A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium

Abstract

Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.

Fig. 1.

Genetic organization of the Serratia 39006 gas-vesicle gene cluster. The 16.7-kb DNA region encoding gas-vesicle proteins. Transposon insertions causing colony translucency are indicated by arrows above (TnKRCPN1 insertions) or below the genes (TnDS1028 insertions). A summary of the predicted functions of each gene is listed in Table S1.

Fig. 2.

Gas vesicle production in Serratia 39006. (A) Expression from the gvpA1::uidA transcriptional fusion throughout growth in Serratia 39006 (WT) and gvrA backgrounds. β-Glucuronidase expression was measured by the RFUs per minute, produced from cleavage of 4'-Methylumbelliferyl-β-d-glucuronide, normalized to the optical density of the culture (RFU/min normalized to OD₆₀₀) (values are average of three biological replicates ± SD). Filled symbols indicate subcultures incubated with reduced aeration. (B) Expression from the gvrA::uidA transcriptional fusion throughout growth. (C) TEM of WT cells, (D) pressurized WT cells, and (E) JRGVP cells (harvested from solid media). (F) TEM of individual WT cells showing vesicles of difference widths. (Scale bars, 1 μm unless indicated otherwise.)

Fig. 3.

Quorum-sensing regulated flotation and gas vesicle production. (A) Expression of the gvrA::uidA and (B) gvpA1::uidA reporter fusions in Serratia 39006 (WT), smaI background, and smaI supplemented with BHL (values are average of three biological replicates ± SD). (C) (Upper) Flotation of JRGVP and WT cultures after 24 and 48 h. (Lower) PCM of JRGVP and WT taken from the top layer of cells after 24-h static culture are shown below. TEM of cells harvested from the top white layer of cells formed in the 48-h WT culture is shown below the 48-h culture. (D) Flotation of the smaI strain with and without supplemented BHL, after static culture for 24 h; PCM of cells taken from the top layer of cells are shown below each culture. (E) Flotation of E. coli W3110 transformed with pWEBTNC or pGAS after static culture for 24 h; PCM of cells taken from the top layer of cells are shown below each culture. (F) Opaque morphology of Serratia strains [prodigiosin (−) background]; (Upper Left portion) WT; (Upper Right portion) smaI; (Lower) smaI + 1μM BHL. (G) TEM of E. coli W3110(pGAS). (Scale bars, 1 μm.)

Fig. 4.

Regulation of gas vesicle production and swarming motility by RsmA. (A) Expression of the gvpA1::uidA reporter fusion in Serratia 39006 (WT), rsmA, and rsmB backgrounds (values are average of three biological replicates ± SD). (B) Flotation (i), swarming motility (ii), and PCM of gas vesicles in the rsmA mutant backgrounds carrying vector-only plasmid pJR3XF (Left) or pJRrsmA (Right). (Scale bars, 1 μm.)