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Peptide-based quorum sensing systems in Paenibacillus polymyxa

View ORCID ProfileMaya Voichek, View ORCID ProfileSandra Maaß, Tobias Kroniger, Dörte Becher, View ORCID ProfileRotem Sorek  Correspondence email
Maya Voichek
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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  • ORCID record for Maya Voichek
Sandra Maaß
2Department of Microbial Proteomics, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
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Tobias Kroniger
2Department of Microbial Proteomics, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
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Dörte Becher
2Department of Microbial Proteomics, Institute of Microbiology, Center for Functional Genomics of Microbes, University of Greifswald, Greifswald, Germany
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Rotem Sorek
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
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  • For correspondence: rotem.sorek@weizmann.ac.il
Published 6 August 2020. DOI: 10.26508/lsa.202000847
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  • Figure 1.
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    Figure 1. Alo systems identified in P. polymyxa.

    (A) Schematic representation of aloR-aloP operon organization and putative peptide processing. HTH, helix-turn-helix; TPR, tetratricopeptide repeat. (B) Alo loci identified in P. polymyxa ATCC 842. For the aloR receptor genes, the locus tag in the Integrated Microbial Genomes (IMG) database (49) is specified. Blue sequence in AloP peptides represents the predicted N-terminal signal sequence for secretion, as predicted by Phobius (50) (see the Materials and Methods section). Asterisks (*) mark cases in which the aloP gene was absent or found as a degenerate sequence.

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    Figure 2. Phylogenetic distribution of Alo systems among P. polymyxa strains.

    (A) Phylogenetic tree of 234 AloR homologs found in 14 P. polymyxa strains. Colored segments in the tree circumference define clades of AloR proteins corresponding to the systems found in P. polymyxa ATCC 842 (labeled as arrows). Systems immediately followed by a Spo0E-like protein are indicated. Tree was constructed using IQ-Tree (55, 56, 57) with 1,000 iterations, and ultrafast bootstrap support values are presented for major branches. Tree visualization was done with iTOL (58). Alo1/8 refers to a system duplicated in P. polymyxa ATCC 842 (where it is represented as Alo1 and Alo8). (B) Frequency and distribution of Alo systems in P. polymyxa strains. The number of copies per Alo system found within P. polymyxa strains is shown using colored squares. The absence of an Alo system in a specific strain is marked by a white square. (A) Phylogenetic tree of the Alo systems shown on the vertical axis is derived from the tree in the panel (A). Phylogenetic tree of the strains shown on the horizontal axis is based on the GyrA protein, with GyrA from B. subtilis 168 as an out-group.

  • Figure 3.
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    Figure 3. Mass spectrometry (MS)–based identification of secreted communication peptides.

    (A) Phr peptide fragments detected in the growth media of B. subtilis 168. Peptide fragments identified by MS are underlined, with the cumulative number of spectra detected in all tested repeats indicated (see the Materials and Methods section). Known mature communication peptides are in red (26, 45). The two most abundantly identified fragments are presented for each protein by an underline. The mature peptides which were not directly detected are inferred from the C-terminal sequence of the full peptides, presumably after processing. (B) AloP peptide fragments detected by MS. Same as (A) for AloP peptide fragments detected in the growth media of P. polymyxa ATCC 842.

  • Figure 4.
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    Figure 4. The mature AloP13 peptide elicits an immediate transcriptional response.

    (A) Volcano plot depicting P. polymyxa gene expression after incubation with 5 μM of the peptide SHGRGG for 10 min, as compared to control conditions in which no peptide was added. X-axis, log2 fold expression change (adjusted using the lfcShrink function in the DESeq2 R package (62); see the Materials and Methods section). Y-axis, −log10 P-value. Average of three independent replicates; each dot represents a single gene. Dots appearing in colors are genes that passed the threshold for statistical significance of differential expression (see the Materials and Methods section). Red dots correspond to genes encoded by Alo systems. (B) Differentially expressed genes as appears in panel (A). Fold change represents the average of the three independent replicates. P-adjusted is the P-value after correction for multiple hypothesis testing (see the Materials and Methods section). (C) RNA-seq coverage of the Alo13-Alo14 locus (Scaffold: PPTDRAFT_AFOX01000049_1.49), in control conditions (green) or 10 min after addition of 5 μM of the SHGRGG peptide (black). RNA-seq coverage is in log scale and was normalized by the number of uniquely mapped reads in each condition. Representative of three independent replicates. (D) RNA-seq coverage of the Alo13-Alo14 locus in control conditions (green) or 10 min after addition of 5 μM of a scrambled version (GRGSGH) of the AloP13 peptide. RNA-seq coverage is in log scale and normalized as in the panel (C). Representative of three independent replicates. (E) Schematic representation of the genetic construct used in B. subtilis to verify the activity of AloR13. The start codon of the aloP13 gene (dashed blue arrow) was mutated to inactivate it. GFP was placed as a reporter gene instead of the Spo0E ORF. (F) GFP fluorescence upon addition of the AloP13 peptide SHGRGG in varying concentrations, or the scrambled mature peptide GRGSGH in 5 μM (“scrambled”), or no peptide added. Dashed lines represent GFP fluorescence measured without the addition of IPTG.

