Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis

Bagert, John D.

Bassler, Bonnie L.

Feng, Lihui

Hess, Sonja

Sweredoski, Michael J.

Tirrell, David A.

van Kessel, Julia C.

PubMed

Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism
Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables
measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed
BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors.
We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the
transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both
transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86
known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription
factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis

Bagert, John D

van Kessel, Julia C

Sweredoski, Michael J

Bassler, Bonnie L

Tirrell, David A

Princeton University Open Access Repository

Chemical Science

Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi

Bio-orthogonal non-canonical amino acid tagging allows time-resolved proteomic analysis of quorum sensing inVibrio harveyi.</p

John D. Bagert

Julia C. van Kessel

Michael J. Sweredoski

Lihui Feng

Sonja Hess

Bonnie L. Bassler

David A. Tirrell

Crossref

Caltech Authors - Main

Supplementary figures 
Fig. S1. (A) The structure of L‐azidohomoalanine (Aha). (B) The structure of TAMRA‐alkyne. (C) A combined SILAC‐
BONCAT approach for quantifying differences in protein translation. Reference cultures were not treated with AI‐1 
at time 0 min. Otherwise, they were treated identically to experimental cultures. Experiments were performed in 
triplicate with one isotope label swap experiment. (D) Gel showing enrichment of Aha‐labeled proteins using the 
DADPS tag. F – flow‐through, W1‐5 – washes, E – elution. The band marked by * is monomeric avidin. (E) The 
structures of the alkyne DADPS tag and the alkyne fragment released upon cleavage.  
 
   
Electronic Supplementary Material (ESI) for Chemical Science.
This journal is © The Royal Society of Chemistry 2015
Fig. S2. Confirmation of LuxR peptide (RPRTRLSPLK) quantitation. (A) Masses in the range of 1222.7622 ± 2 ppm; 
the predicted mass of the RPRTRLSPLK peptide. Orange and blue markers represent normalized ratios of peptides 
from label swap experiments. (B) An additive model of polypeptide chromatography accurately predicts the 
retention time of the RPRTRLSPLK peptide. The measured and calculated retention times were 16.02 min and 
15.09 min, respectively. (C) Fragmentation spectra of candidate masses in the 1222.7622 ± 2 ppm range were 
matched to the RPRTRLSPLK peptide by ProteinProspector (v 5.12.4). Red text and lines denote matched 
fragmentation spectra of the RPRTRLSPLK peptide. 
 
   
Fig. S3. Summary of measurements from proteomics experiments. (A) Sorted list of MS intensities for all quantified 
proteins. (B) MS/MS spectra per protein. Number of peptides (C) and quantifications (D) for each protein, 
calculated separately for each time point.  
 
   
Fig. S4. Comparison of BONCAT proteomics data with the previously measured LuxR, AphA, and quorum‐sensing 
regulons. For each regulon, the subset of genes for which proteins were identified with and without significant 
regulation is designated.   
 
   
Fig. S5. Gene ontology analysis. Proteins from the significantly regulated gene ontology groups (Fig. 6B) are shown 
on the PCA plot. Groups were scored based on the average distance of proteins from the origin, and groups with 
fewer than 4 members were excluded. Ontology analysis used a combination of groups from the Gene Ontology 
(GO) database, and the KEGG Orthology (KO) and KEGG Module (KM) databases. 
 
   
Fig. S6. (A) The type VI secretion genes in V. harveyi are organized into five putative operons. The black asterisk 
symbol marks the location of the LuxR binding site previously identified by ChIP. Red askterisk symbols mark newly 
identified LuxR binding sites. (B) Up‐regulation of type VI secretion operons at HCD is LuxR‐dependent and AphA‐
independent. Results are from van Kessel et al. (2013). These data  show relative gene expression values from 
∆aphA (JV48), ∆luxR (KM669), and ∆aphA ∆luxR (STR417) V. harveyi strains relative to wild‐type (BB120; wt) at 
varying cell densities. (C) EMSAs for reaction mixtures containing 0, 10, 100, or 1000 nM LuxR incubated with 1 nM 
radiolabeled DNA substrate corresponding to the three TSSS promoter regions for VIBHAR_05855–56, 
VIBHAR_05865, or VIBHAR_05871–72. 
 
Supplementary tables 
Table S1. Calculation of Aha incorporation based on total evidence counts, MS‐MS counts, and MS intensity 
provides estimates in a range of 13−17%. 
 Measure of 
abundance 
Aha 
peptides 
Met 
peptides 
All 
peptides 
Calculated 
Aha 
incorporation 
Evidence Counts  26,496  131,808  158,304  16.7% 
MS‐MS Counts  23,285  147,918  171,203  13.6% 
MS Intensity  5.09x1011  2.72x1012  3.23x1012  15.8% 
 
