forming units (log10 CFU/mL) quantified after growth inhibition assay at 8 h. Data in panel (A,B) represent mean ± SEM of four independent experiments (n = 4), each averaged with three technical repeats. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test. (C) Plasmid degradation assay performed by E. coli DH10B cells harboring pTrc200 and pJN105 plasmids (vector) or the derivatives induced with (+) or without (−) Ara for 2 h. Plasmid DNA was extracted, and the degradation pattern was observed in agarose gel. (D) In vitro DNase activity assay performed by using pTrc200HA plasmid DNA as DNA substrate and incubated with purified V2c or its variants. Nuclease-free water (ddH2O), protein elution buffer (elution buffer), and eluate from empty vector expression (pET28a) served as negative controls, while Benzonase nuclease served as a positive control. The digestion of the DNA substrate was determined by agarose gel electrophoresis. The reaction samples of V2c or its variants were detected by Western blotting using an anti-V2c antibody (α-V2c). The results of panel (C,D) are representative of three independent experiments.CC BY · PubMed Central
Figure 3 V2c Tox-SHH DNase exhibits the cell elongation phenotype. (A) Images of E. coli DH10B cells harboring vector(s) and the derivatives expressing v2c or its variants in the presence or absence of its cognate immunity gene v3c with (+Ara) and without (-Ara) Arabinose induction stained with FM 4–64 (red) and Hoechst stain (blue). The micrographs from left to right are FM 4–64, Hoechst, phase contrast, and a merged image of FM 4–64 and Hoechst stains. Scale bar, 2 μm. Representative images of two independent experiments are shown. (B) Normalized Hoechst intensity (intensity/unit area of cells) and (C) cell length (μm) as measured from the indicated number of cells (n) per treatment. The graph shows a combined count from three frames of a representative result of two independent experiments. The red line in panel (C) shows the median with an interquartile range. Statistics were performed with the mean ± SEM of three frames. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.
Figure 3 V2c Tox-SHH DNase exhibits the cell elongation phenotype. (A) Images of E. coli DH10B cells harboring vector(s) and the derivatives expressing v2c or its variants in the presence or absence of its cognate immunity gene v3c with (+Ara) and without (-Ara) Arabinose induction stained with FM 4–64 (red) and Hoechst stain (blue). The micrographs from left to right are FM 4–64, Hoechst, phase contrast, and a merged image of FM 4–64 and Hoechst stains. Scale bar, 2 μm. Representative images of two independent experiments are shown. (B) Normalized Hoechst intensity (intensity/unit area of cells) and (C) cell length (μm) as measured from the indicated number of cells (n) per treatment. The graph shows a combined count from three frames of a representative result of two independent experiments. The red line in panel (C) shows the median with an interquartile range. Statistics were performed with the mean ± SEM of three frames. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.CC BY · PubMed Central
Figure 4 V2a Tox-AHH DNase exhibits the cell elongation phenotype. (A) A cartoon model of V2a (residues 380–470). The structure of V2a predicted by AlphaFold indicates V2a consisting of a His-Me finger domain with its signature antiparallel β-strands (yellow) connected with an Ω loop (light blue) with a histidine (H386, orange) at the C-terminus of the first β-strand. Two α-helices (pink) with histidine residues (H430, H456 magenta) followed the β-strands. N412 is found in the loop between the β-strands, and H430 is in the first α-helix (E429–G430). The predicted local-distance difference tests (pLDDTs) of the His-Me domains of V2a range from confident to very high; most of the pLDDTs of the residues are >90. (B) Morphological analysis of E. coli DH10B cells harboring vector(s) or its derivatives expressing v2a, catalytic site (H385A) mutant without (-Ara) or with (+Ara) arabinose induction. Cells were stained with FM 4–64 (red) and Hoechst stain (blue). The micrographs from left to right are FM 4–64, Hoechst, phase contrast, and a merged image of the two fluorescent images. Scale bar, 2 μm. (C) Cell length (μm) and (D) Hoechst intensity in different treatments as determined from a combined count of three random frames of a representative result; the number of cells (n) per sample is indicated. The graph shows a combined count from three frames of a representative result of two independent experiments. The red line shows the median with an interquartile range. Statistics were performed with the mean ± SEM of three frames. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.
Figure 4 V2a Tox-AHH DNase exhibits the cell elongation phenotype. (A) A cartoon model of V2a (residues 380–470). The structure of V2a predicted by AlphaFold indicates V2a consisting of a His-Me finger domain with its signature antiparallel β-strands (yellow) connected with an Ω loop (light blue) with a histidine (H386, orange) at the C-terminus of the first β-strand. Two α-helices (pink) with histidine residues (H430, H456 magenta) followed the β-strands. N412 is found in the loop between the β-strands, and H430 is in the first α-helix (E429–G430). The predicted local-distance difference tests (pLDDTs) of the His-Me domains of V2a range from confident to very high; most of the pLDDTs of the residues are >90. (B) Morphological analysis of E. coli DH10B cells harboring vector(s) or its derivatives expressing v2a, catalytic site (H385A) mutant without (-Ara) or with (+Ara) arabinose induction. Cells were stained with FM 4–64 (red) and Hoechst stain (blue). The micrographs from left to right are FM 4–64, Hoechst, phase contrast, and a merged image of the two fluorescent images. Scale bar, 2 μm. (C) Cell length (μm) and (D) Hoechst intensity in different treatments as determined from a combined count of three random frames of a representative result; the number of cells (n) per sample is indicated. The graph shows a combined count from three frames of a representative result of two independent experiments. The red line shows the median with an interquartile range. Statistics were performed with the mean ± SEM of three frames. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.CC BY · PubMed Central
Figure 5 V2a and V2c exhibit different toxicity against A. tumefaciens and D. dadantii. (A) Intra-species competition between A. tumefaciens 1D1609 attacker and prey C58. (B–D) Inter-species competition between A. tumefaciens 1D1609 attacker and prey D. dadantii 3937 ∆imp. The plasmid pRL-GFP(S65T) plasmid (GmR) was transformed into prey cells, which were mixed at a 1:10 ratio with each of the attacker strains, 1D1609 WT, or various EI mutant strains as indicated. After co-culture on AK agar plates, cells were collected, serially diluted, and plated on the LB agar plate containing Gm for CFU counting of surviving prey cells (B). Data are mean ± SEM (n = 6 biological repeats from three independent experiments). For imaging, co-cultured D. dadantii Δimp-GFP(S65T) and 1D1609 EI pair mutants were concentrated and spotted on a 2% agarose pad for fluorescence micrographs (C). D. dadantii Δimp-GFP(S65T) indicated as Dd only is included as a control. The inset in phase contrast shows a magnified image of cells in the red box. White arrows point to D. dadantii cells. Scale bar, 2 μm. (D) Cell length after competition between A. tumefaciens 1D1609 EI mutants and D. dadantii 3937 ∆imp-GFP(S65T). Cell length of GFP fluorescent D. dadantii cells was measured using the Fiji analyze particles tool. Cell length (μm) in different treatments as determined from a combined count of at least 10 random frames of two independent experiments. The red line shows the median with an interquartile range. Statistics were performed with the mean ± SEM of total counts. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.
Figure 5 V2a and V2c exhibit different toxicity against A. tumefaciens and D. dadantii. (A) Intra-species competition between A. tumefaciens 1D1609 attacker and prey C58. (B–D) Inter-species competition between A. tumefaciens 1D1609 attacker and prey D. dadantii 3937 ∆imp. The plasmid pRL-GFP(S65T) plasmid (GmR) was transformed into prey cells, which were mixed at a 1:10 ratio with each of the attacker strains, 1D1609 WT, or various EI mutant strains as indicated. After co-culture on AK agar plates, cells were collected, serially diluted, and plated on the LB agar plate containing Gm for CFU counting of surviving prey cells (B). Data are mean ± SEM (n = 6 biological repeats from three independent experiments). For imaging, co-cultured D. dadantii Δimp-GFP(S65T) and 1D1609 EI pair mutants were concentrated and spotted on a 2% agarose pad for fluorescence micrographs (C). D. dadantii Δimp-GFP(S65T) indicated as Dd only is included as a control. The inset in phase contrast shows a magnified image of cells in the red box. White arrows point to D. dadantii cells. Scale bar, 2 μm. (D) Cell length after competition between A. tumefaciens 1D1609 EI mutants and D. dadantii 3937 ∆imp-GFP(S65T). Cell length of GFP fluorescent D. dadantii cells was measured using the Fiji analyze particles tool. Cell length (μm) in different treatments as determined from a combined count of at least 10 random frames of two independent experiments. The red line shows the median with an interquartile range. Statistics were performed with the mean ± SEM of total counts. Different letters above the bar indicate statistically different groups of strains (p < 0.05) determined by Tukey’s HSD test.CC BY · PubMed Central