  • Figure S1.
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    Figure S1. RT-qPCR analysis of the spo0E-like gene expression in P. polymyxa.

    Normalized RNA levels of the spo0E-like gene in P. polymyxa (PPTDRAFT_04974) were measured with RT-qPCR, after treatment with 5 μM AloP13 mature peptide (SHGRGG, gray bars), AloP13 scrambled mature peptide (GRGSGH, blue bars), or no added peptide (green bars). Each experiment was done in two biological replicates and measured with two sets of primers matching different regions of the gene sequence (see the Materials and Methods section). (A) Primers set #1. (B) Primers set #2. For (A, B), error bars indicate SD of 2–3 measurements for each sample, and the housekeeping gene gyrB was used for normalization. Relative mRNA levels were calculated compared to “no peptide repeat A.”

  • Figure S2.
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    Figure S2. Phylogenetic tree of AloR13 and representative RRNPP receptors.

    The aa sequences of AloR13 and representative RRNPP receptors were aligned and phylogenetic tree was constructed as in Fig 2. NCBI accessions of representative RRNPP receptors were taken from Fig S5 of reference 59 as follows: RapE (AAM51168), RapA (AAM51160), RapC (AAT75294), NprR (ABK83928), transcriptional regulator Enterococcus faecalis (T.reg E. faecalis, NP_815038), DNA-binding protein Bacillus anthracis (DNAbd B. anthracis, NP_843644), PlcR (ZP_00739149), PrgX (AAA65845), TraA BAA11197, transcriptional activator L. monocytogenes (T.act L. monocytogenes, YP_013453), transcriptional regulator L. casei (T.reg_Lcas, YP_805489), MutR (AAD56141), and RggD (AAG32546).

Tables

  • Figures
  • Supplementary Materials
  • Primers used in this study.

    NameSequenceUse
    Spo0E_1_FTGGCGAAAGAGGTGGGATTGART-qPCR of spo0E-like gene (PPTDRAFT_04974)—primers set 1
    Spo0E_1_RGGGTACGCTCTGTTGTCTACGART-qPCR of spo0E-like gene (PPTDRAFT_04974)—primers set 1
    Spo0E_2_FGAGTTGGATCGGCTCCTTAATRT-qPCR of spo0E-like gene (PPTDRAFT_04974)—primers set 2
    Spo0E_2_RCTCGTGTTGCTTCTCTTGGART-qPCR of spo0E-like gene (PPTDRAFT_04974)—primers set 2
    gyrB_FGCCAGCGATACATTCCACTATRT-qPCR of gyrB gene for normalization
    gyrB_RCACGAGAGCCTTCGACATAAART-qPCR of gyrB gene for normalization
    Alo13_FATGGAAAATGCAACCACGATTCUsed to amplify the Alo13 locus for cloning (start of AloR13 ORF)
    Alo13_RATCAATAAACCTCCTGTTCGGTGUsed to amplify the Alo13 locus for cloning (end of Spo0E promoter)
    Alo13_Gib_FCACCGAACAGGAGGTTTATTGATatgtcaaaaggagaagaactttttacagUsed for Gibson cloning of Alo13_F and Alo13_R amplified fragment (lower case letters correspond to beginning of sfGFP)
    Alo13_Gib_RGAACGAATCGTGGTTGCATTTTCCATgtttgtcctccttattagttaatcagcUsed for Gibson cloning of Alo13_F and Alo13_R amplified fragment (lower case letters correspond to RBS sequence following hyper-spank promoter)
    AloP13_mut_FTGTGCAAAGACGAAGAAGGTTTTTCUsed for site-directed mutagenesis of AloP13 start codon ATG > ACG
    AloP13_mut_RCCCCCTTAATAATAGATTTATAAATTTCUsed for site-directed mutagenesis of AloP13 start codon ATG > ACG

Supplementary Materials

  • Figures
  • Tables
  • Supplemental Data 1.

    TPRpred analysis results for AloR proteins.[LSA-2020-00847_Supplemental_Data_1.pdf]

  • Table S1 AloR genes and corresponding AloP sequences identified in P. polymyxa strains.

  • Table S2 AloR homologs in microbial genomes.

  • Table S3 Alo systems in P. terrae NRRL B-30644.

  • Table S4 All peptide fragments detected in mass spectrometry analysis.

  • Table S5 Differential gene expression analysis at 10 min mature AloP13, scrambled AloP13 pro-peptide, and no peptide (control) incubation.

  • Table S6 Differential gene expression analysis at 10 min mature AloP13 and scrambled mature AloP13 peptide incubation.

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Quorum sensing systems in P. polymyxa
Maya Voichek, Sandra Maaß, Tobias Kroniger, Dörte Becher, Rotem Sorek
Life Science Alliance Aug 2020, 3 (10) e202000847; DOI: 10.26508/lsa.202000847

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Quorum sensing systems in P. polymyxa
Maya Voichek, Sandra Maaß, Tobias Kroniger, Dörte Becher, Rotem Sorek
Life Science Alliance Aug 2020, 3 (10) e202000847; DOI: 10.26508/lsa.202000847
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Volume 3, No. 10
October 2020
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