The Sinorhizobium meliloti chemoreceptor McpU mediates positive and negative chemotaxis responses to dipeptides and competes with periplasmic solute-binding proteins for ligand binding

Targeting methyl-accepting chemotaxis transmembrane sensory protein inhibits Helicobacter pylori autoinducer-2 quorum sensing system

4-Aminobenzoic acid attenuates Ralstonia solanacearum virulence by suppressing quorum sensing system

ABSTRACT Quorum sensing (QS) is widely involved in the regulation of the biological functions and pathogenic processes of bacteria in a cell density-dependent manner. The anthranilic acid signal is directly sensed by the receptor RaaR, a transcription regulator, to control the expression level of downstream target genes in Ralstonia solanacearum, thus affecting the QS signals generation and the bacterial virulence. Here, we reported that 4-Aminobenzoic acid (PABA), a structural analogue of anthranilic acid, not only significantly inhibited the production of extracellular polysaccharides (EPS), biofilm formation, and motility of R. solanacearum, but also reduced the expression level of QS signaling molecules. We also found that PABA can directly bind to RaaR and prevented it from interacting with the promoters of target genes associated with the QS system and virulence of R. solanacearum. Point mutations in RaaR at residues F137 and I270 abolished the binding between RaaR and PABA, indicating that these two amino acid residues are likely critical for the interaction between RaaR and PABA. These findings indicated that PABA could be a candidate inhibitor of anthranilic acid signaling to interfere with the QS systems of R. solanacearum to attenuate the virulence. Together, our work presented a non-antibiotic-based treatment strategy and provided a theoretical basis for the development of novel antibacterial treatment.

1-Aminocyclopropane-1-Carboxylate and Its Chemoreceptor Drive Metabolic Reprogramming to Enhance Chemotactic Rhizocompetence in Pseudomonas sp. UW4

1-Aminocyclopropane-1-carboxylate (ACC) has been identified as the preferred chemoattractant driving rhizosphere colonization by the model plant growth-promoting rhizobacterium (PGPR) Pseudomonas sp. UW4. However, the regulatory mechanisms governing the expression of its ACC chemoreceptor WP116, as well as how changes in its abundance influence ACC chemotaxis and rhizosphere colonization, remain poorly understood. In this study, we found that the addition of ACC (3.0 mM) during the mid-logarithmic growth phase of UW4 rapidly induced the transcription of WP116 (up to 1.4-fold) and several other chemoreceptor genes, promoted bacterial growth by increasing the maximum specific growth rate by 21.1% (p < 0.05), yet did not significantly alter the protein abundance of chemoreceptors. At the proteome level, however, ACC drove a reprogramming of carbon, nitrogen, and energy metabolism, facilitating the transition of cells from a motile to a colonizing state. Furthermore, trans-overexpression of WP116 disrupted the homeostatic balance of receptor abundance, leading to a marked increase in its protein level (up to 32.3-fold) and enhancing both the ACC chemotactic response (up to 3–5-fold) and the rhizosphere colonization competitiveness (up to 9-fold) of UW4 (p < 0.05). These findings not only deepen our understanding of the molecular mechanisms underlying metabolism-dependent chemotaxis but also provide a novel technological avenue for rhizosphere microbiome engineering aimed at directionally enhancing the chemotactic rhizocompetence of PGPR.

A lettuce receptor-like kinase recognizes the highly conserved heptapeptide motif within microbial Nep1-like proteins.

Plants rely on cell surface immune receptors to detect microbial patterns and initiate effective defense responses. Although the Asteraceae family is one of the largest and economically important plant groups, little information is available about its pattern-triggered immunity signaling. Cultivated lettuce (Lactuca sativa L.) recognizes a 24-amino acid peptide (nlp24) from necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) found in bacteria, fungi, and oomycetes. Here, we perform an extensive characterization of nlp24-induced immune responses in lettuce and identify the LETTUCE nlp24 RECEPTOR (LNR) as the leucine-rich repeat receptor-like kinase mediating its recognition. Remarkably, nlp24 recognition in lettuce and subsequent activation of defenses strongly depend on the conserved heptapeptide motif (GHRHDWE). Structural modeling-guided mutagenesis experiments suggest that residues in the nlp24 heptapeptide interact with a hydrophobic pocket in the LNR solenoid structure. Divergent ligand specificities and the absence of sequence homology between LNR and the Arabidopsis nlp24-recognizing receptor RLP23 indicate that NLP recognition in lettuce and Arabidopsis emerged independently, through convergent evolution. Our phylogenetic analysis shows that LNR is closely related to Arabidopsis MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) but belongs to a distinct, Asteraceae-specific monophyletic subgroup that has undergone a significant expansion in Lactuca. Our findings provide insights into the mechanisms of pattern-triggered immunity in lettuce and present an example of convergent evolution of plant pattern-recognition receptors.

Identification of Quorum Sensing Molecules of N-Acyl-Homoserine Lactone in Leptospira Strains Supernatants

The bacterial Quorum Sensing system refers to the recognition of signaling molecules called autoinducers produced by bacteria when a certain cell density is reached in the environment. Those cell-density-dependent autoinducers regulate and coordinate diverse functional processes, such as bioluminescence, biofilm production, sporulation, and even the expression of some virulence factors, among others. There is a wide variety of autoinducers, and for Gram-negative bacteria, the canonical autoinducers are the N-acyl-homoserine lactones (AI-1). Presently, the production of autoinducers in Leptospira has not been described; therefore, the objective of this study was to detect and identify autoinducers in this bacterial genus. We report here the expression of AI-1 in cultures ≥2.4 × 108 of Leptospira meyeri. Ethyl acetate extracts of Leptospira culture supernatants were capable of activating the β-galactosidase system in the biosensor Agrobacterium tumefaciens strain NTL4. Partial identification of the leptospiral supernatant extracts was done by thin-layer chromatography (TLC), showing a similar retention factor to the synthetic standard N-Octanoyl-DL-homoserine lactone (C8-AHL) in the Leptospira supernatant extracts. In addition, infrared spectroscopy (IR) analysis showed peaks corresponding to the lactone and amide groups in both the C8-AHL standard and the Leptospira meyeri culture extracts. Moreover, High-Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS/MS) confirmed the same retention time (10.7 ± 0.1 min) in both the Leptospira meyeri supernatant extracts and the C8-AHL standard. These results show that Leptospira meyeri synthesizes N-acyl homoserine lactone family autoinducers, particularly the N-Octanoyl-DL-homoserine lactone, and lay the groundwork for future research on Quorum Sensing systems in Leptospira.

Deciphering the Function and Structure of PA1216 as an S‐Adenosyl‐l‐Methionine Binding Protein Using Differential Scanning Fluorimetry and Circular Dichroism

Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration‐dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high‐throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl‐glycine dipeptides, amino esters with aromatic or basic side chains, and N‐Boc‐protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann‐like fold and a glycine‐rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.