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Cited by 1Microbial Natural Products and BiosynthesisBacterial biofilms and quorum sensingAntimicrobial Peptides and Activities

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Quorum sensing regulators and non-ribosomal peptide synthetases govern antibacterial secretions in Xenorhabdus szentirmaii

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Dey, Ritisha, Valle, Domonique Olivia, Chakraborty, Abhijit, Mayer, Kimberly A., Uppala, Jagadeesh Kumar, Chakraborty, Anish, Mirza, Shama, Skwor, Troy, Forst, Steven, Dey, Madhusudan·2025·Frontiers in Microbiology
DOI 10.3389/fmicb.2025.1560663PMC PMC11936946

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Abstract

The decades-long gap in antibiotic discovery has led to a significant health crisis due to antimicrobial resistance (AMR). The bacterial genus Xenorhabdus , which forms symbiotic relationships with the soil nematode Steinernema , are known to secrete a variety of antimicrobial compounds with potential effectiveness against AMR. These antimicrobial compounds are primarily bio-synthesized by non-ribosomal peptide synthetases (NRPS) and polyketide synthase (PKS) genes. In this study, we report that X. szentirmaii produces high levels of antibiotic activity during the stationary phase against diverse bacteria including known antibiotic resistant pathogens. It possesses 17 operons to encode predicted NRPS and PKS enzymes, designated as ste1 through ste17 . The ste15-ste16 and ste17 operons are predicted to produce the known antibiotics Pax peptide and Fabclavine, respectively. Additionally, the newly identified operons ste3 , ste4 , ste5 , ste8, ste9 , and ste14 consist of single genes, each containing two or more NRPS genes. The ste13 operon harbors two NRPS genes, while the ste7 and ste12 operons contain three NRPS genes each. Further, RNA-seq analysis showed that lsrF that encodes a quorum sensing autoinducer-2 (AI-2) thiolase was expressed at high levels during stationary phase. These findings provide evidence that X. szentirmaii uses quorum sensing (QS) to synchronize the expression of multiple NRPS and PKS enzymes responsible for synthesizing various antimicrobial compounds. This study underscores the potential to leverage these regulatory insights for maximizing commercial applications of novel antibiotics combating AMR, as well as broader industrial uses.