 
Table S2. The weights used to transform protein ratios into principal component space. The mean (µ) and standard 
deviation (σ) of each sample were used to standardize the original variables prior to multivariate analysis. 
  10 min  20 min  30 min  40 min  50 min  60 min  70 min  80 min  90 min  Variance accounted for 
PC1  0.128  0.254  0.300  0.343  0.399  0.325  0.378  0.387  0.393  50% 
PC2  0.583  0.407  0.428  0.196  ‐0.018  ‐0.360  ‐0.259  ‐0.220  ‐0.168  13% 
PC3  0.782  ‐0.491  ‐0.258  ‐0.127  0.023  0.224  0.109  0.038  0.018  9% 
PC4  ‐0.162  ‐0.652  0.385  0.526  0.092  0.026  ‐0.257  ‐0.212  0.064  7% 
PC5  0.042  0.296  ‐0.473  0.523  ‐0.273  0.392  ‐0.119  ‐0.386  0.147  6% 
PC6  ‐0.020  0.088  0.446  ‐0.335  ‐0.181  0.736  ‐0.202  ‐0.091  ‐0.239  5% 
PC7  0.029  ‐0.080  0.294  ‐0.100  ‐0.684  ‐0.135  0.406  ‐0.152  0.472  4% 
PC8  ‐0.031  ‐0.047  0.042  0.321  ‐0.175  0.018  0.593  0.032  ‐0.713  3% 
PC9  0.045  ‐0.022  ‐0.039  0.228  ‐0.475  ‐0.015  ‐0.375  0.757  ‐0.061  3% 
µ  0.001  ‐0.004  0.028  0.003  0.050  0.025  0.052  0.047  0.021  ‐ 
σ  0.203  0.250  0.311  0.259  0.451  0.381  0.444  0.575  0.375  ‐ 
   
Table S3. Timings of significantly regulated proteins that are directly regulated by LuxR and the Qrr sRNAs. 
LuxR‐regulated genes  Qrr sRNA‐regulated genes 
Gene 
Time first 
significant 
(min) 
Log2 H/L 
ratio  Gene 
Time first 
significant 
(min) 
Log2 H/L 
ratio 
VIBHAR_06238  40–50  3.11  VIBHAR_00417  0–10  0.66 
VIBHAR_06244  40–50  3.24  VIBHAR_06667  10–20  0.78 
VIBHAR_02988  70–80  ‐0.73  VIBHAR_06666  10–20  0.92 
VIBHAR_06253  70–80  ‐1.07  VIBHAR_02446  10–20  0.75 
VIBHAR_01749  70–80  0.97  VIBHAR_03459  0–10  1.13 
VIBHAR_01762  20–30  ‐0.69  VIBHAR_02959  0–10  0.74 
VIBHAR_00081  50–60  ‐0.85  VIBHAR_00046  10–20  ‐1.22 
VIBHAR_03197  40–50  ‐0.82       
VIBHAR_06838  50–60  ‐3.13       
VIBHAR_05086  40–50  0.96       
VIBHAR_02986  30–40  ‐0.64       
VIBHAR_04809  0–10  0.60       
VIBHAR_02041  70–80  1.08       
VIBHAR_06007  40–50  ‐0.94       
VIBHAR_01133  20–30  ‐0.77       
VIBHAR_02617  20–30  ‐1.00       
VIBHAR_06860  70–80  1.54       
VIBHAR_01256  50–60  ‐1.03       
VIBHAR_03248  70–80  ‐1.63       
VIBHAR_05968  80–90  ‐0.73       
VIBHAR_01398  70–80  ‐0.64       
 
   
Table S4. Oligonucleotides used in this study. 
Name  Sequence
P05855‐56  GGGCGAAAGATATCAAGTCTCTCTT
P05855‐56  ATTTTCCAATTCCAACTGATTATATGAAGG
P05865  GTTGCTCTTCACTAGCGCTCTTG
P05865  CCTTGTTTCAAGGCTGGTATTTAAA
P05871‐22  ATATGCTGGAGTTGGCATCGTTATT
P05871‐22  TTTATTCTTTAGAGGAAAAGAGGTGGTC
aphA qRT‐PCR  ATCCATCAACTCTAGGTGATAAAC
aphA qRT‐PCR  CGTCGCGAGTGCTAAGTACA
luxO qRT‐PCR  GCATTCCTGATCTTATTCTGCTCG
luxO qRT‐PCR  TCCATCCCCGTCATATCAGGTA
luxR qRT‐PCR   GCAAAGAGACCTCGTACTAGG
luxR qRT‐PCR   GCGACGAGCAAACACTTC
02788 qRT‐PCR  TGTTTAACAGTATACGTACTCGAATCG
02788 qRT‐PCR  TCAGTAAATGCATCGGTAGTCAT
05853 qRT‐PCR  CAACAGGCATTACGCCAG
05853 qRT‐PCR  CGCAAAATAACTGGAGAGGATTG
05857 qRT‐PCR  CGATTTTGGTTTCTACCATAGTGG
05857 qRT‐PCR  CAATCCATAGATATAGCTTATCTGCATCT
05861 qRT‐PCR  GTTATCGTCTTTTAGAGCGGATTG
05861 qRT‐PCR  AGGTGAGAATGAATGGATTCGAT
05864 qRT‐PCR  GATTGATATCGAACGTTTGCTTACG
05864 qRT‐PCR  CTTCACTTCGGATTCCATCATTTC
05871 qRT‐PCR  GTGAAACTCAAGGTCACATCAC
05871 qRT‐PCR  AGTTCTTGAACTAGGAACTCATCAA
05872 qRT‐PCR  GTCTCTGAAAAAGCAAACGAAGT
05872 qRT‐PCR  GATTGTCTTAATAAGTTCTCAGAACAATCA
 
 


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Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi

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