Isolation and Complete Genome Sequence Analysis of Kosakonia cowanii Pa82, a Novel Pathogen Causing Bacterial Wilt on Patchouli

Pogostemon cablin (patchouli), an important medicinal and aromatic plant, is widely used in traditional Chinese medicine as well as in perfume industry. Patchouli plants are susceptible to bacterial wilt disease, which causes significant economic losses by reduction in yield and quality of the plant products. However, few studies focus on the pathogens causing bacterial wilt on patchouli. In this study, strain Pa82 was isolated from diseased patchouli plants with typical bacterial wilt symptoms in Guangdong province, China, and was confirmed to be a highly virulent pathogen of patchouli bacterial wilt. Comparative sequence analysis of 16S rRNA gene showed that the strain was closely related to Kosakonia sp. CCTCC M2018092 (99.9% similarity) and Kosakonia cowanii Esp_Z (99.8% similarity). Moreover, phylogenetic tree based on 16S rRNA gene sequences showed that the strain was affiliated with genus Kosakonia. Further, the whole genome of strain Pa82 was sequenced, and the sequences were assembled and annotated. The complete genome of the strain consists of one chromosome and three plasmids. Average nucleotide identity (ANI) and phylogenetic analysis revealed that the strain belongs to Kosakonia cowanii (designated Kosakonia cowanii Pa82). Virulence-related genes of the strain involved in adherence, biofilm formation, endotoxin and other virulence factors were predicted. Among them, vgrG gene that encodes one of the type VI secretion system components was functionally validated as a virulence factor in Kosakonia cowanii Pa82 through construction of Tn5 insertion mutants and identification of mutant defective in virulence.

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

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.

Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae

Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes to new niches. Regulatory architecture is often inferred from transcription factor identification and genome analysis using purely computational approaches. However, there are few examples of phenotypic divergence that arise from the rewiring of bacterial regulatory circuity by mutations in intergenic regions, because locating regulatory elements within regions of DNA that do not code for protein requires genomic and experimental data. We identify a single cis-acting single nucleotide polymorphism (SNP) dramatically alters control of the type VI secretion system (T6), a common weapon for inter-bacterial competition. Tight T6 regulatory control is necessary for adaptation of the waterborne pathogen Vibrio cholerae to in vivo conditions within the human gut, which we show can be altered by this single non-coding SNP that results in constitutive expression in vitro. Our results support a model of pathogen evolution through cis-regulatory mutation and preexisting, active transcription factors, thus conferring different fitness advantages to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

Structure of a bacterial Rhs effector exported by the type VI secretion system

The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ‘cocoon’ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ‘plug’ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

Characterization of a Type VI Secretion System vgrG2 Gene in the Pathogenicity of Burkholderia thailandensis BPM

Burkholderia thailandensis is a clinically underestimated conditional pathogen in the genus Burkholderia, the pathogenicity of the infection caused by B. thailandensis remains poorly understood. According to previous studies, Type-VI secretion system (T6SS) is a protein secreting device widely existing in Gram-negative bacilli. Valine-glycine repeat protein G (VgrG) is not only an important component of T6SS, but also a virulence factor of many Gram-negative bacilli. In one of our previous studies, a unique T6SS vgrG gene (vgrG2 gene) was present in a virulent B. thailandensis strain BPM (BPM), but not in the relatively avirulent B. thailandensis strain E264 (E264). Meanwhile, transcriptome analysis of BPM and E264 showed that the vgrG2 gene was strongly expressed in BPM, but not in E264. Therefore, we identified the function of the vgrG2 gene by constructing the mutant and complemented strains in this study. In vitro, the vgrG2 gene was observed to be involved in the interactions with host cells. The animal model experiment showed that the deletion of vgrG2 gene significantly led to the decrease in the lethality of BPM and impaired its ability to trigger host immune response. In conclusion, our study provides a new perspective for studying the pathogenicity of B. thailandensis and lays the foundation for discovering the potential T6SS effectors.

Backbone chemical shift assignment and dynamics of the N-terminal domain of ClpB from Francisella tularensis type VI secretion system

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

Transcriptomic Analysis Reveals Competitive Growth Advantage of Non-pigmented Serratia marcescens Mutants

Serratia marcescens is a common bacterium well-known for the red secondary metabolite prodigiosin. However, color mutants have long been described. Non-pigmented strains can be found to exist both naturally and under laboratory conditions. It is unclear why S. marcescens loses prodigiosin synthesis capacity in certain conditions. In the present study, we find that the spontaneous color mutants arise within a few generations (about five passages) and rapidly replace the wild-type parent cells (about 24 passages), which indicates a growth advantage of the former. Although, the loss of prodigiosin synthesis genes (pigA-N) is frequently reported as the major reason for pigment deficiency, it was unexpected that the whole gene cluster is completely preserved in the different color morphotypes. Comparative transcriptomic analysis indicates a dramatic variation at the transcriptional level. Most of the pig genes are significantly downregulated in the color morphotypes which directly lead to prodigiosin dyssynthesis. Besides, the transcriptional changes of several other genes have been noticed, of which transcriptional regulators, membrane proteins, and nearly all type VI secretion system (T6SS) components are generally downregulated, while both amino acid metabolite and transport systems are activated. In addition, we delete the transcription regulator slyA to generate a non-pigmented mutant. The ΔslyA strain loses prodigiosin synthesis capacity, but has a higher cell density, and surprisingly enhances the virulence as an entomopathogen. These data indicate that S. marcescens shuts down several high-cost systems and activates the amino acid degradation and transport pathways at the transcriptional level to obtain extra resources, which provides new insights into the competitive growth advantage of bacterial spontaneous color mutants.

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

The T6SS (Type VI secretion System) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of S. bongori, showing that it has antibacterial activity. We identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins that contain the DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV2 and TseV3 maintained the ability to bind DNA, but instead cause specific DNA double-strand breaks and induce the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanism regulating the activity of these toxins.

T6SS Accessory Proteins, Including DUF2169 Domain-Containing Protein and Pentapeptide Repeats Protein, Contribute to Bacterial Virulence in T6SS Group_5 of Burkholderia glumae BGR1

Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5.

The influence of Janthinobacterium sp. strain SLB01 on the cell culture of primmorphs of the sponge Lubomirskia baicalensis

Sponges (phylum Porifera) are ancient, multicellular metazoans. Freshwater sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. Over the last years, there have been mass diseases and death of endemic sponges. Previously, the strain Janthinobacterium sp. SLB01 was isolated from the diseased sponge Lubomirskia baicalensis. The studies of the pathogenicity of the strain Janthinobacterium sp. SLB01 for Baikal sponges has not been carried out, therefore we infected experimentally in vitro to determine its pathogenicity by the cell culture of the primmorphs with subsequent isolation, sequencing, and analysis of the genomes. The purpose of the study is to show that the strain Janthinobacterium sp. SLB01 isolated from the diseased sponge L. baicalensis is a pathogen for the cell culture of primmorphs. The bacteria from the infected samples were isolated and identified as strain Janthinobacterium sp. PLB02. A comparative analysis of the genomes of the strains showed that they are practically identical. The genomes of both strains contain genes vioABCDE violacein, flok formation, and strong biofilm, and the type VI secretion system (T6SS), as the primary virulence factor. These bacterial strains based on a comparison of complete genomes showed similarity with strain Janthinobacterium lividum MTR. Isolated strains of Janthinobacterium sp. are pathogens for cell cultures of primmorphs and L. baicalensis sponges. The results of the study will help to expand the understanding of microbial relationships in the development of disease and the death of Baikal sponges.