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Figures

Figure 1 Inactivation of ngrA abolishes the synthesis of antimicrobials. (A) The crystal structure of Acinetobacter baumannii NRPS protein AB3403 (PDB ID = 4zxh). The condensation (C)-domain, adenylate (A)-domain, PCP-domain and thioesterase (TE)-domain are shown in gray, orange, red and blue colors, respectively. (B) The Alpha-Fold predicted structure of X. szentirmaii (X. szn) ngrA protein. (C) The bacterium X. szentirmaii (WT or ΔngrA mutant) was spotted on an LB medium and grown overnight. The indicated tester strain S. saprophyticus was overlaid and grown at 37°C for 24 h. The clearance zones indicate the antimicrobial activity. (D) Amino acid sequences of the PCP-domain. (E) The whole X. szentirmaii genome (GenBank accession numbers NIBV01000001 and NIBV01000002) was used to search for genes encoding homologs of the PCP-domain. After the BLAST search, the E-values and percentage identities were retrieved and graphed on a logarithmic scale.
Figure 1 Inactivation of ngrA abolishes the synthesis of antimicrobials. (A) The crystal structure of Acinetobacter baumannii NRPS protein AB3403 (PDB ID = 4zxh). The condensation (C)-domain, adenylate (A)-domain, PCP-domain and thioesterase (TE)-domain are shown in gray, orange, red and blue colors, respectively. (B) The Alpha-Fold predicted structure of X. szentirmaii (X. szn) ngrA protein. (C) The bacterium X. szentirmaii (WT or ΔngrA mutant) was spotted on an LB medium and grown overnight. The indicated tester strain S. saprophyticus was overlaid and grown at 37°C for 24 h. The clearance zones indicate the antimicrobial activity. (D) Amino acid sequences of the PCP-domain. (E) The whole X. szentirmaii genome (GenBank accession numbers NIBV01000001 and NIBV01000002) was used to search for genes encoding homologs of the PCP-domain. After the BLAST search, the E-values and percentage identities were retrieved and graphed on a logarithmic scale.CC BY · PubMed Central
Figure 2 X. nematophila and X. szentirmaii are natural resources of antibiotics. (A) The bacteria X. nematophila and X. szentirmaii were subjected to overlay assays with four tester strains: S. saprophyticus, E. coli, MRSA and a clinical ESBL-producing UPEC strain. (B) The bacterium X. szentirmaii was grown for 48 h before cross-streaking with the indicated microorganisms. The distance of inhibition was measured and recorded in a bar diagram (C).
Figure 2 X. nematophila and X. szentirmaii are natural resources of antibiotics. (A) The bacteria X. nematophila and X. szentirmaii were subjected to overlay assays with four tester strains: S. saprophyticus, E. coli, MRSA and a clinical ESBL-producing UPEC strain. (B) The bacterium X. szentirmaii was grown for 48 h before cross-streaking with the indicated microorganisms. The distance of inhibition was measured and recorded in a bar diagram (C).CC BY · PubMed Central
Figure 3 X. szentirmaii produces antimicrobials in the stationary phase. (A) The bacterium X. szentirmaii was grown for the indicated time and the optical density (OD) was measured at 600 mm. The experiments were repeated at least three times. One representative data are shown. (B) Cell-free supernatants were collected from both log and stationary phases, filter-sterilized, spotted (5 μL) on an agar plate, and subjected to overlay assay with the bacterium S. saprophyticus. The clearance zone indicates antibacterial activity. (C) The 20 μL or 5 μL of cell-free supernatants containing equal number of cells were collected, concentrated, and spotted on the LB medium and subjected to overlay assay.
Figure 3 X. szentirmaii produces antimicrobials in the stationary phase. (A) The bacterium X. szentirmaii was grown for the indicated time and the optical density (OD) was measured at 600 mm. The experiments were repeated at least three times. One representative data are shown. (B) Cell-free supernatants were collected from both log and stationary phases, filter-sterilized, spotted (5 μL) on an agar plate, and subjected to overlay assay with the bacterium S. saprophyticus. The clearance zone indicates antibacterial activity. (C) The 20 μL or 5 μL of cell-free supernatants containing equal number of cells were collected, concentrated, and spotted on the LB medium and subjected to overlay assay.CC BY · PubMed Central
Figure 4 Analysis of transcripts expressed during log and stationary phases. (A) Four bacterial colonies were grown till the log and stationary phases. Total RNAs were isolated and run in an agarose gel. (B) The principal component analysis (PCA) plot shows two distinct clusters of samples from log-phase (green dots) and stationary-phase (red dots). (C) Heatmap of differentially expressed genes. The heatmap shows the row normalized TPM (Transcripts per million) values of 637 differential genes with FDR < 0.05 and absolute log2FC above >1.
Figure 4 Analysis of transcripts expressed during log and stationary phases. (A) Four bacterial colonies were grown till the log and stationary phases. Total RNAs were isolated and run in an agarose gel. (B) The principal component analysis (PCA) plot shows two distinct clusters of samples from log-phase (green dots) and stationary-phase (red dots). (C) Heatmap of differentially expressed genes. The heatmap shows the row normalized TPM (Transcripts per million) values of 637 differential genes with FDR < 0.05 and absolute log2FC above >1.CC BY · PubMed Central
Figure 5 Upregulation of quorum sensing activities during the stationary phase. (A) The bar diagram shows the number of up-regulating genes during the stationary phase are classified based on their physiological functions. (B) Total RNA was isolated from the log and stationary phases of X. szentirmaii and subjected to RNA-seq analysis. Up and down regulated genes are shown in red and blue color, respectively, in the volcano plot. (C) (Left panel) Total RNA was isolated from the log and stationary phases of X. szentirmaii and run on an agarose gel. (Middle panel) cDNA was synthesized by reverse transcriptase (RT) from total RNA isolated from log and saturated (Sat.) phases and subjected to RT-PCR with the LsrF-specific primers. The PCR products were run in an agarose gel. A control reaction without RT (Mock) was also carried out. (Right panel) The band intensities of the PCR products were measured NIH-image J software and shown in a bar diagram.
Figure 5 Upregulation of quorum sensing activities during the stationary phase. (A) The bar diagram shows the number of up-regulating genes during the stationary phase are classified based on their physiological functions. (B) Total RNA was isolated from the log and stationary phases of X. szentirmaii and subjected to RNA-seq analysis. Up and down regulated genes are shown in red and blue color, respectively, in the volcano plot. (C) (Left panel) Total RNA was isolated from the log and stationary phases of X. szentirmaii and run on an agarose gel. (Middle panel) cDNA was synthesized by reverse transcriptase (RT) from total RNA isolated from log and saturated (Sat.) phases and subjected to RT-PCR with the LsrF-specific primers. The PCR products were run in an agarose gel. A control reaction without RT (Mock) was also carried out. (Right panel) The band intensities of the PCR products were measured NIH-image J software and shown in a bar diagram.CC BY · PubMed Central
Figure 6 Upregulation of operons ste2, ste9, ste10, ste14A and ste17A during the stationary phase. (A) The average FPKM counts of the indicated ste genes are shown in a bar diagram with standard error (**p-value<0.001, paired t-test). (B) cDNA was synthesized by reverse transcriptase (RT) from total RNA isolated from log (L) and saturated (S) phases and subjected to RT-PCR with primers designed from the indicated ngrA and ste2 operons. (C) The PCR-amplified DNA bands were measured by ImageJ software and indicated in a bar diagram with standard error (*p-value<0.001, paired t-test). (D) Wild type (WT) and Δste2 strains of X. szentirmaii were subjected to overlay with the tester strain S. saprophyticus. The experiments were repeated at least three times. Results from one representative experiment data are shown.
Figure 6 Upregulation of operons ste2, ste9, ste10, ste14A and ste17A during the stationary phase. (A) The average FPKM counts of the indicated ste genes are shown in a bar diagram with standard error (**p-value<0.001, paired t-test). (B) cDNA was synthesized by reverse transcriptase (RT) from total RNA isolated from log (L) and saturated (S) phases and subjected to RT-PCR with primers designed from the indicated ngrA and ste2 operons. (C) The PCR-amplified DNA bands were measured by ImageJ software and indicated in a bar diagram with standard error (*p-value<0.001, paired t-test). (D) Wild type (WT) and Δste2 strains of X. szentirmaii were subjected to overlay with the tester strain S. saprophyticus. The experiments were repeated at least three times. Results from one representative experiment data are shown.CC BY · PubMed Central

