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Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

Babin, Brett M. and Atangcho, Lydia and van Eldijk, Mark B. and Sweredoski, Michael J. and Moradian, Annie and Hess, Sonja and Tolker-Nielsen, Tim and Newman, Dianne K. and Tirrell, David A. (2017) Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms. mBio, 8 (5). Art. No. e01593-17. ISSN 2150-7511. PMCID PMC5654934.

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Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Babin, Brett M.0000-0002-4133-6665
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Newman, Dianne K.0000-0003-1647-1918
Tirrell, David A.0000-0003-3175-4596
Additional Information:Copyright © 2017 Babin et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Received 6 September 2017 . Accepted 20 September 2017. Published 24 October 2017. We thank Geoff Smith and Roxana Eggleston-Rangel for assistance with liquid chromatography-tandem mass spectrometry and Mustafa Fazli for assistance with flow cell biofilm cultures. We appreciate the constructive feedback on the manuscript provided by members of the D.K.N. and D.A.T. laboratories. D.K.N. helped support this research with grants from the Howard Hughes Medical Institute and NIH (5R01HL117328-03). T.T.-N. was supported by a grant from the Danish Council for Independent Research (DFF–1323-00177). M.B.V.E. was supported by the Netherlands Organization for Scientific Research (Rubicon fellowship 680-50-1407). The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775, the Beckman Institute, and NIH S10RR029594. This work was also supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the U.S. Government, and no official endorsement should be inferred.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Danish Council for Independent ResearchDFF–1323-00177
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)680-50-1407
Gordon and Betty Moore FoundationGBMF775
Beckman InstituteUNSPECIFIED
Army Research Office (ARO)W911NF-09-0001
Issue or Number:5
PubMed Central ID:PMC5654934
Record Number:CaltechAUTHORS:20171030-071731344
Persistent URL:
Official Citation:Babin BM, Atangcho L, van Eldijk MB, Sweredoski MJ, Moradian A, Hess S, TolkerNielsen T, Newman DK, Tirrell DA. 2017. Selective proteomic analysis of antibiotictolerant cellular subpopulations in Pseudomonas aeruginosa biofilms. mBio 8:e01593-17.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:82764
Deposited By: Donna Wrublewski
Deposited On:30 Oct 2017 16:28
Last Modified:22 Nov 2019 09:58

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