A Serratia marcescens PigP homolog controls prodigiosin biosynthesis, swarming motility and hemolysis and is regulated by cAMP-CRP and HexS

Abstract

Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.

Figure 1. S. marcescens PigP positively regulates prodigiosin production.

A. Complementation analysis of a pigP mutant strain. The vector is pMQ132, and ppigP refers to pMQ221 (Table 1). Error bars = one standard deviation. * = p<0.05 compared to WT by ANOVA with Tukey’s post-test. B. Genetic organization of the chromosome proximal to the pigP gene including the predicted pigP promoter. Enumerated bars under the genes indicate the regions amplified for operon analysis in panel D. C. Growth curve analysis shows similar growth rates for the WT and isogenic pigP mutant strains. The average of four biological replicates is shown. D. Analysis of the pigP operon supports that pigP is in a polycistronic message with SMA3565 and SMA3566. RNA isolated from stationary phase cells was treated with reverse transcriptase (+RT) or without reverse transcriptase (−RT) as a negative control. Positive control DNA and experimental samples were assessed with PCR for the presence of amplicons internal to pigP as a positive control and that span the genes indicated in the figure. Regions amplified by primers are indicated in by numbered brackets in panel B. Primers for analysis of pigP-SMA3565 co-expression extend 144 base pairs into the SMA3565 open frame; those for SMA3565-SMA3566 extend 407 base pairs into the SMA3566 open reading frame.

Figure 2. PigP transcriptional regulation of the pigment biosynthetic operon.

A. Expression of the pigA promoter measured using a chromosomal lacZ transcriptional fusion at early stationary phase. The average of 4 biological replicates is shown. Error bars indicate one standard deviation. One asterisk indicates a significant difference from (p<0.05, ANOVA Tukey’s post-test). B. RT-PCR of cDNA from stationary phase cells (OD₆₀₀ = 3.5) with the 16S target as a control to show equal input cDNA. Genotypes are listed from left to right, and target cDNAs are listed from top to bottom, with 16S rDNA serving as an internal loading control. Representative images are shown. Asterisk indicates that there is no signal here because the pigP gene is deleted in this strain; this experiment serves as a negative control. C. EMSA assay with biotinylated pigA promoter DNA (4 ng) with or without recombinant PigP protein (His₉-PigP) and with (+) or without (−) unlabeled competitor pigA promoter DNA (500 ng). D. Chromatin affinity purification (ChAP) enrichment of pigA promoter DNA, but not oxyR promoter DNA in cells expressing a functional His₉-PigP (+), but not the vector alone negative control (−). “Input” indicates sheared DNA before affinity purification and shows similar levels of starting DNA.

Figure 3. Direct regulation of pigP expression by PigP. A.

Expression of a chromosomal pigP-lacZ transcriptional reporter shows reduced expression in the ΔpigP mutant strain (n = 6 biological replicates per time point). B. EMSA assay with biotinylated pigP promoter DNA (2 ng) as a target that had been incubated with or without recombinant PigP protein (His₉-PigP) and with (+) or without (−) unlabeled competitor pigP promoter DNA (500 ng).

Figure 4. Impact of cAMP-CRP on pigP transcription.

A. ß-galactosidase activity as expressed from the chromosomal pigP promoter as a function of culture density. This representative experiment shows the average of 3 biological replicates per genotype. B. ß-galactosidase activity from a chromosomal pigP reporter in early stationary phase. WT and crp strains were grown without exogenous cAMP. The isogenic cyaA mutant was grown with 0, 5, or 10 mM cAMP dissolved in the growth medium. This experiment shows the average of 6 biological replicates per cAMP concentration, performed on two different days. Asterisk = significantly different than WT (p<0.05). In this figure “crp” refers to the crp-23 transposon mutant. Error bars = one standard deviation.

Figure 5. PigP is necessary for swarming motility and serratamolide production, but not swimming motility.

A. Swarming motility plates show that the pigP mutant is defective in surface motility. Mutation of the serratamolide inhibitor hexS restores swarming to the pigP mutant (hexS pigP), and hexS requires serratamolide to swarm (hexS swrW). B. The pigP swarming defect can be complemented by the wild-type pigP gene provided in trans. Vector refers to pMQ132, and ppigP is pMQ221. C. Swimming motility plates show similar zones (in all cases “zones” indicates the measurement from the edge of the colony to the outer edge of the observed phenotype in mm) between the WT and pigP mutant. D. Surfactant zones are absent in strains without pigP and the serratamolide biosynthetic gene swrW. Mutation of hexS restores surfactant zones to the pigP mutant. N ≥6 biological replicates per strain. E. Arabinose-induced expression of swrW is sufficient to restore swarming to a swrW and pigP mutant. pMQ200+ swrW refers to pMQ368. F. Purified serratamolide is sufficient to restore swarming to different strain backgrounds with pigP mutations, whereas the serratamolide vehicle, DMSO, is not.

Figure 6. PigP is necessary for hemolysis in laboratory and clinical isolates. A

. Hemolytic strains grown on TSA plates with sheep blood show a zone of clearing around colonies indicative of hemolysis. Isogenic pigP mutant strains show highly reduced zones of hemolysis. B. The hemolysis defect of pigP mutants can be complemented by wild-type pigP on a plasmid (pMQ221); vector refers to pMQ132. C. Arabinose inducible expression of swrW is sufficient to restore hemolysis to the pigP mutant. The swrW gene was expressed from plasmid pMQ367 (pswrW), and vector refers to pMQ125. D. Mutation of pigP reduces the hyper-hemolytic phenotypes of crp and hexS mutants.

Figure 7. HexS regulates pigP expression.

A. Mutation of hexS leads to an increase in output from a chromosomal pigP reporter (strains CMS1785 and CMS3408 respectively). Cells were harvested at OD₆₀₀ = 4.0 and ß-gal activity is reported. Data is the mean from 7 biological replicates per genotype. Error bars = one standard-deviation and the asterisk indicates a statistical difference from the WT (p<0.05, Student’s t-test). B. EMSA analysis indicates that a recombinant maltose binding protein-HexS fusion (MBP-HexS, 25 µg) binds to the labeled pigP promoter (2 ng) in vitro, whereas the recombinant maltose binding protein (MBP, 33 µg) control does not bind to the pigP promoter. Unlabeled pigP promoter region competitor DNA (500 ng) was able to inhibit the MBP-HexS induced shift suggesting a specific interaction.

Figure 8. PigP mediates rugose colony morphology. A.

The CHASM rugose phenotype is absent in the pigP mutant (CMS2981) and can be complemented by wild-type pigP on a plasmid (ppigP = pMQ221). The vector alone is pMQ132. B. The rugose colony morphology defect of the CHASM pigP mutant (CMS2981) can be restored through expression of swrW from a plasmid (pMQ367), but not from the vector alone (pMQ125).

Figure 9. Model for regulation of secondary metabolite biosynthesis genes by the transcription factors described in this study.

The secondary metabolite genes (pigA-N for prodigiosin and swrW for serratamolide) and the pigP regulator gene (not shown) are negatively (bar) and directly (solid line) regulated by HexS. They are negatively and indirectly (dashed line) regulated by CRP. The pigA-N operon and pigP are positively (arrow) and directly regulated by PigP, whereas swrW is indirectly regulated by PigP. The asterisk indicates that the same pattern of regulation for the pigA-N operon is observed for the pigP gene.