Biology subjects

Sankari, S.

Publications and source records attributed to Sankari, S..

4 recordsLinked to original sources

bioRxiv2025

A CRISPR Interference System for Inducible Gene Knockdown in soil bacterium Sinorhizobium meliloti

Symbiotic relationships have an important role in most life forms, but the molecular and cellular processes that establish and maintain these harmonious interactions remain largely unknown. The relationship between leguminous plants and rhizobial bacteria is a classic example of symbiosis, where the bacterium converts atmospheric nitrogen to plant-usable ammonia in exchange for fixed carbon and nutrients. Some legumes such as Medicago truncatula has evolved a set of small peptides that exploit this relationship, turning its bacterial partner, Sinorhizobium meliloti, into a terminally differentiated bacterium that loses its capability to survive outside the host. However, the mechanisms of how this transformation happens remain elusive due to the absence of high-throughput tools for targeted gene knockdowns in the bacterium. To overcome these limitations in the plant-rhizobia field, we developed an inducible CRISPR-interference knockdown system which can reversibly block the transcription of a target gene through the combined action of a deactivated-Cas9 (dCas9) and single-guide RNAs (sgRNAs). We used a taurine-inducible promoter to achieve fine-tunable expression levels of dCas9 in free-living S. meliloti and demonstrated that this tool is suitable for the study of essential genes that could be involved in the symbiotic process, including hemH, dnaN and ctrA. Our cost-effective inducible CRISPRi strategy will contribute to understanding the molecular mechanisms underlying legume-rhizobia symbiosis, ultimately allowing soil improvement and reducing chemical fertilizers usage while meeting global food demands.

genetics↗

bioRxiv2025

Exploiting Peptide Chirality and Transport to Dissect the Complex Mechanism of Action of Host Peptides on Bacteria

Elucidation of the complex mechanisms of action of antimicrobial peptides (AMPs) is critical for improving their efficacy. A major challenge in AMP research is distinguishing AMP effects resulting from various protein interactions from those caused by membrane disruption. Moreover, since AMPs often act in multiple cellular compartments, it is challenging to pinpoint where their distinct activities occur. Nodule-specific cysteine-rich (NCR) peptides secreted by legumes, including NCR247, have evolved from AMPs to regulate differentiation of their nitrogen-fixing bacterial partner during symbiosis as well as to exert antimicrobial actions. At sub-lethal concentrations, NCR247 exhibits strikingly pleiotropic effects on Sinorhizobium meliloti. We used the L- and D-enantiomeric forms of NCR247 to distinguish between phenotypes resulting from stereospecific, protein-targeted interactions and those caused by achiral interactions such as membrane disruption. In addition, we utilized an S. meliloti strain lacking BacA, the transporter that imports NCR peptides into the cytoplasm. BacA plays critical symbiotic roles by reducing periplasmic peptide accumulation and fine-tuning symbiotic signaling. Use of the BacA-deficient strain made it possible to distinguish between phenotypes resulting from peptide interactions in the periplasm and those occurring in the cytoplasm. At high concentrations, both L- and D-NCR247 permeabilize bacterial membranes, consistent with nonspecific cationic AMP activity. In the cytoplasm, both NCR247 enantiomers sequester heme and trigger iron starvation in an achiral but BacA-dependent manner. However, only L-NCR247 activates bacterial two-component systems via stereospecific periplasmic interactions. By combining stereochemistry and genetics, this work disentangles the spatial and molecular complexity of NCR247 action. This approach provides critical mechanistic insights into how host peptides with pleiotropic functions modulate bacterial physiology. Author SummaryMany organisms produce antimicrobial peptides (AMPs) to fight infections, but legumes have uniquely co-opted these molecules to control their symbiotic partners. During symbiosis between Medicago truncatula and Sinorhizobium meliloti, the plant secreted Nodule-specific Cysteine-Rich (NCR) peptides, transforms free-living bacteria into differentiated bacteroids that fix nitrogen but cannot reproduce outside the host. One such peptide, NCR247, exerts pleiotropic effects on the bacteria, acting on different subcellular locations, including membrane, heme, and proteins. Using a mirror-image (D-form) peptide, we disentangled peptide effects arising from generic physiochemical interactions versus stereospecific binding. The inner membrane protein BacA is known to play a protective role by importing NCR peptides into the cytoplasm. Using a bacterium lacking BacA, we were able to distinguish the effects of the peptide within and outside the cytoplasm. It was thought that BacA safeguards symbiotic bacteria by internalizing NCR peptides, thereby limiting their toxic membrane lytic effects, yet this has not been demonstrated. We show that BacA prevents lethal overstimulation of signaling pathways in the periplasm by internalizing the peptides. Our methods provide a framework for testing mechanism of action of new peptide-based antibiotics to combat multidrug-resistant bacteria.

