Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665T

Abstract

Pantoea ananatis LMG 2665^T synthesizes and utilizes acyl homoserine lactones (AHLs) for signalling. The complete set of genes regulated by the EanI/R quorum sensing (QS) system in this strain is still not fully known. In this study, RNA-sequencing (RNA-seq) was used to identify the EanI/R regulon in LMG 2665^T. Pairwise comparisons of LMG 2665^T in the absence of AHLs (Optical density (OD)₆₀₀ = 0.2) and in the presence of AHLs (OD₆₀₀ = 0.5) were performed. Additionally, pairwise comparisons of LMG 2665^T and its QS mutant at OD₆₀₀ = 0.5 were undertaken. In total, 608 genes were differentially expressed between LMG 2665^T at OD₆₀₀ = 0.5 versus the same strain at OD₆₀₀ = 0.2 and 701 genes were differentially expressed between LMG 2665^T versus its QS mutant at OD₆₀₀ = 0.5. A total of 196 genes were commonly differentially expressed between the two approaches. These constituted approximately 4.5% of the whole transcriptome under the experimental conditions used in this study. The RNA-seq data was validated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Genes found to be regulated by EanI/R QS were those coding for redox sensing, metabolism, flagella formation, flagella dependent motility, cell adhesion, biofilm formation, regulators, transport, chemotaxis, methyl accepting proteins, membrane proteins, cell wall synthesis, stress response and a large number of hypothetical proteins. The results of this study give insight into the genes that are regulated by the EanI/R system in LMG 2665^T. Functional characterization of the QS regulated genes in LMG 2665^T could assist in the formulation of control strategies for this plant pathogen.

Keywords: LMG 2665T; Pantoea ananatis; RNA-seq; acyl homoserine lactones; quorum sensing; regulon.

Figure 1

CV026 bioassay results from samples of the wild-type collected at OD₆₀₀ = 0.2 and OD₆₀₀ = 0.5. The purple halo indicates presence of acyl homoserine lactones (AHLs) in culture supernatant at OD₆₀₀ = 0.5. The purple colour is a result of violacein pigment production by the CV026 bio reporter strain. The absence of purple colour at OD₆₀₀ = 0.2 shows absence of AHLs and absence of quorum sensing.

Figure 2

RT-qPCR validation of RNA-sequencing (RNA-seq) data using six selected genes and wild-type, P. ananatis LMG 2665^T RNA samples. The fold change in gene expression levels between sampling points, before Quorum Sensing (QS) (wild-type strain at OD₆₀₀ = 0.2) and in the presence of QS (wild-type strain at OD₆₀₀ = 0.5) were calculated using the comparative cycle threshold (C_T) method [24] and data normalization was done using the ffh gene. The RT-qPCR results indicate fold change as cells shifted from before quorum sensing (OD₆₀₀ = 0.2) to during quorum sensing (OD₆₀₀ = 0.5) in LMG 2665^T. The fold change in RNA-seq data were calculated using 2^{(log₂FoldChange)}. Error bars represent the range of relative expression calculated using 2^{−(ΔΔC_T ± StandardDeviation)}. Triplicates were used per biological sample. The genes aroE and the one encoding for glycoside hydrolase were not differentially expressed in RNA-seq data (M5 versus W5).

Figure 3

Identification of the EanI/R QS regulon. The genes that were differentially expressed between the sampling points before QS and during QS (W2 versus W5) that were also differentially expressed between the wild type and its QS mutants (M5 versus W5) were considered to be under QS regulation. A total of 196 genes were found to be influenced by EanI/R QS system. The number of genes in W2 versus W5 in the Venn diagram is 593 since 15 genes in W2 versus W5 data set encode for proteins with unknown protein ID. The 15 genes have protein IDs indicated as not available (N/A) in Table S2.

Figure 4

Functional categories of genes in the EanI/R QS regulon. The figure represents those genes in the EanI/R regulon that were differentially expressed in the wild type strain, LMG 2665^T between the two sampling points before QS and during QS. The 194 genes were grouped into different functional categories. The up regulated groups included those for flagella formation and flagella mediated motility, methyl accepting proteins and chemotaxis, redox sensing and cell adhesion. The groups with genes that were either up or down regulated included hypothetical proteins, transport, stress response, metabolism, cell wall synthesis, outer membrane proteins and regulators.