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Authors
Santos, M. N. M., Pintor, K. L., Hsieh, P.-Y., Cheung, Y.-W., Sung, L.-K., Shih, Y.-L., & Lai, E.-M.
Affiliations
  • National Chung Hsing University (TW)
  • Institute of Plant and Microbial Biology, Academia Sinica (TW)
  • Institute of Biological Chemistry, Academia Sinica (TW)
Corresponding author
Lai, Erh-Min
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2024-02-14
Volume
15
Pages
1351590-1351590
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2024.1351590
PMC
PMC10902643
OpenAlex
W4391842643
Cited by
5
References
45
Citations by year
2026: 3 · 2025: 2
FWCI
1.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Academia Sinica (AS‐IA‐107‐L01)
  • Ministry of Science and Technology, Taiwan (MOST110-2311-B001-011)
  • National Science and Technology Council
  • Directorate for Biological Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Pathogenic Bacteria Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
Agrobacterium tumefaciens, type VI secretion system, HNH nuclease, V2C, immunity proteins, DNase activity, bacterial competition, antibacterial activity, Dickeya dadantii, plasmid DNA degradation, cell elongation

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Santos, M. N. M., Pintor, K. L., Hsieh, P.-Y., Cheung, Y.-W., Sung, L.-K., Shih, Y.-L., & Lai, E.-M. (2024). Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2024.1351590

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