RsmC of Erwinia carotovora subsp. carotovora negatively controls motility, extracellular protein production, and virulence by binding FlhD and modulating transcriptional activity of the master regulator, FlhDC

Abstract

RsmC and FlhDC are global regulators controlling extracellular proteins/enzymes, rsmB RNA, motility, and virulence of Erwinia carotovora subsp. carotovora. FlhDC, the master regulator of flagellar genes, controls these traits by positively regulating gacA, fliA, and rsmC and negatively regulating hexA. RsmC, on the other hand, is a negative regulator of extracellular proteins/enzymes, motility, and virulence since the deficiency of RsmC in FlhDC(+) strain results in overproduction of extracellular proteins/enzymes, hypermotility, and hypervirulence. These phenotypes are abolished in an RsmC(-) FlhDC(-) double mutant. We show that RsmC interferes with FlhDC action. Indeed, the expression of all three targets (i.e., gacA, rsmC, and fliA) positively regulated in E. carotovora subsp. carotovora by FlhDC is inhibited by RsmC. RsmC also partly relieves the inhibition of hexA expression by FlhDC. The results of yeast two-hybrid analysis revealed that RsmC binds FlhD and FlhDC, but not FlhC. We propose that binding of RsmC with FlhD/FlhDC interferes with its regulatory functions and that RsmC acts as an anti-FlhD(4)FlhC(2) factor. We document here for the first time that RsmC interferes with activation of fliA and motility in several members of the Enterobacteriaceae family. The extent of E. carotovora subsp. carotovora RsmC-mediated inhibition of FlhDC-dependent expression of fliA and motility varies depending upon enterobacterial species. The data presented here support the idea that differences in structural features in enterobacterial FlhD are responsible for differential susceptibility to E. carotovora subsp. carotovora RsmC action.

FIG. 1.

A model depicting the regulatory network controlling extracellular protein and AHL production and bacterial motility in E. carotovora subsp. carotovora. The regulatory steps indicated by broken lines with arrows are based upon the results presented in this report. In this model, RsmA, a small RNA binding protein, promotes mRNA decay, and rsmB specifies an untranslated regulatory RNA that binds RsmA and neutralizes its negative regulatory effect. The two-component system GacS (the putative sensor kinase)-GacA (the cognate response regulator) controls exoprotein production mainly by regulating rsmB. HexA negatively controls exoprotein via rsmB, and it also controls AHL production and motility. KdgR, an IcII-like protein, negatively controls exoproteins by inhibiting the transcription of rsmB by a “road-block” mechanism. RpoS, an alternate sigma factor, negatively affects the production of exoprotein by stimulating rsmA transcription. The two LuxR homologs ExpR1 and ExpR2 activate rsmA transcription in the absence of AHL. FlhDC controls extracellular protein production and bacterial motility by positively regulating gacA, fliA, and rsmC. FlhDC also negatively regulates hexA. RsmC acts as an anti-FlhDC factor by binding to FlhD or FlhDC complex, and it interferes with FlhDC action.

FIG. 2.

Characteristics of an FlhDC⁻ RsmC⁻ mutant of E. carotovora subsp. carotovora strain AC5006 and reversal of the pleiotropic phenotype of this mutant by flhDC⁺ DNA. (A) Agarose plate assays are shown of Pel, Peh, Prt, and Cel activities of AC5006 (FlhDC⁺ RsmC⁺) and its mutants. Soft-rot disease symptoms caused by those strains in celery petiole. Water (W) was injected in celery petiole for control. (C) Agarose plate assays of Pel, Peh, Prt, and Cel activities of the FlhDC⁻ RsmC⁻ strain carrying vector pDK7 and the flhDC⁺ plasmid pAKC1255.

FIG. 3.

Characteristics of an RsmC⁻ mutant of E. carotovora subsp. carotovora strain Ecc7N2. (A) Agarose plate assays of Pel, Peh, Prt, and Cel activities. (B) Soft-rot disease symptoms in celery petiole. (C) Motility on tryptone swarm agar. The inoculated plates were incubated at 28°C for 18 h. (D) Northern blot analysis of fliA, fliZ, fliC, and fliE. Each lane contained 15 μg of total RNA. For fliA, fliZ, and fliE, X-ray films were exposed for 24 h at −80°C, and for fliC, X-ray film was exposed for 1 h.

FIG. 4.

Northern blot analysis of fliA, fliZ, fliE, and fliC and of several regulatory genes in Ecc71 (FlhDC⁺ RsmC⁺) and its RsmC⁻ mutant (A and B) as well as in AC5006 (FlhDC⁺ RsmC⁺) and its mutants, as indicated (C). Each lane contained 15 μg of total RNA. For fliA, fliZ, fliE, gacA, flhD, flhDC, and hexA in panels A and B, X-ray films were exposed for 24 h at −80°C, and for fliC in panel A, X-ray film was exposed for 1 h. X-ray films for gacA and fliA in panel C were exposed for 48 h.

FIG. 5.

(A) Primer extension experiment to determine the transcriptional start site of hexA. Lane 1, experiments with 20 μg of Ecc71 total RNA; lane 2, no RNA added. The nucleotides at the left of the panel refer to the nucleotide sequences beyond the transcriptional start site. The asterisk indicates the residue at which transcription was initiated. (B) Nucleotide sequence of the upstream region of Ecc71 hexA. The transcriptional start site is indicated by +1. The potential FlhDC binding sequences are indicated by double underlining. The putative −10 and −35 sequences are underlined, and the putative translational start site is indicated by a star.

FIG. 6.

(A) Effects of the rsmC⁺ plasmid pAKC975 on motility of E. carotovora subsp. atroseptica strain Eca12, E. carotovora subsp. carotovora strain Ecc7N2, E. chrysanthemi strain 3937, E. cloacae strain EC1, E. herbicola strain EH4, S. marcescens strain SM274, Y. enterocolitica strain YE1, S. Typhimurium strain LT2, C. fruendii strain CF, E. coli strain K12, and S. flexneri strain Sf1. Bacterial cells were inoculated in tryptone swarm agar and incubated at 28°C. Strains EH4, SM274, and CF carrying plasmids were incubated for 11 h; strains Ecc7N2, EC1, LT2, and YE1 carrying plasmids were incubated for 24 h; and strains 3937, Sf1, K12, and Sf1 carrying plasmids were incubated for 48 h. (B) Effects of the rsmC⁺ plasmid pAKC975 on transcript levels of fliA in strains Eca12, Ecc7N2, 3937, YE1, LT2, CF, K12, and Sf1. Each lane contained 15 μg of total RNA.