Point Mutations Lead to Increased Levels of c-di-GMP and Phenotypic Changes to the Colony Biofilm Morphology in Alcanivorax borkumensis SK2

Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.


Supplementary Information
Percentages of R and S type: To estimate the ratio of appearance of R type to S types, we streaked 8 plates on marine agar supplemented with pyruvate from the original stock and cultured for 48 h. We took 5 representative images from each plate and counted the number of each type of colony. We counted a total of 933 colonies, where 778 were R colonies and 155 were S colonies. The average and standard deviation of the percentage on these 8 plates for R and S types are 83 ± 6% and 17 ± 6%, respectively.
India Ink Exclusion Staining: To image the excluded region surrounding the S type cells, we mix 2 µL of bacterial culture with 2 µL of commercially available Bokueki Sumi Ink (Sun Note, Osaka, Japan) on a glass slide. We then cover the sample with cover glass and image using phase contrast microscopy at 100x magnification.
Full genome resequencing: We extracted genomic DNAs from S-and R-type cells using QIAGEN Genomic-tip 100/G (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. Next generation sequencing and data analysis were supported by Macrogen Japan Co. (Macrogen Japan, Tokyo, Japan). The libraries were prepared using TruSeq DNA PCR-free kit (Illumina, San Diego, CA, USA). Each sample was sequenced using 100-bp paired end reads using the Illumina platform. The reads were mapped to 100% of the reference sequence A. borkumensis SK2 genome (Genbank accession number AM286690.1) at a mean depth of over 620. We found 9 SNPs at a depth of at least 433 using SAMTools.
Quantification of c-di-GMP by LC-MS: Cells were grown in ONR7Aa for two days and then sub-cultured for another two days to reach stationary phase. Bacteria were collected from 8 mL of culture media, washed, then lysed using sonication (Biorupter, Tokyo, Japan). c-di-GMP was extracted using a previously reported protocol (26). Briefly, we washed the lysate in 63% v/v ethanol in phosphate buffered saline (PBS) twice and kept the supernatant.
We then evaporated the supernatant containing the c-di-GMP overnight under vacuum. The residue was stored at -80° C until ready to be measured. The residue was resuspended in 100 µL of DI water. We quantified the amount of c-di-GMP with an LC-ESI-MS with a Shimadzu Nexera X2 system equipped with a XSelect HSS C18 column, 4.6 x 150 mm (Waters, Milford MA, USA) under the following conditions: column temperature, 40° C; gradient elution; mobile-phase solvent A [10mM ammonium acetate in DI H2O] and solvent B [methanol]; mobile-phase composition, 0% solvent B at 0-10 min, 0-30% solvent B at 18-20 min, 30-0% solvent B at 20-24 min, and 0% solvent B at 24-30 min; and flow rate, 0.5 mL min -1 ; and photodiode array detector, 190~600 nm. We obtained the MS analysis data using a mass spectrometer with ESI in the positive ion multiple reaction monitoring mode.
The monitored product ions were m/z 248, m/z 540, and m/z 152. We used commercially available cyclic-di-GMP (CAS No. 61093-23-0) with a purity of ≥ 95%, purchased from Cayman Chemical (Ann Arbor, MI, USA) as a reference for the LC-MS. We quantify the amount of protein per sample after sonication with the Pierce bicinchoninic acid (BCA) protein assay kit (ThermoFisher, Waltham MA, USA) and measure the output using