scholarly journals Species boundaries in the Agrobacterium tumefaciens complex and multi-level modular evolution of their antibacterial type VI secretion system and tumor-inducing plasmids

2021 ◽  
Author(s):  
Lin Chou ◽  
Yu-Chen Lin ◽  
Mindia Haryono ◽  
Mary Nia M. Santos ◽  
Shu-Ting Cho ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Pathogenic Bacteria ◽  
Secretion System ◽  
Species Boundaries ◽  
Type Vi Secretion System ◽  
Type Iv ◽  
Type Vi Secretion ◽  
Modular Evolution ◽  
Species Complexes ◽  
Genome Phylogeny

Many pathogenic bacteria are recognized as species complexes and uncertainties regarding the organization of their genetic diversity are challenges for research efforts. Within Agrobacterium tumefaciens, multiple genomospecies have been identified; however, the exact species boundaries are unclear, which causes chaos in nomenclature and hampers communication. In this work, we conducted targeted genome sequencing to achieve a comprehensive and balanced taxon sampling within this complex. Our results from genome-wide sequence identity, core genome phylogeny, and gene content not only supported that those recognized genomospecies are distinct biological entities but also identified novel genomospecies. Based on the fully resolved phylogeny, we further investigated the evolution of genes critical in Agrobacterium fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition, multiple losses and one horizontal gene transfer (HGT) event were inferred. For the tumor-inducing plasmids (pTi) and the pTi-encoded type IV secretion system (T4SS) that determine Agrobacterium phytopathogenicity, the evolution of these accessory replicons was decoupled from the chromosomes, thus contributing to another level of complexity. Intriguingly, for both T6SS and T4SS, genes that encode the structural components are highly conserved, whereas extensive diversity exists at multiple levels (i.e., between-species, within-species, intra-genome, and intra-gene) for genes that encode effectors and associated proteins. These findings suggest that opposite modes of selection may act on components conferring different functions within a system. In conclusion, this work provides insights into the genomic diversification of these bacteria and sheds light on the modularity of their molecular evolution.

scholarly journals Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System in Agrobacterium tumefaciens

2008 ◽  
Vol 190 (8) ◽  
pp. 2841-2850 ◽  
Author(s):  
Hung-Yi Wu ◽  
Pei-Che Chung ◽  
Hsiao-Wei Shih ◽  
Sy-Ray Wen ◽  
Erh-Min Lai
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pathogenic Bacteria ◽  
Secretion System ◽  
Secretory Proteins ◽  
Consistent Difference ◽  
Type Vi Secretion System ◽  
Type Vi Secretion

ABSTRACT Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.

scholarly journals Redundancy and specificity of type VI secretion vgrG loci in antibacterial activity of Agrobacterium tumefaciens 1D1609 strain

2019 ◽  
Author(s):  
Mary Nia Santos ◽  
Shu-Ting Cho ◽  
Chih-Feng Wu ◽  
Chun-Ju Chang ◽  
Chih-Horng Kuo ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Agrobacterium Tumefaciens ◽  
Biological Significance ◽  
Gene Clusters ◽  
Functional Redundancy ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Multiple Copies ◽  
Close Relatives

AbstractType VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found ∼70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e. vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609’s antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58’s VgrG2 shares 99% amino acid sequence identity with 1D1609’s VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition.Author’s SummarySelective pressure drives bacteria to develop adaptive strategies, which include competitive and cooperative behaviors. Type VI secretion system (T6SS) is one powerful antibacterial and anti-host nanoweapon employed by many Gram-negative bacteria for growth advantages or pathogenesis. A T6SS-harboring bacterium can encode one to multiple VgrG proteins for delivery of cognate effector(s) but the prevalence and biological significance of having sequence redundant vgrGs have not been comprehensively explored. In this study, we investigated the extensiveness of having multicopy vgrG genes for effector delivery among diverse Proteobacteria with T6SS. Moreover, a plant pathogenic bacterium Agrobacterium tumefaciens strain 1D1609 with highest vgrG redundancy was selected for detailed characterization of the roles of multiple VgrGs in T6SS secretion and antibacterial activity. We revealed that the majority of Proteobacterial genomes harbor multiple copies of vgrG and the expansion of vgrG gene clusters contributed to effector diversity and functional redundancy. Furthermore, the near identical VgrG proteins between 1D1609 and its sibling strain C58 can be exchanged for effector delivery in killing another competing bacterium. Such strategy in using exchangeable effector carriers injected from its isogenic sibling or close relatives during T6SS attacks may be a beneficial strategy for agrobacteria to compete in their ecological niche.