microbiology↗

bioRxiv2025

The BacA(SbmA) Importer of Symbiotically Important Legume Nodule Cysteine-Rich Peptides: Insights into Protein Architecture, Function, and Evolutionary Implications

Some legumes encode families of NCR (Nodule-Cysteine-Rich) peptides that cause their rhizobial partners to terminally differentiate during the development of a nitrogen-fixing symbiosis. Sinorhizobium meliloti, whose plant hosts Medicago truncatula and M. sativa express ca. 600 NCR peptides during root nodule development, possesses a symbiotically essential BacASm protein that imports certain NCR peptides into the cytoplasm. This import permits proteolytic degradation of the NCR peptides, thereby protecting the endocytosed bacteria from their antimicrobial peptide-like lethality, while also allowing certain NCR peptides to undergo their symbiotically critical interactions with cytoplasmic components, for example heme-sequestration in the case of NCR247. BacAs Escherichia coli ortholog SbmAEc can restore a wildtype phenotype to a{Delta} bacASm mutant. Our study employed 54 S. meliloti bacASm missense mutants (35 to cysteine and 19 to glycine) that we tested for protein production, ability to establish a nitrogen-fixing symbiosis, and their susceptibility to killing by higher levels of the NCR247 and the Bac7(1-35) peptides. We also used the Single Cysteine Accessibility Method to make topological inferences. Our detailed genetic, biochemical, structural, and physiological analyses have revealed that BacASm and SbmA homodimers function as finely tuned import machines, whose structures can be relatively easily disrupted by single amino acid changes. Our discovery that several mutations that differentially separate nitrogen-fixation, NCR247 import, and Bac7(1-35) import map to the lining of the peptide-binding cavity in the outward-open SbmA/BacA conformation suggests a molecular explanation the other otherwise paradoxical observation that SbmA/BacAs from pathogens can fully replace BacASm, whereas BacAs from other rhizobia cannot. Significance StatementSinorhizobium meliloti BacASm and Escherichia coli SbmAEc are closely related proteins that function as homodimeric transporters to import peptides and other cargos through the cytoplasmic membrane into the cytoplasm. BacA is critical for S. meliloti to establish a nitrogen-fixing symbiosis with its legume hosts because of its ability to import Nodule Cysteine-Rich (NCR) nodule-specific plant peptides. This import protects the bacteria inside the nodule from the potentially lethal effects of these NCR peptides while also enabling NCRs to make their intracellular interactions that are necessary for symbiosis. Our extensive multidisciplinary studies offer new insights into function of BacA/SbmA transporters and provide a molecular explanation for why BacA/SbmA orthologs from mammalian pathogens can replace BacASm but those from other rhizobia cannot.

biochemistry↗

bioRxiv2025

Structure-based phylogenetic analysis reveals multiple events of convergent evolution of cysteine-rich antimicrobial peptides in legume-rhizobium symbiosis

