An IgaA/UmoB Family Protein from Serratia marcescens Regulates Motility, Capsular Polysaccharide Biosynthesis, and Secondary Metabolite Production

Abstract

Secondary metabolites are an important source of pharmaceuticals and key modulators of microbe-microbe interactions. The bacterium Serratia marcescens is part of the Enterobacteriaceae family of eubacteria and produces a number of biologically active secondary metabolites. In this study, we screened for novel regulators of secondary metabolites synthesized by a clinical isolate of S. marcescens and found mutations in a gene for an uncharacterized UmoB/IgaA family member here named gumB Mutation of gumB conferred a severe loss of the secondary metabolites prodigiosin and serratamolide. The gumB mutation conferred pleiotropic phenotypes, including altered biofilm formation, highly increased capsular polysaccharide production, and loss of swimming and swarming motility. These phenotypes corresponded to transcriptional changes in fimA, wecA, and flhD Unlike other UmoB/IgaA family members, gumB was found to be not essential for growth in S. marcescens, yet igaA from Salmonella enterica, yrfF from Escherichia coli, and an uncharacterized predicted ortholog from Klebsiella pneumoniae complemented the gumB mutant secondary metabolite defects, suggesting highly conserved function. These data support the idea that UmoB/IgaA family proteins are functionally conserved and extend the known regulatory influence of UmoB/IgaA family proteins to the control of competition-associated secondary metabolites and biofilm formation.IMPORTANCE IgaA/UmoB family proteins are found in members of the Enterobacteriaceae family of bacteria, which are of environmental and public health importance. IgaA/UmoB family proteins are thought to be inner membrane proteins that report extracellular stresses to intracellular signaling pathways that respond to environmental challenge. This study introduces a new member of the IgaA/UmoB family and demonstrates a high degree of functional similarity between IgaA/UmoB family proteins. Moreover, this study extends the phenomena controlled by IgaA/UmoB family proteins to include the biosynthesis of antimicrobial secondary metabolites.

Keywords: biofilm; capsular polysaccharide; competition; flagella; motility; secondary metabolite; surfactant.

FIG 1

Isolation of mutants with secondary metabolite biosynthesis defects and a distinct colony morphology. (A) Representative images of the wild-type (WT) strain K904 and the two gummy mutants. The colonies were top-lit and demonstrate a lack of prodigiosin and altered colony morphology. (B) Representative images show a hemolysis zone (white arrow) on blood agar plates around K904 and a lack of hemolysis zones around two gumB transposon mutants and a defined gumB mutant. The plate was lighted from below to highlight the hemolysis zone, obscuring colony color. (C) Genetic map of the gumB gene and surrounding genes. Transposon insertion sites are noted by gray arrow points. (D) Phylogram of amino acid sequences of known and predicted IgaA/UmoB family proteins. The dendrogram was made using the “one-click” mode of the tree rendering program from www.Phylogeny.fr, using default settings. The number of substitutions per site are proportional to the branch length.

FIG 2

Complementation of gumB mutant defects and growth analysis. (A) The gumB mutant colony and color phenotypes are complemented by wild-type gumB on a plasmid under the control of the P_lac promoter. Representative images are of the strain K904 and the ΔgumB mutant with vector control plasmid pMQ132 and complementation plasmid pgumB (pMQ480). Bacteria were incubated for 48 h at 30°C on LB or blood agar plates and lighted from above. (B and C) Growth curves of the wild-type strain K904 and the ΔgumB mutant in LB (B) or M9 minimal medium supplemented with glucose (C). Means ± standard deviation (SD) are shown, n ≥ 3.

FIG 3

Complementation of gumB mutant defects by IgaA/UmoB family genes from several genera. (A) Image of the ΔgumB mutant with vector alone (pMQ132) or pMQ132 with various IgaA/UmoB family genes under the control of P_lac to test for complementation. (B) Zoomed-in view of panel A to highlight the colony morphology complementation of the gumB mutant with a predicted IgaA/UmoB gene from K. pneumoniae (kumO).

FIG 4

S. marcescens GumB is necessary for swimming and swarming motility. (A) Representative images of the swimming and swarming defects of the ΔgumB mutant. (B) Representative images of complementation of the ΔgumB swarming defect. (C) Complementation of the ΔgumB swimming phenotype. n ≥ 5 independent plates per genotype from 2 separate days. Asterisks indicate a significant difference from all of the other genotypes (P < 0.001). Mean and SD are shown. Vector, pMQ132; pgumB, pMQ480. (D) Fold change of both flagellum-related gene transcript levels. qRT-PCR of fliC and flhD gene expression, n ≥ 4. Mean and SD are shown. *, P < 0.05; **, P < 0.01.

FIG 5

Prodigiosin pigmentation is severely reduced in a ΔgumB mutant. (A) The gumB mutant grown in LB medium is defective in prodigiosin production (75-fold reduction compared to strain K904; n = 3, P < 0.01). Extracted prodigiosin was measured by absorbance, and the defect could be complemented by wild-type gumB in trans. The vector control plasmid is pMQ132, and pgumB is pMQ480. (B) qRT-PCR analysis revealed reduced expression from the prodigiosin biosynthetic locus; pigA gene expression in the ΔgumB mutant was down 103-fold compared to the wild type (P = 0.029, n = 4). Means and standard deviations are shown. *, P < 0.05; **, P < 0.01.

FIG 6

Serratamolide is severely reduced in a ΔgumB mutant. (A) Image of blood agar plate, lit from below. Serratamolide is responsible for hemolysis zones generated by strain K904. The ΔgumB mutant was defective for hemolysis and could be complemented by wild-type gumB in trans. The vector control plasmid is pMQ132, and pgumB is pMQ480. (B) Representative top-lit image. Inhibition of S. aureus bacterial growth (black arrow) by S. marcescens is serratamolide dependent and is eliminated in the ΔgumB mutant. (C) Mass spectrometry reveals a >50-fold significant reduction of serratamolide in the ΔgumB mutant (P < 0.01, n = 3). (D) qRT-PCR analysis demonstrates reduced expression of the serratamolide biosynthetic gene swrW, which was reduced >1,000-fold in the relative transcript levels for the mutant compared to the wild type (P = 0.029, n = 4). Mean and SD are shown. *, P < 0.05; **, P < 0.01.

FIG 7

Gummy phenotype of the ΔgumB mutant requires the capsular polysaccharide operon. (A) Suppressor mutants of the rugose colony morphology phenotype of the ΔgumB mutant map to and wza genes of the capsular polysaccharide operon. (B) Extracellular polysaccharide (EPS) was extracted and measured from liquid cultures. Mean and SD are shown, n = 4. Asterisks indicate a significant increase relative to other groups (ANOVA, Tukey's posttest, P < 0.01). (C) qRT-PCR analysis revealed elevated relative wecA gene expression levels in the ΔgumB mutant (P = 0.016, n = 5). Mean and SD are shown. *, P < 0.05. (D) Biofilm formation in LB medium on borosilicate glass tubes grown under high-sheer conditions for 6 and 20 h and stained with crystal violet. The ΔgumB mutant had an elevated biofilm that was absent in a ΔgumB wza mutant. Mean and SD are shown, n = 12 biological replicates.