scholarly journals Identification of Escherichia coli ClpAP in regulating susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens

2019 ◽  
Author(s):  
Hsiao-Han Lin (林筱涵) ◽  
Manda Yu (余文廸) ◽  
Manoj Kumar Sriramoju ◽  
Shang-Te Danny Hsu (徐尚德) ◽  
Chi-Te Liu (劉啟德) ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Competition Assay ◽  
E Coli ◽  
Type Vi Secretion ◽  
In Trans

AbstractType VI secretion system (T6SS) is an effector delivery system used by gram-negative bacteria to kill other bacteria or eukaryotic host to gain fitness. In Agrobacterium tumefaciens, T6SS has been shown to kill other bacteria such as Escherichia coli. Interestingly, the A. tumefaciens T6SS killing efficiency differs when using different E. coli strains as recipient cells. Thus, we hypothesize that a successful T6SS killing not only relies on attacker T6SS activity but also depends on recipient factors. A high-throughput interbacterial competition assay was employed to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. From the 3909 E. coli Keio mutants screened, 16 candidate mutants were filtered out. One strain, ΔclpP::Kan, showed ten times more resistant to T6SS-mediating killing but restored its susceptibility when complemented with clpP in trans. ClpP is a universal and highly conserved protease that exists in both prokaryotes and eukaryotic organelles. In E. coli, ClpP uses either ClpA or ClpX as an adaptor for substrate specificity. Therefore, the susceptibility of the ΔclpA::Kan and ΔclpX::Kan was also tested. The T6SS attack susceptibility of ΔclpA::Kan is at the same level as ΔclpP::Kan, while ΔclpX::Kan showed no difference as compared to that of wild-type E. coli BW25113. The data also suggest that ClpA-ClpP interaction, rather than its protease activity, is responsible for enhancing susceptibility to T6SS killing. This study highlights the importance of recipient factors in determining the outcome of T6SS killing.

scholarly journals The structure of VgrG1 fromPseudomonas aeruginosa, the needle tip of the bacterial type VI secretion system

2016 ◽  
Vol 72 (1) ◽  
pp. 22-33 ◽  
Author(s):  
Mercedes Spínola-Amilibia ◽  
Irene Davó-Siguero ◽  
Federico M. Ruiz ◽  
Elena Santillana ◽  
Francisco Javier Medrano ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Neck Region ◽  
Structural Features ◽  
Secretion System ◽  
Host Cells ◽  
Type Vi Secretion System ◽  
Competitive Environments ◽  
Type Vi Secretion ◽  
Target Membrane ◽  
T4 Phage

The type VI secretion system (T6SS) is a mechanism that is commonly used by pathogenic bacteria to infect host cells and for survival in competitive environments. This system assembles on a core baseplate and elongates like a phage puncturing device; it is thought to penetrate the target membrane and deliver effectors into the host or competing bacteria. Valine–glycine repeat protein G1 (VgrG1) forms the spike at the tip of the elongating tube formed by haemolysin co-regulated protein 1 (Hcp1); it is structurally similar to the T4 phage (gp27)3–(gp5)3puncturing complex. Here, the crystal structure of full-length VgrG1 fromPseudomonas aeruginosais reported at a resolution of 2.0 Å, which through a trimeric arrangement generates a needle-like shape composed of two main parts, the head and the spike, connectedviaa small neck region. The structure reveals several remarkable structural features pointing to the possible roles of the two main segments of VgrG1: the head as a scaffold cargo domain and the β-roll spike with implications in the cell-membrane puncturing process and as a carrier of cognate toxins.

scholarly journals Modular evolution of secretion systems and virulence plasmids in a bacterial species complex

