Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand

Abstract

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

FIG. 1.

Sequence of the inverse PCR product containing the putative promoter for pra, a structural gene encoding the PA bioemulsifying protein. An 8-bp region homologous to the pra structural gene (GenBank accession no. L08966) is italicized. The Shine-Dalgarno region (S/D) is underlined. PCR primers as described in the text are in boldface.

FIG. 2.

OD at 660 nm (circles), liquid surface tension (squares), and headspace parts per thousand carbon dioxide (triangles) (data for PG201 and PG201pra⁻ only) with either ammonium (closed symbols) or nitrate (open symbols) in liquid cultures of P. aeruginosa. Strains are either wild type (PG201) or deficient in PA protein synthesis (PG201pra⁻), in rhamnolipid synthesis (PG201rhlA⁻), or in both (PG201pra⁻rhlA⁻). Hexadecane is the carbon source. Each point represents the average of three or more independent replicates.

FIG. 3.

Depletion of hexadecane (closed symbols) and accumulation of CO₂ (open symbols) by four strains of P. aeruginosa PG201 in sand culture. Hexadecane is the sole C source; N is provided as either NO₃⁻ (top) or NH₄⁺ (bottom). Strains are either wild type (PG201) or deficient in PA protein synthesis (PG201pra⁻), in rhamnolipid synthesis (PG201rhlA⁻), or in both (PG201pra⁻rhlA⁻). Points represent either the average of three independent replicates (NO₃⁻) or single measurements (NH₄⁺) at each time point.

FIG. 4.

ESEM images of P. aeruginosa PG201 in moist sand after 2 days of cultivation. NO₃⁻ was the N source and hexadecane was the carbon source. Magnifications, ×2,500 (A) and ×3,500 (B). Arrows point to typical cells or cell clusters.

FIG. 5.

CLSM of drop slides containing P buffer, hexadecane, and P. aeruginosa strains PG201(pHX1) (A) and PG201(pRhlPGB) (B). Top frames are plan views; bottom frames are x-z sections. Fluorescence is from transcription of plasmid-borne gfp controlled by the putative promoters for pra (A) and rhlR (B). Bars, 20 μm.