Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Cs1-4

Abstract

The bacterium Delftia sp. Cs1-4 produces novel extracellular structures (nanopods) in conjunction with its growth on phenanthrene. While a full genome sequence is available for strain Cs1-4, genetic tools that could be applied to study phenanthrene degradation/nanopod production have not been reported. Thus, the objectives of this study were to establish such tools, and apply them for molecular analysis of nanopod formation or phenanthrene degradation. Three types of tools were developed or validated. First, we developed a new expression system based on a strong promoter controlling expression of a surface layer protein (NpdA) from Delftia sp. Cs1-4, which was ca. 2,500-fold stronger than the widely used lactose promoter. Second, the Cre-loxP system was validated for generation of markerless, in-frame, gene deletions, and for in-frame gene insertions. The gene deletion function was applied to examine potential roles in nanopod formation of three genes (omp32, lasI, and hcp), while the gene insertion function was used for reporter gene tagging of npdA. Lastly, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis. Application of the latter to strain Cs1-4, revealed several new genes with potential roles in phenanthrene degradation or npdA expression. Collectively, the availability of these tools has opened new avenues of investigation in Delftia sp. Cs1-4 and other related genera/species with importance in environmental toxicology.

Keywords: Delftia sp. Cs1-4; biodegradation; genetic manipulation; nanopods; phenanthrene; polynuclear aromatic hydrocarbons; surface layer protein.

Figure 1

Analysis of npdA promoter regions. (A) Putative −10- and −35-bp motifs are indicated with P1, P2 and P3. Transcription start points are capitalized and underlined. Arrows indicate positions of deletions (D1-7). (B) Effect of serial deletion on rluc expression. Results were normalized to Rluc activity of the wild-type. Reactions were done in triplicate, and standard deviations are indicated by error bars.

Figure 2

Growth and fluorescence characteristics of the GFP and RFP reporter strains. (A) SCH481 (PnpdA:gfp, circle), SCH482 (PnpdA:mStrawberry, diamond), and WT (square) were adjusted to the same cell density and cultured for 30 h. (B) Fluorescence determination for GFP (circle) and RFP (diamond) reporter strains. Values in (A) and (B) are means of measurements made from triplicate cultures, and error bars indicate standard deviation) (C) Stability test of the expression vector (pSCH477, PnpdA:gfp) in Delftia sp. Cs1-4. The strain SCH481 (PnpdA:gfp) was serially transferred in LB medium and the CFU were determined at generations of 16, 36 and 56. The blank bar is without tetracycline addition and the black one is supplemented with tetracycline.

Figure 3

Characterization of the Delftia sp. Cs1-4 Δomp32 mutant. (A) Protein profiles of the wild type (WT) and mutant. Boxed area indicates the region of the Omp32 band in the WT. In the mutant, arrows indicate two bands identified as different porins, which were not detected in the WT. (B) Transmission electron micrographs of strain Cs1-4 biofilm cells grown on phenanthrene illustrating the mutant’s cellular deformities. (C) and (D) Growth of the WT (diamonds) and mutant (circles) on the indicated substrate.