Matthew L. Cope-Arguello

Type IV secretion systems (T4SSs) are versatile machines with variable functions including DNA uptake and release, protein translocation, and DNA conjugation. However, the diversity, distribution, and functional roles of the T4SS in the Ralstonia genus remain poorly understood. The Ralstonia solanacearum species complex (RSSC) comprises three species of plant-pathogenic bacteria that cause bacterial wilt disease. The Ralstonia genus also includes non-RSSC species that are primarily environmental bacteria and rare opportunistic human pathogens. This study compared the diversity and phylogenetic distribution of T4SSs in the RSSC phytopathogens vs. non-RSSC environmentals. Phylogenetic analysis of VirB4 sequences and synteny analysis revealed 16 distinct T4SS clusters in Ralstonia , with 10 clusters found in RSSC phytopathogen genomes, 12 in non-RSSC environmental genomes, and 6 clusters in both groups. Collectively, these gene clusters were more prevalent in non-RSSC environmental genomes. The presence of type IV coupling protein and relaxase genes suggests that at least 14 of these T4SS gene clusters are putative DNA-conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements on the chromosome or megaplasmid. The putative regions of transfer for T4SS gene clusters in the RSSC phytopathogen genomes often contained type III effectors, type VI secretion toxin/antitoxin clusters, and haemagglutinin gene clusters. In contrast, the non-RSSC environmentals were enriched in heavy metal metabolism and resistance genes. One of the 16 T4SS clusters, cluster i, exhibited evidence of specialization for the RSSC phytopathogens. These findings shed light on the eco-evolutionary differences within the genus Ralstonia .

Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocol’s efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium.

Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocol's efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium.

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta, we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers “biofilm mobility”, which allows wild-type RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia . In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics.

Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocol's efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium.

Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocol’s efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium. Data summary: Supplemental File S1 is the preliminary version of this chemical competence protocol. Supplemental Figures S1 and S2 show transformation efficiency data for pUFR80 knockout plasmids, and heat shock number and temperature variation, respectively.

