Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression

Abstract

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA(-) FlhDC(-) double mutant and multiple copies of rsmB(+) DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora.

FIG. 1.

Model depicting the regulatory network controlling the production of extracellular enzymes, HrpL, Harpin, and AHL, as well as motility, pathogenicity, and the hypersensitive response in E. carotovora subsp. carotovora (see the text for the details). The regulatory steps indicated by broken lines with arrows are based upon the results presented in this report.

FIG. 2.

Characteristics of an FlhDC^- mutant (1). Ecc71 and (2) its FlhDC^- mutant AC5140. (A) Northern blot analysis of flhD and flhC; (B) Pel activities; (C) agarose plate assays of Peh, Prt, and Cel activities; (D) Northern blot analysis of pel-1, peh-1, celV, hrpL, and hrpN. For panels A and D, each lane contained 15 μg of total RNA. The arrows show the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel. (E) Soft-rot disease symptoms in celery petiole; (F) Western blot analysis of Harpin_Ecc protein. Each lane contained 20 μg of total protein.

FIG. 3.

Reversal of the pleiotropic phenotype of the FlhDC^- mutant by flhDC⁺ DNA. (A) Pel activities; (B) agarose plate assays of Peh, Prt, and Cel activities; (C) Northern blot analysis of pel-1, peh-1, celV, hrpN, hrpL, and rsmC. Each lane contained 15 μg of total RNA. The arrow shows the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel. (D) Western blot analysis of Harpin_Ecc of the FlhDC^- mutant AC5140 carrying pCL1920 (cloning vector) (lane 1), pAKC1241 (flhD⁺) (lane 2), or pAKC1242 (flhDC⁺) (lane 3). Each lane contained 20 μg of total protein.

FIG. 4.

FlhDC controls regulatory genes for exoprotein production and motility. (A) Northern blot analysis of gacA, rsmA, rsmC, hexA, and fliA in Ecc71 (lane 1) and its FlhDC^- mutant AC5140 (lane 2). Each lane contained 15 μg of total RNA. The arrow shows the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel. (B and C) β-Galactosidase activities of transcriptional gacA-lacZ fusion pAKC1243 and rsmC-lacZ fusion pAKC1244 in AC5006 and AC5141, respectively.

FIG. 5.

Reversal of the pleiotropic phenotype of the FlhDC^- mutant by gacA⁺ DNA, as well as by HexA deficiency. (A) Pel activities; (B) agarose plate assays of Peh, Prt, and Cel activities; (C) Northern blot analysis of pel-1, peh-1, celV, hrpN, hrpL, rsmB, and rsmC. Each lane contained 15 μg of total RNA. The arrow shows the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel. (D) Western blot analysis of Harpin_Ecc in the FlhDC^- mutant AC5140 carrying pAKC1242 (flhDC⁺) (lane 1) or pAKC1245 (gacA⁺) (lane 2). Each lane contained 20 μg of total protein. (E) Agarose plate assays of Pel, Peh, Prt, and Cel activities in FlhDC^- HexA⁺ strain AC5140 (column 1) and FlhDC^- HexA^- strain AC5145 (column 2).

FIG. 6.

Restoration of extracellular enzyme production by gacA in an FlhDC^- GacA^- double mutant, AC5143. (A and B) Pel activities (A) and agarose plate assays of Peh, Prt, and Cel activities (B) in AC5143 carrying the cloning vector pCL1920 (column 1), the flhDC⁺ plasmid pAKC1242 (column 2), or the gacA⁺ plasmid pAKC1245 (column 3).

FIG. 7.

Reversal of extracellular enzyme production of the FlhDC^- mutant by rsmB⁺ DNA. (A and B) Pel activities (A) and agarose plate assays of Peh, Prt, and Cel activities (B) in FlhDC^- mutant AC5141 carrying pCL1920 (column 1) or pAKC1049 (rsmB⁺) (column 2).

FIG. 8.

FlhDC controls rsmB RNA via their effects on gacA and hexA. Northern blot analysis of rsmB RNA in wild-type strain Ecc71 (A1) and its FlhDC^- mutant AC5140 (A2); in FlhDC^- GacA^- strain AC5143 carrying pCL1920 (B1), pAKC1242 (flhDC⁺) (B2), or pAKC1245 (gacA⁺) (B3); in FlhDC^- GacA⁺ strain AC5140 carrying pCL1920 (C1), pAKC1241 (flhD⁺) (C2), or pAKC1242 (C3); and in FlhDC^- HexA⁺ strain AC5140 (D1) and FlhDC^- HexA^- strain AC5145 (D2). Each lane contained 10 μg of total RNA. The arrows show the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel.

FIG. 9.

Stabilities of pel-1, peh-1, and hrpL transcripts in Ecc71 (lanes 1 to 7) and its FlhDC^- mutant AC5140 (lanes 8 to 14). Samples were collected at 0, 2.5, 5, 7.5, 10, 12.5, and 15 min after the addition of rifampin. For lanes 1 to 7, each lane contained 15 μg of total RNA, and for lanes 8 to 14, each lane contained 30 μg of total RNA. The arrow shows the levels of total RNA as revealed by ethidium bromide staining of denatured agarose gel.

FIG. 10.

RsmA is responsible for the pleiotropic phenotype resulting from FlhDC deficiency. (A to C) Pel activities (A), agarose plate assays of Peh, Prt, and Cel activities (B), and pel-1, peh-1, and celV transcripts (C) in FlhDC⁺ RsmA⁺ strain AC5006 (column 1), FlhDC^- RsmA⁺ strain AC5141 (column 2), FlhDC^- RsmA^- strain AC5144 (column 3), FlhDC⁺ RsmA⁺ strain AC5047 (column 4), and FlhDC⁺ RsmA^- strain AC5070 (column 5). (D) rsmB RNA levels in AC5070 (column 1) and AC5144 (column 2). (E and F) Pel, Peh, Prt, and Cel activities in AC5144 carrying the cloning vector pCL1920Gm^r (column 1) or the rsmB⁺ plasmid pAKC1049Gm^r (column 2) and AC5070 carrying pCL1920Gm^r (column 3).

FIG. 11.

FliA does not control exoprotein production in E. carotovora subsp. carotovora. (A) Northern blot analysis of fliA and (B) agarose plate assays of Pel, Peh, Prt, and Cel activities in AC5006 (FlhDC⁺) carrying the cloning vector pCL1920 (column 1) or the fliA⁺ plasmid pAKC1246 (column 2), as well as AC5141 (FlhDC^-) carrying pCL1920 (column 3) or pAKC1246 (column 4).