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Authors
Dey, R., Valle, D. O., Chakraborty, A., Mayer, K. A., Uppala, J. K., Chakraborty, A., Mirza, S., Skwor, T., Forst, S., & Dey, M.
Affiliations
  • University of Wisconsin–Milwaukee (US)
  • La Jolla Institute for Immunology (US)
Corresponding author
Dey, Madhusudan
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2025-03-12
Volume
16
Pages
1560663-1560663
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2025.1560663
PMC
PMC11936946
OpenAlex
W4408379288
Cited by
1
References
61
Citations by year
2026: 1
FWCI
0.57
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Immunology and Microbiology › Microbiology › Antimicrobial Peptides and Activities
Keywords
Xenorhabdus szentirmaii, quorum sensing, nonribosomal peptide synthetase, polyketide synthase, antimicrobial peptides, antibiotic resistance, AI-2 signaling, stationary phase gene expression, novel antibiotics

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CoChat. (2026). Citation check: Quorum sensing regulators and non-ribosomal peptide synthetases govern antibacterial secretions in Xenorhabdus szentirmaii. https://app.cochat.ai/papers/10.3389/fmicb.2025.1560663
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Dey, R., Valle, D. O., Chakraborty, A., Mayer, K. A., Uppala, J. K., Chakraborty, A., Mirza, S., Skwor, T., Forst, S., & Dey, M. (2025). Quorum sensing regulators and non-ribosomal peptide synthetases govern antibacterial secretions in Xenorhabdus szentirmaii. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2025.1560663

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