Virulence and prodigiosin antibiotic biosynthesis in Serratia are regulated pleiotropically by the GGDEF/EAL domain protein, PigX

Abstract

Gram-negative bacteria of the genus Serratia are opportunistic human, plant, and insect pathogens. Serratia sp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin antibiotic biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site of pigX was determined, and pigX expression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein in Serratia and the first example of the regulation of antibiotic production by a GGDEF/EAL domain protein.

FIG. 1.

The pigX gene encodes a GGDEF/EAL domain protein. (A) Genomic location of pigX, indicating the transcriptional start site (+1) and the insertion sites of transposons (black triangles) in strains HSPIG66 (1), ROP4S (2), and ROP4 (3). (B) Predicted GGDEF/EAL protein domain structure and inner membrane (IM) localization of PigX. OM, outer membrane; PL, periplasmic loop.

FIG. 2.

PigX represses Pig production by controlling transcription of the Pig biosynthetic operon, pigA-pigO. (A) Pig levels in WT, HSPIG66 (pigXpro), and ROP4 (pigX) strains throughout growth in LB. (B) β-Galactosidase activity was measured from a chromosomal pigA::lacZ fusion in an otherwise WT background (MCP2L) or in a strain containing a mini-Tn5Sm/Sp chromosomal insertion in pigX (4SPAL). (C) Primer extension analysis of the pigA-pigO transcript from the WT and ROP4S (pigX) strains throughout the time of growth in LB. (D) yhdH-pigX intergenic region showing the transcription start site of pigX (+1) and the predicted −10 and −35 elements. (E) β-Galactosidase activity was measured from chromosomal pigX::lacZ and pigXpro::lacZ fusion strains throughout growth in LB. Solid symbols and lines represent the results of either Pig or β-galactosidase assays, whereas the open symbols and dashed lines represent the growth curves of the corresponding strains. Data shown are the means ± standard deviations of the results of at least three independent experiments. β-gal, β-galactosidase.

FIG. 3.

The 2D-DiGE intracellular protein profile in the pigX mutant strain is altered in comparison to that in WT Serratia strain ATCC 39006. Cy2 image of the pooled sample of the 2D gel (24 cm, pI 4 to 7, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel), with proteins which were picked for identification by MS circled. NS means that no significant hits were obtained by MS analysis, and M identifies the protein spot as a mixed hit. M1, M2, and M3 contained FabB (M1 and M2) and FliC (M3) mixed with elongation factor Tu. A list of proteins that were altered in abundance is provided in Table 3. MW, molecular weight.

FIG. 4.

PigX represses Pel activity and virulence in a potato tuber-rotting virulence assay. (A) Pel activities of the WT (LacA), a pigX mutant (ROP4S), an opgG mutant (NW34), and a pigX opgG double mutant (4SOPG) strain. Two halos of enzyme activity were observed and are shown as black (inner halo) and white (outer halo). (B) Cel activity of the WT (LacA), a pigX mutant (ROP4S), an opgG mutant (NW34), and a pigX opgG double mutant (4SOPG) strain. (C) Pel activity of the pigX mutant strain plus vector (ROP4 and pQE-80L) and the pigX mutant strain plus PigX (ROP4 and pTA40). (D) Results of potato tuber-rotting assays comparing the amount of rot caused by the WT with the amount caused by the pigX mutant (ROP4S), the opgG mutant (NW34), and the pigX opgG double mutant (4SOPG) strain, using 10³ CFU as inocula. The difference between the WT and each mutant was statistically significant using a two-tailed paired t test with a P value of either <0.05 (*) or <0.001 (**), and the data shown are the means ± standard errors of the means of the results of at least seven independent experiments. (E) Swimming motility of the WT (LacA), a pigX mutant (ROP4S), an opgG mutant (NW34), and a pigX opgG double mutant (4SOPG) strain. Data shown are the means ± standard deviations of the results of at least three independent experiments (unless otherwise stated).

FIG. 5.

PigX is predicted to function as a c-di-GMP PDE. (A) Schematic of domain organization of PigX and regions cloned into pQE-80L. The sequences of the divergent motifs in the GGDEF and EAL domains are shown (YHSDF and ELI, respectively). Also shown (bottom) is the domain of the E. coli PDE YahA that was cloned into pQE-80L. PL, periplasmic loop. (B) Pig assays of WT and pigX mutant (ROP4) strains in the presence of the PigX plasmids and YahA plasmid shown in panel A. Data shown are the means ± standard deviations of the results of at least three independent experiments.