SlyA regulates type III secretion system (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937

Abstract

The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.

Fig 1

The slyA locus of D. dadantii 3937. The MiniHimar RB1 of four transposon mutants was inserted at bp 120 (Ech4396), 122 (Ech4380), 177 (Ech4120), and 392 (Ech3049) of the 441-bp nucleotide in the slyA ORF.

Fig 2

(A) Expression ratios of hrpA, hrpN, hrpS, and hrpL mRNAs in slyA mutant Ech163 compared to those of D. dadantii 3937. (B) The relative mRNA levels of slyA in the expI mutant Ech164 and the expR mutant Ech165 compared to that of D. dadantii 3937. Bacterial strains were grown in minimal medium for 12 h. Total RNA was isolated, and the mRNA of target genes was measured by real-time RT-PCR. Asterisks indicate statistically significant differences in mRNA levels of the mutants compared to that of the wild type (P < 0.05). Similar results were observed in two independent experiments.

Fig 3

(A) Northern blot analysis of rsmA in D. dadantii 3937 (lane 1), slyA mutant Ech163 (lane 2), and slyA chromosomal complementation strain Ech166 (lane 3). (B) Northern blot analysis of rsmB mRNA in D. dadantii 3937 (lane 1) and Ech163 (lane 2). Similar results were observed in two independent experiments.

Fig 4

(A) Northern blot analysis of hrpA in wild-type D. dadantii 3937 (lane 1), slyA mutant Ech163 (lane 2), and Ech166, the chromosomal complemented strain of Ech163 (lane 3). (B) Northern blot analysis of hrpA mRNA in D. dadantii 3937 carrying empty vector pML123 (lane 1), D. dadantii 3937 carrying hrpL overexpression plasmid pMLhrpL (lane 2), slyA mutant Ech163 carrying pML123 (lane3), Ech163 carrying pMLhrpL (lane 4), hrpS mutant Ech188 carrying pML123 (lane 5), and Ech188 carrying pMLhrpL (lane 6). Similar results were observed in two independent experiments.

Fig 5

(A) Northern blot analysis of rsmA mRNA in D. dadantii 3937 (lane 1), the expI mutant Ech164 (lane 2), and the expR mutant Ech165 (lane 3). (B) Northern blot analysis of hrpA in D. dadantii 3937 (lane 1), expI mutant Ech164 (lane 2), expR mutant Ech165 (lane3), and hrpY mutant WPP92 (lane 4). Similar results were observed in two independent experiments.

Fig 6

(A) Swimming motility of D. dadantii 3937 (lane 1), slyA mutant Ech163 (lane 2), and Ech166 (lane 3), a slyA chromosomal complementation strain of Ech163. (B) Pellicle formation of D. dadantii 3937 (lane 1), Ech163 (lane 2), and Ech166 (lane 3). Similar results were observed in two independent experiments with two biological replicates in each experiment.

Fig 7

The regulation of SlyA on the T3SS expression and extracellular enzyme production. SlyA negatively regulates the transcription of hrpS, through an HrpX/HrpY-independent pathway. It also upregulates the expression of rsmA. The influence of SlyA on hrpS and rsmA leads to a downregulation of hrpL. However, SlyA upregulates the expression of the HrpL regulon genes, such as hrpA and hrpN, in parallel with HrpL. The lines with bars and arrows represent negative and positive regulations, respectively.