rsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin(Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA)

Abstract

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin(Ecc) (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219-234, 1998). We have cloned and characterized a novel regulatory gene, rsmC, that activates RsmA production and represses extracellular enzyme and harpin(Ecc) production, rsmB transcription, and virulence in E. carotovora subsp. carotovora. In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA(+) allele, the basal levels of Pel, Peh, Cel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, and hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC(-) mutant than in the RsmC(+) parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC(-) mutant than in the RsmC(+) parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmA(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutant is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora. These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.

FIG. 1

(A) Agarose plate assays for Pel, Peh, Prt, and Cel activities of E. carotovora subsp. carotovora AC5047 (column 1), the Ohl⁻ derivative of AC5047 (AC5091; column 2), the RsmC⁻ mutant (AC5050; column 3), the Ohl⁻ derivative of AC5050 (AC5051; column 4), and the RsmA⁻ mutant of AC5047 (AC5070; column 5). Bacteria were grown at 28°C in minimal salts medium plus sucrose and harvested at an A₆₀₀ value of 2.5. Culture supernatants (10 μl) were added to each well. After 18 h of incubation at 28°C, the Pel and Peh assay plates were developed with 4 N HCl, and the Cel assay plate was developed with Congo red and NaCl solutions. Halos around the wells in the Prt assay plate became visible within 24 h without any further treatment. (B) Northern blot analysis of pel-1, peh-1, celV, and hrpN_ECC mRNA in E. carotovora subsp. carotovora AC5047 (RsmA⁺ RsmC⁺ Ohl⁺; lane 1), AC5091 (RsmA⁺ RsmC⁺ Ohl⁻; lane 2), AC5050 (RsmA⁺ RsmC⁻ Ohl⁺; lane 3), AC5051 (RsmA⁺ RsmC⁻ Ohl⁻; lane 4), and AC5070 (RsmA⁻ RsmC⁺ Ohl⁺; lane 5). Bacteria were grown at 28°C in minimal salts medium plus sucrose to an A₆₀₀ of 2.0 for total RNA extraction. Each lane contained 10 μg of total RNA. (C) Western blot analysis of harpin_Ecc produced by E. carotovora subsp. carotovora Ecc71 (RsmA⁺ RsmC⁺; lane 1), AC5053 (RsmA⁺ RsmC⁻; lane 2), AC5071 (RsmA⁻ RsmC⁺; lane 3), and AC5054 (RsmA⁻ RsmC⁻; lane 4). Each lane contained 20 μg of total bacterial protein. (D) Northern blot analysis of pel-1, peh-1, celV, hrpN_ECC, and rsmB transcripts in E. carotovora subsp. carotovora Ecc71 (RsmA⁺ RsmC⁺; lane 1), AC5053 (RsmA⁺ RsmC⁻; lane 2), AC5071 (RsmA⁻ RsmC⁺; lane 3), and AC5054 (RsmA⁻ RsmC⁻; lane 4). Bacteria were grown at 28°C in minimal salts medium plus sucrose to an A₆₀₀ of 2.0 for total RNA extraction. Each lane contained 10 μg of total RNA.

FIG. 2

(A) Agarose plate assays for Prt and Cel activities of E. carotovora subsp. carotovora AC5047 (a and c) and its RsmC⁻ mutant (AC5050; b and d) carrying the cloning vector, pCL1920 (column 1) or the RsmC⁺ plasmid, pAKC975 (column 2). Bacteria were grown at 28°C in minimal salts medium plus sucrose and spectinomycin and harvested at an A₆₀₀ of 2.5. Culture supernatants (10 μl) were added to each well. (B) Northern blot analysis of pel-1, peh-1, celV, hrpN_Ecc, and rsmB transcripts produced by E. carotovora subsp. carotovora AC5047 (RsmC⁺) and AC5050 (RsmC⁻) carrying the cloning vector pCL1920 or the RsmC⁺ plasmid pAKC975. Lane 1, AC5047 carrying pCL1920; lane 2, AC5047 carrying pAKC975; lane 3, AC5050 carrying pCL1920; lane 4, AC5050 carrying pAKC975. Total RNAs were isolated from bacteria grown at 28°C in minimal salts medium plus sucrose and spectinomycin to an A₆₀₀ of 2.0. Each lane contained 10 μg of total RNA. (C) Northern blot analysis of pel-1, peh-1, celV, and hrpN_Ecc mRNA produced by E. carotovora subsp. carotovora Ecc71 (RsmA⁺ RsmC⁺), AC5053 (RsmA⁺ RsmC⁻), AC5071 (RsmA⁻ RsmC⁺), and AC5054 (RsmA⁻ RsmC⁻) carrying the cloning vector pCL1920Gm^r or the rsmB⁺ plasmid pAKC1004Gm^r. Lane 1, Ecc71/pCL1920Gm^r; lane 2, Ecc71/pAKC1004Gm^r; lane 3, AC5053/pCL1920Gm^r; lane 4, AC5053/pAKC1004Gm^r; lane 5, AC5071/pCL1920Gm^r; lane 6, AC5071/pAKC1004Gm^r; lane 7, AC5054/pCL1920Gm^r; lane 8, AC5054/pAKC1004Gm^r. Total RNAs were isolated from bacteria grown at 28°C in minimal salts medium plus sucrose and gentamicin to an A₆₀₀ of 2.0. Each lane contained 10 μg of total RNA.