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Lin Chou ◽  
Yu-Chen Lin ◽  
Mindia Haryono ◽  
Mary Nia M. Santos ◽  
Shu-Ting Cho ◽  
...  
Keyword(s):  
Species Complex ◽  
Bacterial Species ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Bacterial Taxonomy ◽  
Plant Host ◽  
Modular Evolution ◽  
Species Complexes ◽  
Genes Encoding

Abstract Background Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. Results We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. Conclusions We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

scholarly journals Crystal structure of the type VI immunity protein Tdi1 (Atu4351) from Agrobacterium tumefaciens

2019 ◽  
Vol 75 (3) ◽  
pp. 153-158
Author(s):  
Lingling Shi ◽  
Zengqiang Gao ◽  
Tianyi Zhang ◽  
Heng Zhang ◽  
Yuhui Dong
Keyword(s):  
Crystal Structure ◽  
Agrobacterium Tumefaciens ◽  
Active Site ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Type Vi Secretion System ◽  
Dispersion Method ◽  
Interspecies Competition ◽  
Immunity Protein ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a novel multiprotein needle-like apparatus that is distributed widely in Gram-negative bacteria. Bacteria harboring T6SSs inject various effectors into both eukaryotic and prokaryotic cells for interspecies competition or virulence-related processes. The toxicities of the effectors can be neutralized by their cognate immunity proteins. Tde1 (Atu4350)–Tdi1 (Atu4351) has recently been characterized as a T6SS effector–immunity pair in the soil bacterium Agrobacterium tumefaciens and the neutralization mechanism remains unknown. Here, the crystal structure of the immunity protein Tdi1 was determined at 2.40 Å resolution by the single-wavelength anomalous dispersion method. Structural analysis suggested that it is composed of a GAD-like domain and an inserted DUF1851 domain, and both domains show low structural similarities to known structures. There is a positive groove mainly located in the GAD-like domain that may be associated with nucleotide binding. The structure provides a basis for further study of the positive groove as a potential active site.

scholarly journals An IcmF Family Protein, ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL, and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (13) ◽  
pp. 4316-4329 ◽  
Author(s):  
Lay-Sun Ma ◽  
Jer-Sheng Lin ◽  
Erh-Min Lai
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Membrane Protein ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Type Vi Secretion ◽  
Family Protein ◽  
Biochemical Fractionation ◽  
Inner Membrane Protein

ABSTRACT An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We have previously identified ImpLM, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpLM and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpLM is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impLM mutants with substitutions or deletions in the Walker A motif failed to complement the impLM deletion mutant for Hcp secretion, which provided evidence that ImpLM may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpLM and another essential T6SS component, ImpKL. Topology and biochemical fractionation analyses suggested that ImpKL is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpLM-ImpKL interaction domains suggested that ImpLM interacts with ImpKL via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpLM interacts with ImpKL, and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens.

scholarly journals Effector loading onto VgrG spike proteins is critical for the assembly of the type VI secretion system inAgrobacterium tumefaciens

2019 ◽  
Author(s):  
Yun-Wei Lien ◽  
Chih-Feng Wu ◽  
Devanand Bondage ◽  
Jer-Sheng Lin ◽  
Yu-Ling Shih ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Microbial Communities ◽  
Social Behaviors ◽  
Secretion System ◽  
Type Vi Secretion System ◽  
Associated Bacteria ◽  
Type Vi Secretion ◽  
Spike Proteins

AbstractThe type VI secretion system (T6SS) is used by many bacteria to engage in social behaviors with others and can directly or indirectly affect the health of plants and animals. Because activities associated with T6SS are often costly, the assembly and activation of the T6SS must be highly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Here we show that the loading of effectors onto their cognate carriers is critical for the assembly of a functional T6SS inAgrobacterium tumefaciens. A. tumefaciensstrain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. We found that loading of Tde effectors onto their cognate carrier, the VgrG spike, is required for active T6SS secretion. Our data also suggest the assembly of the TssBC contractile sheath occurs only after Tde effectors are loaded onto the VgrG spike. The requirement of effector loading for efficient T6SS secretion was also validated in otherA. tumefaciensstrains. Such a mechanism may be used by bacteria as a strategy for efficacious T6SS firing. Given the prevalence of T6SS-encoding loci in host-associated bacteria, these findings inform on mechanisms that influence the composition of microbial communities and the services provided to hosts.

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