Genome-Wide Identification of Genes Regulated by the Rcs Phosphorelay System in Erwinia amylovora

Abstract

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.

Differential regulation of phenazine biosynthesis by RpeA and RpeB in Pseudomonas chlororaphis 30-84

“…For instance, the expression of cupD in P. aeruginosa PA14 is activated by the RcsB response regulator but repressed by its putative cognate sensor RcsC (Nicastro et al, 2009). In the bacterium Erwinia amylovora, RcsB positively controls the expression of amylovoran biosynthetic genes both in vitro and in vivo; whereas RcsC acts as a positive regulator in vivo, but as a negative regulator in vitro (Wang et al, 2009(Wang et al, , 2011b(Wang et al, , 2012. It has been well-documented that the sensor kinase protein has both kinase and phosphatase activities (Krell et al, 2010).…”

“…qPCR was performed at the Gene Expression Service Center at Texas A&M University using a previously described method with a few modifications (Wang et al, 2011a(Wang et al, , 2012. SYBR Green reactions were performed using the ABI 7900 HT Fast System (Applied Biosystems) in 384-well optical reaction plates.…”

“…Primer pairs rpeART1-rpeART2, rpeBRT1-rpeBRT2, pipRT1-pipRT2, phzRRT1-phzRRT2, phzIRT1-phzIRT2, phzBRT1-phzBRT2, hfqRT1-hfqRT2, rpoDRT1-rpoDRT2, csaIRT1-csaIRT2, csaRRT1-csaRRT2, 16RT1-16RT2 and rpoSRT1-rpoSRT2 were used to detect the expression of rpeA, rpeB, pip, phzR, phzI, hfq, rpoD, csaI, csaR, 16S rDNA and rpoS genes, respectively (Table 2). qPCR amplifications were carried out at 50 uC for 2 min and (Wang et al, 2011a(Wang et al, , 2012.…”

Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA

“…Reads were mapped on 5,892 annotated coding sequences (CDS) of strain PA14 using CLC Genomic Workbench 7.5 software. Transcript abundance and differential-expression results between the three replicates of each sample (PA14/PJ01, PA14/PJ01ΔmexT, and PJ01/PJ01ΔmexT) were determined with the Cufflinks and Cuffdiff algorithms (48). A difference in gene expression was considered significant when the false-discovery rate (q value) was Յ0.05 and the expression ratio was Յ0.3-or Ն3.0-fold.…”

Global Small RNA Chaperone Hfq and Regulatory Small RNAs Are Important Virulence Regulators in Erwinia amylovora

“…In order to successfully establish infections, E. amylovora utilizes a complicated regulatory network involving two-component signal transduction systems, alternate sigma factors, quorum sensing, and the second messenger cyclic di-GMP to collectively control the expression of virulence genes at different stages of infection (1,4,10,(15)(16)(17)(18). In addition, the requirement at different times of infection for the T3SS and biofilm formation and the importance of cellular motility suggest that E. amylovora cells must be able to rapidly alter the production of distinct virulence factors in response to specific host cues.…”