Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluorescens for secretion and fluorescence of green fluorescent protein

Abstract

Background: TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter.

Results: Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA.

Conclusion: LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ.

Figure 1

The features and structures of LARDs. A. The secretion/chaperone domain of TliA (residues 301–476). The residues and PCR amplification sites of LARDs are also shown overlapped. TliA has a calcium-binding domain (373–416) comprised of four GGxGxDxUx repeats, hydrophobic five-sequence motif and an extreme C-terminal motif, EGVLIS. The three-dimensional structures of each LARDs were predicted by SWISS-MODEL structural modeling according to their residues and displayed by UCSF Chimera (http://www.cgl.ucsf.edu/chimera/) [30]. The RTX motif is indicated by the arrowhead. B. TliA, C-G. LARD1 to 5. Note that LARD3 has the RTX motif exposed at the N-terminus. The protein data bank (PDB) IDs for the templates used to predict each structure are as follows: TliA, 2z8z_A; LARD1, 2z8z_A; LARD2, 2qub_A; LARD3, 2zvd_C; LARD4, 2qub_A; LARD5; 2zvd_C.

Figure 2

The intracellular expression of EGF-LARDs inE. coliXL1-Blue. Each E. coli XL1-Blue culture harboring one of pEGF-LARD1-4 (lanes 1–4) or pEGF-TliA (lane A) with pEcPrtDEF (+) or pACYC-184 (−) was incubated with 0.05 mM IPTG, 50 μg/ml ampicillin, and 34 μg/ml chloramphenicol in 2 ml glass tubes at 37°C. The cells were isolated from the extracellular medium by double centrifugation, underwent SDS-PAGE in 15% gel, and analyzed by western blotting using anti-LARD antibodies. The plasmid pACYC-184 was used as a negative control for pEcPrtDEF which encodes PrtDEF. Purified TliA at a concentration of 99 μg/ml was used as an index after dilution. The arrowheads indicate the size of each EGF-LARD.

Figure 3

The extracellular secretion of EGF-LARDs inE. coliXL1-Blue and MM294. A. The secretion of EGF-LARDs in E. coli XL1-Blue. Each culture harboring one of pEGF-LARD1-4 (lanes 1–4) or pEGF-TliA (lane A) with pEcPrtDEF was centrifuged twice, and the supernatant was collected. The growth conditions were identical to those in Figure 2. SDS-PAGE with 15% gel and western blotting with anti-LARD antibodies were performed on the supernatant. The (+) sign indicates the presence of PrtDEF. The (−) sign indicates the absence of PrtDEF (pACYC-184 was used as a negative control). Purified TliA at 99 μg/ml concentration was used as an index after dilution. B. The secretion of EGF-LARDs in E. coli MM294. The methods used here were identical to those used in (A).

Figure 4

The intracellular expression of GFP-LARDs inE. coliXL1-Blue. Each E. coli XL1-Blue culture harboring one of pGFP-LARD1-4 (lanes 1–4), pGFP-TliA (lane A) or pGFP-223 (lane None) with pEcPrtDEF (+) or pACYC-184 (−) was grown with 50 μg/ml ampicillin and 34 μg/ml chloramphenicol in a 2 ml glass tube at 37°C (IPTG was not added). The culture was centrifuged twice, and the pellet was isolated. SDS-PAGE with 10% gel and western blotting with anti-LARD antibodies were performed on the pellet. Purified TliA at a concentration of 99 μg/ml was used as an index after dilution.

Figure 5

The extracellular secretion of GFP-LARDs inE. coliXL1-Blue and MM294. A. The secretion of GFP-LARDs in E. coli XL1-Blue. Each E. coli XL1-Blue culture harboring one of pGFP-LARD1-5 (lanes 1–5), pGFP-TliA (lane A) or pGFP-223 (lane None) with pEcPrtDEF (+) or pACYC-184 (−) was centrifuged twice, and the supernatant was collected. The growth conditions were identical to those listed in Figure 4. SDS-PAGE with 10% gel and western blotting with anti-LARD antibodies were performed on the supernatant. Purified TliA at a concentration of 99 μg/ml was used as an index after dilution. B. The secretion of GFP-LARDs in E. coli MM294. The methods used here were identical to those listed in A. The signs are also identical to those of A.

Figure 6

The GFP fluorescence of recombinantE. colicolonies at different temperatures. A. GFP fluorescence detected on LB plates. Single colonies of E. coli XL1-Blue and MM294, each harboring one of the designated plasmids, were streaked on LB plates and grown at 25°C or 37°C. They were subsequently analyzed under UV light. Starting from the culture designated by the arrowhead in a clockwise direction, the plasmids content is as follows: pGFP-223; pGFP-LARD1-5; and pGFP-TliA. B. GFP fluorescence assay. The cultures of E. coli XL1-Blue and MM294 in A were grown in liquid LB. The cells were washed and analyzed under excitation of 485 nm and emission of 535 nm. The relative fluorescence of each culture compared to that of the culture harboring pGFP-223 was plotted on the graph in a percentage scale (%).

Figure 7

The GFP fluorescence of recombinantP. fluorescenscolonies. The recombinant P. fluorescens cells harboring appropriate plasmids were cultured on an LB plate, and the GFP fluorescence was analyzed using UV light. Label 1, pDSK-TliDEF-GFP-LARD3 (pDX-GFP-LARD3); label 2, pDX-GFP-223; label 3, pDX-TliA; label 4, pDX-GFP-TliA.

Figure 8

The expression and secretion of the recombinant proteins inP. fluorescens. A. The intracellular expression and secretion of GFP-TliA and GFP-LARD3 in P. fluorescens cultured in LB were detected by western blotting using anti-LARD antibodies. The P. fluorescens cells expressing the designated proteins with (+) or without (−) TliDEF were isolated from two different extracellular media by double centrifugation. B. The same expression was performed in 2× LB (double concentrated LB). Lane 1, pDSK-TliA; lane 2, pDX-TliA; lane 3, pDX-GFP-LARD3; lane 4, pDX-GFP-TliA. The arrows indicate the sizes of GFP-LARD3 (38.2 kDa), TliA (49.9 kDa), and GFP-TliA (77.4 kDa). Notice the intrinsic TliA indicated by the arrows.

Figure 9

The expression and secretion of the recombinant proteins inP. fluorescens ΔtliA. The intracellular expression and secretion of GFP-TliA and GFP-LARD3 were analyzed by western blotting using anti-LARD antibodies. The cells were isolated from the growth medium by double centrifugation. Lane 1, pDSK-TliA; lane 2, pDX-TliA; lane 3, pDX-GFP-LARD3; lane 4, pDX-GFP-TliA. The arrows indicate the sizes of GFP-LARD3 (38.2 kDa), TliA (49.9 kDa), and GFP-TliA (77.4 kDa).