bioRxiv2025
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 statementThe 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 summarySupplemental 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). O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/675681v2_fig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1640850org.highwire.dtl.DTLVardef@5de6f5org.highwire.dtl.DTLVardef@e605feorg.highwire.dtl.DTLVardef@1937bac_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Sequence diversity of the T4SS marker VirB4 in the Ralstonia genus. VirB4 protein sequences (n=496) were identified by a custom hidden Markov model (HMM) search, and an approximately-maximum-likelihood tree was built with FastTree 2 from a MUSCLE multiple sequence alignment (MSA) of the VirB4 sequences. Bootstrap values (0 to 1) from 1,000 resamples are shown for key nodes. The unrooted tree was visualized in iTOL and labeled using Affinity Designer. The 16 distinct T4SS gene clusters (a-p) are outlined and labeled. Circles denote clusters that are present in the RSSC phytopathogens and triangles denote clusters that are present in the non-RSSC environmentals. Colored clusters were found in RSSC phytopathogen genomes, and clusters with dashed outlines were not found in the RSSC phytopathogens. Supplemental files contain the virB4 gene list (Supplemental Table S2A), the VirB4 protein sequence MSA (Supplemental File S5), the Newick file for the tree (File S6), a PDF of the unrooted tree with branch labels (File S7), and PDFs of the rectangular tree with branch labels, without and with colors (Files S8 and S9). C_FIG This studys 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.