The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

Andreas Dötsch,Susanne Häussler,Maren Scharfe,Vanessa Jensen,Ariane Zimmermann,Denitsa Eckweiler,Robert Geffers,Monika Schniederjans,Szabolcs Semsey

doi:10.1371/journal.pone.0031092

Andreas Dötsch, Susanne Häussler + Show 7 more

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https://doi.org/10.1371/journal.pone.0031092

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Abstract

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

Highlights

The study of gene expression as a function of its adaptation to a specific niche reveals important insights into how bacteria sense and respond to varied habitats
Sequencing of the P. aeruginosa PA14 transcriptome In this study we aimed at using RNA sequencing for the identification of the global gene expression pattern in P. aeruginosa when grown within biofilms
Total RNA was isolated from two biological replicates of planktonic cultures of P. aeruginosa PA14 in the late exponential (P-4) and stationary growth phase (P12) as well as from static biofilm cultures that were harvested after 24 h (B-24) and 48 h (B-48), respectively

Summary

Introduction

The study of gene expression as a function of its adaptation to a specific niche reveals important insights into how bacteria sense and respond to varied habitats. Thereby, the question of how gene expression differs in mature biofilms as opposed to planktonic cells has become a major research focus in the last decade [1,2,3,4], because the biofilm mode of growth is intimately connected to chronic persistent infections [5,6,7]. One example of a chronic infection of major clinical importance is the persistent colonization of the lungs of cystic fibrosis patients with Pseudomonas aeruginosa [11,12]. Once established, these chronic infections cannot be eradicated despite even intensified antimicrobial therapy [8,12,13]. The commercially available and widely used PaeG1a microarray (Affymetrix) is almost entirely restricted to genes that are present in the PAO1 genome and lacks non-coding genes especially those of small regulatory RNAs (sRNAs), a limitation that can be overcome by sequencing based transcriptome analysis

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