Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum

Abstract

Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.

Figure 1

Map of the R. erythropolis R138 genome. The innermost and second circles highlight GC skew and GC content (%), respectively. The third circle shows RNA genes location (tRNAs in blue color, rRNAs in red, other RNAs in black). The fourth and fifth circles show the distribution of genes on the reverse (orange) and forward (green) strand, respectively.

Figure 2

Colonization of potato tubers by R. erythopolis and P. atrosepticum. (a) Growth (c.f.u. per tuber slice) of R. erythropolis R138 (square) and P. atrosepticum CFBP6276 (triangle) populations colonizing the potato tuber slices; (b) Quantification of the NAHL signals (fmol per tuber slice) emitted by P. atrosepticum CFBP6276 (c); Symptoms observed 24 h after inoculation of R. erythropolis R138, P. atrosepticum CFBP6276 and a combination of both the populations on potato tuber slices. All assays for cell growth, NAHL and symptom monitoring were performed in triplicates.

Figure 3

Highly expressed genes of R. erythropolis R138 colonizing the potato tubers. Distribution and relative expression level of the highly expressed genes when R. erythropolis R138 colonized alone the potato tuber slices. We showed only genes exhibiting a RPKM median value which was over by three times the RPKM median value of all genes. All these genes are listed in Supplementary Table S3. RNA extraction and sequencing were performed in triplicates.

Figure 4

In planta expression of alkylsulfatases ORF4771, ORF4816 and ORF4877. Gene expression was measured using RNAseq (a) and qRT-PCR (b) when R. erythropolis R138 colonized alone the potato tuber slices (gray bars) and with P. atrosepticum CFBP6276 (white bars). Relative gene expression is normalized using that of recA gene. Experiments were performed with three biological triplicates.

Figure 5

In vitro expression of alkylsulfatases ORF4771, ORF4816 and ORF4877. R. erythropolis R138 was cultivated in the presence of high (gray bars) and low (white bars) concentration of MgSO₄ or methionine. Relative gene expression is normalized using that of recA gene. Experiments were performed with three biological replicates. Statistical differences were assessed using the Mann–Whitney test (P<0.05).