Supplemental Materials and Methods

Bacterial strains and growth conditions

The bacterial strains, plasmids, and oligonuceltoides used in this study are listed in Table S1. Bacterial growth experiments were performed in M9 minimal media containing 1.7 mM MgSO4, 0.117 mM CaCl2, 0.03 mM and FeSO4 and a carbon source as indicated (1). All strains were grown at 37˚ C, except for V. fischeri which was grown at 22˚C. Media was added to 96-well F96 microtiter plates (Nunc) to a final volume of 200 µL, and culture was added to an OD600 of 0.05. Glucose or LPC 18:0 were supplemented at 0.2% or 2 mM, respectively. For V. fischeri, an artificial seawater-based minimal media (2) was used. All growth media contained 6 mM Brij-58 to promote LPC solubility. For growth curves, OD600 readings were taken every half hour using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). For single read experiments, an OD600 reading was taken after 24 hours.

Isolation and characterization of phospholipids

Phospholipids were isolated using Bligh/Dyer extractions and analyzed by TLC or by liquid chromatography/ESI-mass spectrometry as previously described (3).

RNA Extraction and Quantitative RT-PCR

Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) from cultures grown in minimal media to a final OD600 of 0. 6. RNA was treated with RQ1 RNAse-free DNAse (Promega) to eliminate genomic DNA contamination. cDNA template was generated using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). To verify the transcription of specific genes, a standard PCR was performed; vca0862 was amplified from cDNA using primers 62F and 62R; vca0863 was amplified using primers 63F and 63R while the region spanning the non-coding sequence between vca0862 and vca0863 was amplified with primers 62bF and 63bR (Table S1). The PCR products were visualized by electrophoresis on 1% agarose gel. For quantitative PCR, the 2X SYBR Green PCR Master Mix (AB Applied Biosystems) was used. Data analysis was performed using ABI 7900HT Fast Real Time PCR System and the Software Sequence Detection Systems (SDS) version 2.4 (AB Applied Biosystems). The relative expression ratio of the target transcript was calculated in comparison to the gyrA transcript as the reference gene following the Pfaffl method (Pfaffl, 2001) using primers gyrAF and gyrAR.

Generation of expression plasmids.

The vca0863 complementation construct was generated in the low-copy-number plasmid pWSK29 (Table S1). Oligonucleotides for plasmid construction are listed in Table S2. Initially, vca0863 was amplified from C6706 genomic DNA using primers 0863NF and 0863BR, or 0863HR which incorporates a C-terminal 6x histidine tag. The vector was digested with BamHI and NdeI and ligated into high-copy-plasmid pET21a. The insert was digested out of pET21a using XbaI and XhoI and subsequently ligated into pWSK29. Other genes were inserted into pWSK29 in a similar manner. The vca0863 G20D mutant was generated using primers G20DF and G20DR; ampC was amplified using primers BLF and BLR; ompT was amplified using primers OmptF and OmptR.

Generation of VcA0863-specific polyclonal antibody

VcA0863-specific polyclonal antibody was generated by the Genscript corporation. Briefly, a fifteen amino acid peptide (CGERSLDSTRSANSD) predicted to be solvent-exposed was selected from the primary sequence of VcA0863 and used to generate VcA0863-specific antibody from rabbits.

Whole cell labeling with NHS-LC-LC biotin

Labeling of the cell surface with NHS-LC-LC biotin was performed as previously described (4). Proteins were purified via affinity chromatography using HisPur Cobalt Resin (Pierce). Cells were lysed in a buffer of 10 mM Tris-HCl, 250 mM NaCl, 2.5% Triton X-100 and 20 mM imidazole; clarified cell extract was incubated on the cobalt resin and purified protein was eluted using 500 mM imidazole. Western blot analysis was carried out as described above using a 1:600000 dilution of a 1.25 mg/mL solution of streptavidin-conjugated horseradish peroxidase (Pierce).

Immunogold electron microscopy

Strains of wild-type, vca0863::TnFGL3, and pVcA0863-carrying V. cholerae were grown to mid-log phase in 5 mL of LB medium. 1.5 mL of mid-log culture was harvested at 2.8 x 1000 rpm for 5 min and re-suspended in 500 µL of PBS. Cells were pelleted and re-suspended in a final volume of 100 µL PBS. Bacteria were adsorbed to formvar/carbon coated nickel grids and blocked for 20 min in 0.3% skimmed milk in PBS (M-PBS). The grids were then incubated with a 1:50 dilution of the anti-VcA0863 antibody for 30 min, followed by three 2-min washes in PBS. Washed grids were incubated with a 1:20 dilution of gold-conjugated goat anti-rabbit IgG in M-PBS. After a 30 min incubation, grids were washed three times for 2 min in PBS, followed by three 2-min washes in sterile water. Grids were visualized in a FEI TecnaiTransmission Electron Microscopeat 80 KeV.

Supplemental References

1. Sambrook J, Russell DW. 2001. Molecular cloning : a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

2. Lupp C, Hancock RE, Ruby EG. 2002. The Vibrio fischeri sapABCDF locus is required for normal growth, both in culture and in symbiosis. Archives of microbiology 179:57-65.

3. Giles DK, Hankins JV, Guan Z, Trent MS. 2011. Remodelling of the Vibrio cholerae membrane by incorporation of exogenous fatty acids from host and aquatic environments. Molecular microbiology 79:716-728.

4. Cowles CE, Li YF, Semmelhack MF, Cristea IM, Silhavy TJ. 2011. The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli. Molecular microbiology 79:1168-1181.

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