Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes

Abstract

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.

FIG. 1.

PC biosynthesis pathways in A. tumefaciens C58. As indicated by the question marks, the choline uptake system is unknown. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; PmtA, phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase.

FIG. 2.

Activity of agrobacterial PmtA and Pcs after expression in E. coli. (A) Detection of agrobacterial phospholipid N-methyltransferase (PmtA) and PC synthase (Pcs) in crude extracts of E. coli cells via SDS-PAGE. E. coli BL21(DE3) cells with pET_PmtA, pET_Pcs, or pET24b were cultivated in LB complex medium, and protein expression was induced with IPTG. Protein bands were visualized by Coomassie blue staining. M, BenchMark protein standard (Invitrogen). +, present; −, absent. (B) Lipid formation after expression of agrobacterial PmtA and Pcs in E. coli BL21(DE3). Lipids of BL21(DE3) derivatives were extracted and separated by one-dimensional TLC. Phospholipids were specifically stained with molybdenum blue spray and compared to phospholipid standards in lane M (PE, MMPE, DMPE, and PC).

FIG. 3.

RT-PCR of the pmtA and pcs gene regions. The PCR strategy is outlined in panels A and C. The positions of primers used for the reverse transcription and PCRs are given below the corresponding gene regions. Putative promoters and constructed lacZ fusions are indicated. dnaN encodes β-chain of DNA polymerase III; pmtA encodes phospholipid N-methyltransferase; pyrF encodes orotidine 5′-monophosphate decarboxylase; atu0299, atu0297, and atu0296 are hypothetical open reading frames; ubiH encodes 2-octaprenyl-6-methoxyphenol hydroxylase; pcs encodes PC synthase; abc1 encodes a nucleotide binding ABC transporter; abc2 and abc3 encode membrane-spanning proteins forming an ABC transporter; and abc4 encodes a lipoprotein ABC transporter. In panels B and D, the results of RT-PCRs with RNA from wild-type A. tumefaciens C58 cells are presented. The primer pairs used and lengths of PCR products are indicated. c, PCR products using chromosomal DNA as template; +, standard RT-PCR; −, negative control in which no RT had been added to the reaction mixture.

FIG. 4.

β-Galactosidase activities of plasmid-encoded transcriptional lacZ fusions in wild-type A. tumefaciens C58 and PC biosynthesis mutants. Cells were grown in AB minimal medium in the absence or presence of 0.1 mM choline at 30°C. The error bars indicate standard deviations of the results from three independent assays. The plasmid pAC01 containing the promoterless lacZ gene was used as negative control, and the background activity was below 3 Miller units (MU). 1, wild-type; 2, ΔpmtA; 3, Δpcs; 4, ΔpmtA Δpcs.

FIG. 5.

PC formation in the A. tumefaciens abc1-4 mutant. A. tumefaciens wild-type, ΔpmtA, ΔpmtA Δpcs, and ΔpmtA Δabc cells were grown in YEB complex medium at 30°C. Lipids were extracted, separated by one-dimensional TLC, and visualized by Cu₂SO₄ staining.

FIG. 6.

Localization of PC in membranes of wild-type A. tumefaciens C58. (A) Schematic representation of bands observed after centrifugation. (B) Separation of agrobacterial inner and outer membrane by discontinuous sucrose density gradient centrifugation. Fractions were collected as 500-μl aliquots from the top of the sucrose gradient. Protein concentrations (•) and sugar densities (▴) of the gradient fractions are shown. (C) Fractions 5 and 6 (IM), fraction 12, and fractions 20 and 21 (OM) were analyzed by SDS-PAGE and visualized with Coomassie blue staining. (D) Lipids were extracted, and fractions 3 to 7 and 19 to 21 were pooled, followed by one-dimensional TLC analysis and molybdenum blue staining. Barely detectable lipids are marked with arrows. PE, MMPE, DMPE, and PC were used as standards. M, phospholipid standard; IM, inner membrane; OM, outer membrane.

FIG. 7.

Motility assay of an agrobacterial PC-deficient mutant and complemented strains. (A) Motility of the wild type, the PC-deficient mutant (ΔpmtA Δpcs), and the mutant complemented with either pVS_pmtA or pVS_pcs was assayed on AB minimal medium plate containing 0.3% (wt/vol) agar. (B) Cell lysates of wild type and PC-deficient mutant were analyzed by SDS-PAGE and Western blotting using a flagellin antibody (1:30,000).

FIG. 8.

Biofilm formation of a PC-deficient mutant of A. tumefaciens C58. Flow cell biofilms of wild-type and mutant (ΔpmtA Δpcs) expressing GFP were grown in AB minimal medium at 30°C. Microscopic visualization of biofilms was carried out using an inverted Zeiss LSM510 confocal laser-scanning microscope equipped with 20×/0.5 W Achroplan objective. Image data were obtained after 24 h (A) and 48 h (B) and further processed using the IMARIS software package (Bitplane AG, Zürich, Switzerland) and Adobe Photoshop.