Articles
MICROARRAY CHARACTERIZATION OF THE HRPL REGULON OF THE FIRE BLIGHT PATHOGEN ERWINIA AMYLOVORA
Article number
896_36
Pages
263 – 270
Language
English
Abstract
Microarrays represent a powerful tool for the rapid and comprehensive identification of differentially-regulated genes within a genome.
We developed an oligo microarray for the annotated genes of the fire blight pathogen E. amylovora Ea273, including plasmids pEA29 and plasmid 2, and including additional genes from plasmids pEL60 and pEU30 found in related strains.
The microarray was validated through analysis of differential gene expression of a wild-type and ΔhrpL mutant of E. amylovora Ea1189. HrpL is the cognate sigma factor of hypersensitive and pathogenicity (hrp) promoters, and the goal of this work was to define the HrpL regulon of E. amylovora. Both Ea1189 and Ea1189ΔhrpL were cultured in hrp-inducing minimal medium, and total RNA was isolated after 6 and 18 h incubation.
Results revealed that 24 genes were significantly and differentially regulated in Ea1189ΔhrpL with fold-change expression ratios greater than 1.5. Our analysis uncovered 19 genes that may be directly or indirectly under the positive regulation of HrpL and 5 genes that were negatively-regulated.
The type III secretion system (T3SS) pilus subunit gene hrpA exhibited the largest expression fold-change and is part of a group of HrpL-regulated genes unveiled in our analysis with known roles in T3SS including hrpN, hrpW, dspA/E, and eopB. In addition to T3SS genes, we also discovered HrpL-dependent regulation of EAM_2938, a new gene located outside of the T3SS pathogenicity island.
To uncover new hrp promoters important for the direct HrpL-mediated positive regulation of genes like EAM_2938, the genome of E. amylovora Ea273 was examined using a hidden Markov model assembled from known Erwinia spp. hrp promoters using T-coffee multiple alignment software and HMMer 2.3.2. Preliminary bioinformatic results support the existence of a novel hrp promoter upstream of EAM_2938 as well as additional new hrp promoters that may help reveal the broader HrpL regulon.
We developed an oligo microarray for the annotated genes of the fire blight pathogen E. amylovora Ea273, including plasmids pEA29 and plasmid 2, and including additional genes from plasmids pEL60 and pEU30 found in related strains.
The microarray was validated through analysis of differential gene expression of a wild-type and ΔhrpL mutant of E. amylovora Ea1189. HrpL is the cognate sigma factor of hypersensitive and pathogenicity (hrp) promoters, and the goal of this work was to define the HrpL regulon of E. amylovora. Both Ea1189 and Ea1189ΔhrpL were cultured in hrp-inducing minimal medium, and total RNA was isolated after 6 and 18 h incubation.
Results revealed that 24 genes were significantly and differentially regulated in Ea1189ΔhrpL with fold-change expression ratios greater than 1.5. Our analysis uncovered 19 genes that may be directly or indirectly under the positive regulation of HrpL and 5 genes that were negatively-regulated.
The type III secretion system (T3SS) pilus subunit gene hrpA exhibited the largest expression fold-change and is part of a group of HrpL-regulated genes unveiled in our analysis with known roles in T3SS including hrpN, hrpW, dspA/E, and eopB. In addition to T3SS genes, we also discovered HrpL-dependent regulation of EAM_2938, a new gene located outside of the T3SS pathogenicity island.
To uncover new hrp promoters important for the direct HrpL-mediated positive regulation of genes like EAM_2938, the genome of E. amylovora Ea273 was examined using a hidden Markov model assembled from known Erwinia spp. hrp promoters using T-coffee multiple alignment software and HMMer 2.3.2. Preliminary bioinformatic results support the existence of a novel hrp promoter upstream of EAM_2938 as well as additional new hrp promoters that may help reveal the broader HrpL regulon.
Publication
Authors
R.R. McNally, G.W. Sundin, Y.F. Zhao, I.K. Toth, P.J.A. Cock, P.E. Hedley
Keywords
type III secretion, hrp box, alternate sigma factor
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