Supporting Information
B12 regulates photosystem gene expression via the CrtJ antirepressor AerR
Zhuo Cheng1, Keran Li1, Loubna A. Hammad2, Jonathan A. Karty2 and Carl E. Bauer1,*
Department of 1Molecular and Cellular Biochemistry and 2Chemistry, Indiana University,
Bloomington, IN 47405, USA
*Corresponding
author:
Carl Bauer
Address: Simon Hall MSB, Room 305A, 212 S. Hawthorne Drive, Bloomington, IN 47405
Telephone: 812-855-6595
Fax: 812-856-5710
Email: [email protected]
Supplemental Figures
Fig. S1. In vivo Western blot analysis of CrtJ-FLAG expression levels in a ΔaerR strain and
in the parent WT strain SB1003.
Fig. S2. AerR is a B12 binding protein.
A. Alignment of AerR homologues, CarH and methionine synthase B12 binding domain.
[National Center for Biotechnology Information (NCBI) accession numbers are
AAF24277.1 (PpaA-R.sph), BAG34613.1 (AerR-R.cen), YP_005438182.1 (AerR-R.gel),
CAA79965.2 (CarH-M.xan), ZP_03033495.1 (Meth-E.coi). The conserved residue
Asp/Glu-X-His-X-X-Gly-(41)-Ser(Thr)-X-Leu-(26-28)-Gly-Gly are labeled below each row.
Red background represents 100% conserved residues, yellow background represents
the 80% conserved residues and grey background represents 60% conserved residues.
The position of conserved cobalt coordinating histidine residue is pointed by a blue
arrow. His10 in AerR-R.cap are labeled in blue background. Sequences were aligned
using the MEGA alignment program and modified using GENEDOC. R.cap: Rhodobacter
capsulatus; R.sph: Rhodobacter sphaeroides; R.cen: Rhodospirillum centenum; R.gel:
Rubrivivax gelatinosus; M.xan: Myxococcus xanthus; E.coi: Escherichia coli F11.
B. Structure model of AerR (residues 45~249), using the crystal structure of the B12
binding domain of methionine synthase (MetH) (residues 659~866) in E. coli as a
template (PDB: 1bmt) (Drennan et al., 1994) by SwissModel Automatic Modelling Mode
(Arnold et al., 2006, Guex and Peitsch, 1997, Schwede et al., 2003). AerR structure
model is shown in blue (the histidine residue coordinating to the cobalt atom is shown
in a sticks-representation) and the B12 binding domain of MetH structure is shown in
pink (B12 is shown in a sticks-representation).
Fig. S3. LC-ESI-MS/MS analyses of a B12 containing peptide (residue 1~16:
MRDISVELEHGSTGAL) obtained by chymotrypsin digested AerR C15A protein.
A. Extracted ion chromatogram (EIC) of the triply-charged peptide containing B12 at m/z
1014.8 (* indicates the peak of interest); the corresponding UV-Vis spectrum of this
peptide is shown in the inset.
B. High-resolution mass spectrum of this B12 containing peptide ion at m/z 1014.8 (3
ppm mass error).
C. Collision induced dissociation (CID) spectrum of this B12 containing peptide peptide
ion at m/z 1014.8.
(A) and (C) were obtained using the Bruker HCT ion trap MS instrument whereas (B) was
obtained using the Thermo LTQ-Orbitrap MS instrument.
Fig. S4. X-GAL spotting assays
Left: R. capsulatus SB1003 carrying aerR::lacZ reporter plasmid;
Right: R. capsulatus SB1003 carrying aerRstop::lacZ reporter plasmid.
Supplemental Table
Table S1. Primers used in this study.
Target
aerR
upstream
aerR
downstream
aerR
Use
deletion
Primer Name
Sequence (5’-3’)
aerRupSacI-F
GCGCGAGCTCAGCCTCGTAACCCTCTCCG
aerRupEcoRV-R
GCGCGATATCCATTCGAGTCCCCGAAACC
deletion
aerRdownEcoRV-F
GCGCGATATCTGACTGAAAACGGTTGTGG
aerRdownXbaI-R
GCGCTCTAGACGAGCAGCACGCGATAGC
overexpression aerRSUMOBbsI-f
GCGCGAAGACTGAGGTATGCGGGATATAAGTGTTGAAC
aerRSUMOBamHI-r
GCGCGGATCCCAGTCATACCAGAGAAAATTCG
aerR H145A mutagenesis
aerRH145A-f
GATTGTGCCGCCCGGAGAGCAGGCCACGCTGGGCGCGTTGATCGTGG
aerRH145A-r
CCACGATCAACGCGCCCAGCGTGGCCTGCTCTCCGGGCGGCACAATC
aerR H10A
mutagenesis
aerRH10A-f
AAGTGTTGAACTCGAGGCTGGCTCGACGGGATGC
aerRH10A-r
GCATCCCGTCGAGCCAGCCTCGAGTTCAACACTT
aerR C15A
mutagenesis
aerRC15A-f
AGCATGGCTCGACGGGAGCCCTTGATGGTTTCG
aerRC15A-r
CGAAACCATCAAGGGCTCCCGTCGAGCCATGCT
footprint
bchC5HEX-f
/5HEX/CGGTGATCGAGGCCGAAC
bchC
promoter
bchC6FAM-r
/6-FAM/AACAATTTCTCGGTGCCAGTCG
Underlined sequences indicate restriction sites;
Highlighted sequences in red indicate an alanine codon in place of the original codon.
Supplemental experimental procedures
Determination of the aerR Start Codon
Based on annotation of photosynthesis gene cluster (EMBL Nucleotide Sequence
Database accession number Z11165), aerR gene was annotated to span 576bp, with a
gene product of 192 amino acids (Dong et al., 2002). However, comparison to AerR
homologues from related photosynthetic bacteria indicates that an upstream ATG is
most likely where the aerR open reading frame initiates. This longer version of aerR has
252 amino acids. To determine the correct start site, an aerR::LacZ translation fusion
was constructed in which a mutation forms a stop codon TGCTGA between these two
ATG’s (aerRstop::LacZ). Both wild type and stop codon derivatives were introduced to R.
capsulatus SB1003 (wild type) and assayed for β-galactosidase activity by the addition of
2% X-Gal onto colonies. Colonies of SB1003/aerR::LacZ turned blue within 10 minutes
indicating the presence of the synthesis of an aerR::lacZ translational fusion. However,
colonies of SB1003/aerRstop::lacZ did not turn blue indicating the presence of a stop
codon between these two ATG sites inhibits production of β-galactosidase (Fig. S4). This
indicates that the aerR gene indeed starts from the upstream ATG and that the gene
product extends 252 amino acids.
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