Supplemental Information

Supplemental Table

Table S1. Bacterial strains and plasmids used in this study, related to Experimental Procedures.Strain or Plasmid Description ReferenceStrains

E. coliDH10B Used for cloning of plasmids and protein expression (Durfee et al., 2008)

M. xanthusDK1622 Wild-type strain (Chen et al., 1990)LS2442 DK1622 bearing clean deletion of csgA (MXAN_RS06255) This study

PlasmidspUC57 Derivative of pUC19, E. coli vector (Yanisch-Perron et al.,

1985)pTOB10 Synthetic clone of M. xanthus csgA codon optimized for E. coli in pUC57 This studypTrcHisB E. coli Expression vector (Amann et al., 1983)pTOB11 Codon optimized csgA sequence inserted into pTrcHisB This studypTOB25 p17 sequence of csgA inserted into pTrcHisB This studypTOB57 pTOB11 with the K155R mutation in CsgA coding sequence This studypLEE112 SocA sequence in pET100/D-TOPO (Avadhani et al., 2006)Sni-DROME D. melongaster sniffer coding sequence in pET-15b (Martin et al., 2011)HSD10 H. sapiens HSD10 coding sequence in pNIC28-Bsa4 Gift of Albert AmbergerHSD10D86G As HSD10, but bearing the D86G mutation Gift of Albert AmbergerHSD10Q165H As HSD10, but bearing the Q165H mutation Gift of Albert AmbergerHSD10R130C As HSD10, but bearing the R130C mutation Gift of Albert Amberger

Supplemental Figures

Figure S1. Codon optimized sequence of csgA (MXAN_RS06225) used to create CsgA expression vectors. Changed codons are shown in red. Sequence was optimized for expression and secondary structure in E. coli by Genscript (Piscataway, NJ, USA). The clone for p17 is denoted by the p17 start site.

Figure S2. Recombinant CsgA purifies as an active enzyme and is capable of restoring development in mutant cells. A. SDS-PAGE analysis of purified recombinant CsgA proteins containing N-terminal 6xhis epitope tags and expressed in E. coli. p25 rCsgA, left; p17 rCsgA, right. Molecular weight markers are shown. Each protein appears as a single band consistent with expected recombinant sizes. B. p25 rCsgA but not p17 is able to complement LS2442, a CsgA mutant through extracellular supplementation. LS2442 is unable to develop in submerged culture, but addition of 1 μM p25 rCsgA restores this development. Bar; 200 μm.

Figure S3. Sample assays of NAD+ versus NADP+ cofactor usage in CsgA-like enzymes. A. CsgA (), SocA (), HSD10 (), and Sniffer () are able capable of utilizing NAD+ in sample reactions using CL as the substrate. B. In contrast, only SocA () and Sniffer () are able to use NADP in the same reactions. CsgA () and HSD10 () show no activity.

Figure S4. Lipid structures tested for CsgA enzymatic activity. Bolded are the glycerol moieties. All lipids contain C14:0 myristoyl fatty acids. MAG, monoacylglycerol; DAG, diacylglycerol; Lyso-PE, lysophosphatidylethanolamine; CL, cardiolipin; PG, phosphatidylglycerol.

Figure S5. Sample mass spectrometry identification of the lipid product of CsgA reaction using cardiolipin as a substrate. A. Positive LC/MS ESI spectra obtained from representative reaction identifying the product as having a mass value 535.4202, in line with a Na+-adduct of C14:0 diacylglycerol. B. Positive LC/MS ESI spectra obtained from an otherwise identical reaction lacking enzyme.

Supplemental References

Amann, E, Brosius, J, and Ptashne, M. 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25: 167-178.

Chen, H, Keseler, IM, and Shimkets, LJ. 1990. Genome size of Myxococcus xanthus determined by pulsed-field gel electrophoresis. Journal of Bacteriology 172: 4206-4213.

Durfee, T, Nelson, R, Baldwin, S, Plunkett, G, Burland, V, Mau, B, Petrosino, JF, Qin, X, Muzny, DM, Ayele, M, et al. 2008. The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. Journal of Bacteriology 190: 2597-2606.

Yanisch-Perron, C, Vieira, J, and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119.