Type IV secretion systems (T4SSs) are versatile machines with variable functions including DNA uptake and release, protein translocation, and DNA conjugation. However, the diversity, distribution, and functional roles of the T4SS in the Ralstonia genus remain poorly understood. The Ralstonia solanacearum species complex (RSSC) comprises three species of plant-pathogenic bacteria that cause bacterial wilt disease. The Ralstonia genus also includes non-RSSC species that are primarily environmental bacteria and rare opportunistic human pathogens. This study compared the diversity and phylogenetic distribution of T4SSs in the RSSC phytopathogens vs. non-RSSC environmentals. Phylogenetic analysis of VirB4 sequences and synteny analysis revealed 16 distinct T4SS clusters in Ralstonia , with ten clusters found in RSSC phytopathogen genomes, twelve in non-RSSC environmental genomes, and six clusters in both groups. Collectively, these gene clusters were more prevalent in non-RSSC environmental genomes. The presence of type IV coupling protein and relaxase genes suggests that at least 14 of these T4SS gene clusters could be putative DNA conjugation systems. The clusters were encoded on accessory plasmids of various sizes or as integrative and conjugative elements (ICEs) on the chromosome or megaplasmid. The putative regions of transfer for T4SS gene clusters in the RSSC phytopathogen genomes often contained type III effectors, type VI secretion toxin/antitoxin clusters, and hemagglutinin gene clusters. In contrast, the non-RSSC environmentals were enriched in heavy metal metabolism and resistance genes. One of the 16 T4SS clusters, cluster i, exhibited evidence of specialization for the RSSC phytopathogens. These findings shed light on the eco-evolutionary differences in the Ralstonia genus. Impact statement: The Ralstonia genus contains the Ralstonia solanacearum species complex (RSSC), a group of globally important plant pathogens that impact food security. These pathogens are known to have an expansive, open pangenome with high levels of gene flow. To shed light on the eco-evolutionary differences between RSSC phytopathogens and closely related environmental species, we explored the potential role of type IV secretion systems (T4SSs) in horizontal gene flow within the Ralstonia genus. Surprisingly, these mobile genetic elements were less common in the phytopathogens than the environmentals. Nevertheless, we identified a particular T4SS gene cluster encoded on an accessory plasmid that appears to be specialized to the pathogenic lifestyle of the RSSC. This specialized cluster harbors genes that drive RSSC host range (type III effectors) and others that likely allow these pathogens to antagonize the plant microbiota (type VI secretion toxins and hemagglutinin two partner secretion systems). Moreover, this study sheds light on the cryptic lifestyles of the poorly studied environmental Ralstonia species, revealing that their conjugative T4SSs frequently carry heavy metal metabolism and resistance genes. Overall, this study provides a foundation to investigate the functional roles of T4SS clusters and their cargo genes in the fitness of phytopathogenic and environmental Ralstonia . Data summary: Supplemental Table S1 lists details of the RSSC phytopathogen, non-RSSC environmental, and Burkholderiaceae family genomes used in this study. Supplemental Table S2A is the final list of 501 VirB4 sequences collectively identified in 636 RSSC phytopathogen genomes and 143 non-RSSC environmental genomes. Supplemental Table S2B includes the annotations and NCBI accessions for every gene and protein sequence in reference clusters a-p. Supplemental Table S2C is the list of putative T4SS cargo genes identified in complete Ralstonia genomes. Supplemental Table S2D is the list of putative T4SS cargo genes identified in cluster i regions from draft and complete Ralstonia genomes. Supplemental File S1 is the multiple sequence alignment (MSA) input for HMMER, made from the 753 VirB4 protein sequences from the Burkholderiaceae family genomes. Supplemental Files S2 and S3 are PDF-format versions of the species trees displaying the presence of T4SS gene clusters in RSSC phytopathogens and non-RSSC environmentals, respectively. Supplemental File S4 is the species tree displaying the presence of RSp0179 and RSp1521 in the Ralstonia genus. Supplemental File S5 is the MSA input for FastTree 2, made from the VirB4 protein sequences from Ralstonia genomes. Supplemental Files S6-S9 are various formats of the VirB4 protein tree in Figure 1. Supplemental Files S10-S25 are the GBK files for reference clusters a-p. Supplemental File S26 is the clinker output comparing the reference clusters. Supplemental Files S27-S36 are the clinker outputs comparing the putative regions of transfer of T4SSs in complete Ralstonia genomes. Supplemental File S37 is an MSA analysis comparing RSp1521 to AcvB homologs. Supplemental File S38 is the clustered average nucleotide identity (ANI) matrix for the putative regions of transfer of cluster i T4SSs in draft and complete Ralstonia genomes. Supplemental File S39 is the clinker output comparing the cluster i regions in draft and complete Ralstonia genomes. All Supplemental Tables and Files are available through Zenodo (DOI: 10.5281/zenodo.14873518). This study’s newly sequenced genomes, Gazipur 4 and Gazipur 5, are available on NCBI with the following accessions: GCF_049860735.1 and GCF_049860725.1, respectively. The two contigs containing the cluster j T4SS were submitted to NCBI through the Third Party Annotation (TPA) section of the DDBJ/ENA/ GenBank databases with the following accessions: BK072068 - BK072069 . These contigs were assembled from SRA reads under the accession: SRR18649448.

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta , we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers “biofilm mobility”, which allows wildtype RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia . In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics. Significance Statement: Ralstonia solanacearum species complex (RSSC) pathogens threaten global food security by fatally wilting plants. A soft matter physics lens demystified the cryptic role of a major virulence factor, the EPS-I exopolysaccharide. EPS-I transforms RSSC biofilms into viscoelastic fluids, a mechanical behavior not previously described for other microbial biofilms that are almost always viscoelastic solids. We demonstrate that the development of fluid biofilms was a key evolutionary innovation that enabled pathogenic success of these aggressive pathogens that rapidly wilt plants.

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