Nitrogen is essential for plant growth, yet its availability often limits agricultural productivity. Some legumes have evolved a unique ability to form symbiotic relationships with nitrogen-fixing soil bacteria called rhizobia, enabling them to thrive in nitrogen-deficient soils. In five legume clades, an exploitive strategy has evolved in which rhizobia undergo Terminal Bacteroid Differentiation (TBD), where the bacteria become larger, polyploid, and have a permeabilized membrane. Terminally differentiated bacteria are associated with higher N2-fixation and, thus, a higher return on investment to the plant. In several members of the IRLC (Inverted Repeat-Lacking Clade) and the Dalbergioid clades of legumes, this differentiation process is triggered by a set of apparently unrelated plant antimicrobial peptides with membrane-damaging activity, known as Nodule-specific Cysteine-Rich (NCR) peptides. However, whether NCR peptides are also implicated in symbiotic TBD in other legume clades and whether they are evolutionarily related remains unknown. Here, to address the molecular identity of NCR peptides and their evolution in different legume clades, we performed inter- and intra-clade comparisons of NCR peptides in representative species of four TBD-inducing legume clades. First, we collected genomic and proteomic data of species for which NCR peptides are known (1523 NCR peptides). We then used sequence similarity-based clustering to regroup the NCR peptides, resulting in over 400 different NCR clusters, each clade-specific. We obtained Hidden Markov Models for each cluster and used them to predict NCR peptides in 21 legume genomes (6 clades), including newly generated deep-sequenced root and nodule RNA-seq data of Indigofera argentea (Indigoferoid clade) and newly assembled high-quality transcriptomes of Lupinus luteus and Lupinus mariae-josephae (Genistoid clade), using tailored gene prediction pipeline and transcriptome matching. This resulted in 3710 NCR peptides in species that induce TBD. To date, the rapid diversification of NCR peptides that reduces the sequence similarities has masked the origin of NCR peptide evolution. We obtained high-confidence structural models for one sequence of each cluster. We performed structure-based clustering and phylogenetics, which resulted in 23 superclusters (14 inter-clade and nine clade-specific) that we represent in a structural distance-based tree. Our study revealed that the evolution of NCR peptides is a mix of divergent and convergent processes within each clade. We further chose nine independently evolved NCR peptides to test in vitro whether they are functional analogs in symbiosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=145 HEIGHT=200 SRC="FIGDIR/small/675119v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8d1698org.highwire.dtl.DTLVardef@c65b98org.highwire.dtl.DTLVardef@a75994org.highwire.dtl.DTLVardef@ea1a73_HPS_FORMAT_FIGEXP M_FIG Overview of the experimental and computational workflow for NCR peptide detection, characterization, and structural analysis. Nodule and root samples from Indigofera argentea (8 weeks post-inoculation) were collected and subjected to RNA extraction, library preparation, and Illumina PE150 sequencing. Raw RNA-seq reads from two Lupinus species were also included (Lupinus luteus and Lupinus mariae-josephae). Bacteroid differentiation of I. argentea was assessed by flow cytometry and confocal microscopy. Transcriptomes were assembled de novo and analyzed for differential gene expression between root and nodule tissues. NCR peptides were identified from them and other legume genomes and transcriptomes using the SPADA pipeline and HMM profiles from NCR clusters of the known NCR peptides. The putative NCR peptides were filtered based on conserved cysteine motifs, length, and nodule expression to build an exhaustive NCR peptide database. 3D structural predictions of NCR clusters were performed using AlphaFold2 (pLDDT >70), followed by structural clustering (Foldseek) and phylogenetic analysis (Foldtree). Functional validation involved flow cytometry and antimicrobial assays (against Eschericha coli, Sinorhizobium meliloti, and Bacillus subtilis), enabling structural and evolutionary characterization of NCR peptides. The green box at the top represents the experimental analysis, the blue box represents the sequence-based computational pipeline, the red box represents the structure-based computational pipeline, and the grey box at the bottom left represents the functional validation and interpretation of the results. C_FIG

evolutionary biology↗