MCE domain proteins

75 Pages·2017·4.38 MB·English

MCE domain proteins: conserved inner membrane lipid-
binding proteins required for outer membrane homeostasis
Georgia L. Isom1, Nathaniel J. Davies2, Zhi-Soon Chong3, Jack A. Bryant1,
Mohammed Jamshad1, Maria Sharif1, Adam F. Cunningham1, Timothy J. Knowles1,
Shu-Sin Chng3, Jeffrey A. Cole1, Ian R. Henderson1*

1. Institute of Microbiology and Infection, University of Birmingham
2. Institute of Immunology and Immunotherapy, University of Birmingham
3. Department of Chemistry, National University of Singapore
* Corresponding author: [email protected]

Supplementary figure S1. A heat map showing the distribution of MCE protein
architectures across the major classes of Proteobacteria. For architecture information
see supplementary table 1. The colours are based on percentages ranging from 0%
(white) to 100% (bright red). The classes are displayed on the right of the heat map
and the number of species is in brackets.
Supplementary figure S2. A heat map showing the distribution of MCE proteins
across Metazoa and Viridiplantae. The colours are based on percentages ranging from
0% (white) to 100% (bright red). The classes are displayed on the right of the heat
map and the number of species is in brackets.
Supplementary figure S3. A) Cleavable signal peptide predictions of PqiB and
YebT using SignalP 4.1 server and B) the transmembrane helix predictions of PqiB
and YebT using the TMHMM server.
Supplementary figure S4. Dilutions of the WT strain and ΔmlaD, ΔpqiAB and
ΔyebST deletion strains on LB agar supplemented with 0.5% SDS and 1.1 mM
EDTA. The plates were prepared as described previously.
Supplementary figure S5. Complementation of the phenotypes of mlaD, pqiAB and
pqiAB yebST mutants on 1% lauryl sulfobetaine: (A) LB agar, (B) 1% lauryl
sulfobetaine. The plates were prepared as described previously.
O
A N+ S
O
O-
O
N+ S
B
O
O-
O
C N+ S
O
O-

Supplementary figure S6. Chemical structures of A) caprylyl sulfobetaine B) lauryl
sulfobetaine and C) myristyl sulfobetaine.
Supplementary figure S7. Dilutions of the WT strain and all mutants on LB agar
supplemented with 1% SDS. The plates were prepared as described previously.
Supplementary figure S8. Outer membrane lipid profiles of the parent strain and the
mlaD pqiAB yebST mutant. Lipids were extracted from outer membranes and
separated by TLC in 65:25:4 chloroform:methanol:water (direction 1) and 80:12:15:4
chloroform:methanol: acetic acid:water (direction 2).

8
T T
b b
e e
WWT T ΔpqiB P ΔyebT Y PΔpqiB ΔyY WWT T ΔpqiB P ΔyebT Y ΔpqiB ΔyPY
OM IM OM IM OM IM OM IM OM IM OM IM OM IM OM IM
(kDa)
180 a-TolC a-TolC
135
100 a-AcrB a-AcrB
75

63 a-PqiB a-PqiB

48 a-YebT a-YebT

35

anti-PqiB anti-YebT
(kDa)
180

135
100
75

63

48

35
anti-AcrB anti-TolC

Supplementary figure S9. Full western blots to identify the locations of PqiB and
YebT in E. coli K-12 BW25113.

9
Supplementary table S1: Architectures for MCE proteins
Architecture
no. No. proteins Description
1 11012 MCE
2 2718 MCE, DUF3407
3 2026 MCE, MCE, MCE
4 1042 MCE, MCE, MCE, MCE, MCE, MCE, MCE
5 236 MCE, MCE, MCE, MCE, MCE, MCE
6 125 MCE, MCE
7 35 MCE, DUF330
8 20 MCE, MCE, MCE, MCE
9 15 MCE, MCE, MCE, MCE, MCE
10 8 MCE, MCE, DUF3407
11 7 MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE
12 4 MCE, DUF3407, MCE
13 4 MCE, MCE, MCE, DUF330
14 2 MCE, AP2
15 2 PqiA, PqiA, MCE, MCE, MCE, DUF330
16 2 PqiA, MCE, MCE
17 2 MCE, PLU-1
18 2 MCE, OmpA
19 1 MCE, MCE, MCE, MCE, Molybdopterin
20 1 PqiA, PqiA, MCE, MCE, MCE, MCE, MCE, MCE, MCE, Nol1_Nop2_Fmu,
Methyltransf_31, Methyltransf_18, Methyltransf_26, Nol1_Nop2_Fmu, Nol1_Nop2_Fmu_2
21 1 MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE, MCE
22 1 MCE, DUF4455
23 1 OTCace_N, OTCace, MCE
24 1 DUF3407, MCE
25 1 MCE, Inositol_P

10