FIG. 3

(A) Nucleotide and deduced amino acid sequences of E. carotovora subsp. carotovora rsmC. The putative ribosome binding site is double underlined. The transcriptional start site at the thymine residue is indicated with an asterisk. The −10 consensus sequences are shown in boldface. The nucleotide sequence used for the synthesis of a complementary oligonucleotide for the primer extension assay is underlined with a wavy line. The position of mini-Tn5-Km insertion in the RsmC⁻ mutants AC5050 and AC5053 is indicated with an arrowhead. Some restriction endonuclease sites are also shown. Numbers on the right refer to positions of the nucleotides and the amino acid residues for each line. (B) Primer extension analysis of rsmC mRNA. Lane 1, 20 μg of total RNA sample from E. carotovora subsp. carotovora Ecc71 grown at 28°C in minimal salts medium plus sucrose to an A₆₀₀ of 2.0. The products of the primer extension reaction were run alongside a regular sequencing gel. Nucleotides on the left refer to the nucleotide sequence beyond the transcriptional start site; the asterisk denotes the thymine residue at which transcription was initiated. (C) Northern blot analysis of rsmC mRNA in E. carotovora subsp. carotovora Ecc71 (RmsC⁺; lane 1) and its RsmC⁻ derivative, AC5053 (lane 2). Total RNAs were isolated from bacteria grown at 28°C in minimal salts medium plus sucrose to an A₆₀₀ of 2.0. Each lane contained 20 μg of total RNA. (D) Overproduction of RsmC in E. coli strain JM109(DE3). Bacteria carrying the cloning vector pET28a(+) or the rsmC⁺ plasmid pAKC978 were grown in LB medium plus kanamycin with or without IPTG as described in Materials and Methods. Each lane contained 10 μg of total bacterial protein. The arrow indicates the 15-kDa overproduced RsmC protein. Lane 1, pET28(+), no IPTG; lane 2, pET28(+), with IPTG (final concentration, 1 mM); lane 3, pAKC978, no IPTG; lane 4, pAKC978 with IPTG (final concentration, 1 mM).

FIG. 4

(A) Maceration of a celery petiole by E. carotovora subsp. carotovora Ecc71 (RsmA⁺ RsmC⁺; site 1), AC5053 (RsmA⁺ RsmC⁻; site 2), AC5071 (RsmA⁻ RsmC⁺; site 3), and AC5054 (RsmA⁻ RsmC⁻; site 4). (B) Maceration of celery petioles by E. carotovora subsp. carotovora AC5047 (RsmA⁺ RsmC⁺; a) and AC5050 (RsmA⁺ RsmC⁻; b) carrying the cloning vector pCL1920 (site 1) or the RsmC⁺ plasmid pAKC975 (site 2). About 2 × 10⁸ cells were injected into the celery petiole at each inoculation site and covered with petroleum jelly. The inoculated petioles were incubated in a moist chamber at 25°C for 24 h.

FIG. 5

(A) Northern analysis of rsmA transcripts produced by E. carotovora subsp. carotovora Ecc71 (lane 1) and its RsmC⁻ derivative, AC5053 (lane 2). Total RNAs were extracted from bacteria grown in minimal salts medium plus sucrose at 28°C to an A₆₀₀ of 2.0. Each lane contained 10 μg of total RNA. (B) Western blot analysis of RsmA produced by E. carotovora subsp. carotovora Ecc71 (RsmC⁺; lane 1) and AC5053 (RsmC⁻; lane 2). Each lane contained 20 μg of total bacterial protein. (C) Levels of rsmA transcripts in E. carotovora subsp. carotovora Ecc71 (RsmC⁺) carrying the cloning vector pCL1920 (lane 1) or the rsmC⁺ plasmid pAKC975 (lane 2) and AC5053 (RsmC⁻) carrying the cloning vector pCL1920 (lane 3) or the rsmC⁺ plasmid pAKC975 (lane 4). Total RNAs were extracted from bacteria grown in minimal salts medium plus sucrose and spectinomycin at 28°C to an A₆₀₀ of 2.0 and subjected to Northern analysis. Each lane contained 10 μg of total RNA.

FIG. 6

(A) β-Galactosidase assays of E. carotovora subsp. carotovora AC5047 or its RsmC⁻ mutant AC5050 carrying the cloning vector pNM481Sp^r, pAKC887 (rsmA_Ecc-lacZ fusion), pAKC888 (rsmA_Ea-lacZ fusion), pAKC889 (rsmA_Ehg-lacZ fusion), and pAKC890 (csrA-lacZ fusion). Bacteria were grown at 28°C in minimal salts medium plus sucrose and spectinomycin to an A₆₀₀ of 2.0 and assayed for β-galactosidase activity. (B) β-Galactosidase assays of E. carotovora subsp. carotovora AC5047 or its RsmC⁻ mutant AC5050 carrying the cloning vector pMP220 or the rsmB-lacZ plasmid pAKC1002. Bacteria were grown at 28°C in minimal salts medium plus sucrose and tetracycline to an A₆₀₀ of 2.0 and assayed for β-galactosidase activity.

FIG. 7

Southern blot analysis of EcoRI-digested chromosomal DNAs of E. carotovora subspecies with rsmC of E. carotovora subsp. carotovora Ecc71. Lane 1, E. carotovora subsp. atroseptica Eca12; lane 2, E. carotovora subsp. betavasculorum Ecb11129; lanes 3 and 4, E. carotovora subsp. carotovora strains Ecc71 and SCRI193.