Suvendra Kumar Ray

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

Ralstonia pseudosolanacearum ( Rps ) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 10 ⁹ CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::Ω mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::Ω . It indicated that the necrotic lesion caused in tomato leaves was hrp -dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Transformation of foreign DNA, subjecting Escherichia coli to CaCl₂ treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its popularity in E. coli, heat shock transformation is rarely reported in other Gram-negative bacteria. The objective of this research is to demonstrate that the heat shock transformation method can also be performed to introduce genes in different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii. A green fluorescent plasmid, pDSK-GFPuv, having the size of ~ 8.9 kb, was directly transferred to R. pseudosolanacearum by heat shock at 50 °C for 60s. For P. aeruginosa, P. putida, and E. roggenkampii, the cells were made competent using CaCl_2, followed by heat shock transformation at 50 °C for 180s. As the plasmid contains a gene encoding green fluorescence protein, after successful transformation, bacterial strains were demonstrated for colonization in tomato seedlings. The introduced plasmid was re-isolated from R. pseudosolanacearum to prove its stability inside the bacterium after the heat shock transformation. This easy and convenient method of transformation may be used for other Gram-negative bacteria to prove its wider applicability.

1 The Ralstonia solanacearum species complex (RSSC) is a highly diverse plant pathogen whose evolution is shaped by horizontal gene transfer. We generated a complete, gap-free hybrid genome assembly of F1C1, a South Asian Phylotype I strain classified as R. pseudosolanacearum. The closed assembly resolves a bipartite genome (3.73 Mb chromosome; 2.03 Mb megaplasmid), enabling precise localization of mobile genetic elements. Using F1C1 together with 142 complete RSSC genomes, we implemented a lineage-stratified pangenome framework that reveals a hierarchically structured gene repertoire and shows that accessory gene content can discriminate host-associated lineages beyond core-genome. Pangenome-informed mapping of genome plasticity identified 651 conserved integration spots that concentrate accessory turnover and are enriched for adaptive functions, including type III secretion system effectors and antiviral defense systems (e.g., Wadjet and CRISPR-Cas). Genes within these spots display elevated K _a / K _s relative to housekeeping functions, consistent with conflict-driven diversification and/or relaxed constraint. Together, these results link RSSC genome architecture to adaptive potential and provide a spot-based framework for genomic surveillance and resistance breeding in bacterial wilt pathosystems.

The genome G+C content of bacteria varies widely, from 13% to 75%, which is influenced by both environmental and internal mutation pressure; however, the precise determinants of this variability remain unresolved. Mutation-based models, such as Sueoka’s directional mutation hypothesis, suggest that G+C content arises from mutational pressures within an organism without providing any specific advantage to it. Though there are several advantages associated with genome G+C%, there is limited evidence, favoring any selection mechanism for G+C% evolution. Hence, the genome G+C% in organisms is largely studied under the neutral theory of evolution. Cytosine deamination and guanine oxidation are recognized as major contributors to A/T mutational bias, producing frequent substitutions such as C→T transitions and G→T transversions, respectively. While these mechanisms leading to A+T enrichment have been well studied, counteracting processes that promote G+C enrichment in organisms are comparatively less understood. This review mainly highlights adenine as an underexplored contributor: its deamination and oxidation yield A→G and A→C substitutions, respectively, both biased toward increased G+C content. We further consider how the efficiency of DNA repair mechanisms may shape G+C content across evolutionary timescales. Together, these perspectives address a gap in the current understanding of the mutational forces influencing genome composition.

Alcoholic beverages contains ethanol as the main component. Ethanol is metabolized into acetaldehyde, a Group 1 carcinogen, and other reactive by-products such as ROS and hydroxyl radicals. These metabolites can interact with DNA to form adducts and interstrand cross-links, leading to mutations and genomic instability, which are key contributors to alcohol-related cancers. Ethanol metabolism is mediated by critical enzymes - Alcohol Dehydrogenase (ADH), Cytochrome P450 2E1 (CYP2E1), Catalase, and Aldehyde Dehydrogenase (ALDH), which regulate both the generation and detoxification of these harmful intermediates. Every cell in our body owns a toolkit with which it can repair different types of DNA lesions. This review highlights the enzymatic pathways of ethanol metabolism, the formation of genotoxic by-products, and the cellular mechanisms that maintain genomic integrity. Understanding these processes provides crucial insights into the molecular basis of alcohol-induced carcinogenesis and may guide strategies for prevention and therapeutic intervention.

Transformation of foreign DNA, subjecting Escherichia coli to CaCl ₂ treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its large popularity in E. coli , heat shock transformation is rarely reported in other Gram-negative bacteria. Instead, techniques such as natural transformation, conjugation, or electroporation are used in those bacteria. In this study, we have successfully implemented the heat shock transformation in four different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii . The standard heat shock transformation procedure used for E. coli DH5α has been modified. The pDSK-GFPuv plasmid bearing the green fluorescence gene was directly transferred to Ralstonia pseudosolanacearum by heat shock at 50 ºC for 60 seconds. For Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii , the cells were made competent using CaCl _2, followed by performing transformation by heat shock at 50 ºC for 180 seconds. The transformants were resistant to kanamycin as well as exhibited fluorescence. These transformants were used to study the colonization pattern of tomato seedlings. Our study suggested that the heat shock transformation method can also be performed to introduce genes in other Gram-negative bacteria, other than E. coli .

Ralstonia solanacearum, a gram-negative bacterium, is known to cause devastating wilting disease in many plant species. The bacterium’s pathogenesis relies on various virulence determinants, including twitching motility, facilitated by type IV pili appendages. Twitching motility is a density-dependent mechanism enabling bacterial movement on solid surfaces, crucial for biofilm formation and virulence. Understanding this motility is of utmost importance in studying the pathogen’s behavior. In this chapter, we present a simple and reproducible method to assess twitching motility in R. solanacearum. The method involves microliter spotting of a diluted bacterial suspension onto a solid agar growth medium, followed by microscopic observation of the early-stage microcolonies. Twitching motility becomes evident as finger-like projections, which merge and progressively spread in all directions, forming multilayered structures. The examination also reveals cell heterogeneity within colonies and the influence of bacterial concentration on microcolony shape, potentially attributed to quorum sensing. Our simplified protocol for assessing twitching motility in microcolonies using microliter spotting offers significant advantages over existing methods described in the literature, making it an effective tool for investigating twitching motility in bacterial pathogenesis. A detailed step-by-step description of the protocol is provided in this chapter, facilitating its adoption and application in future research.