Table S1. List of strains and plasmids used in this study.
Phenotype, genotype and/or descriptiona
Ref/Source
TOP10
F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74
recA1 araD139 Δ(ara leu)7697 galU galK rpsL (Strr) endA1
nupG
Invitrogen
GM2163
dam-13::Tn 9 dcm6 hsdR2 leuB6 his-4 thi-1 ara-14 lacY1
galK2 galT22 xyl-5 mtl-1 rpsL136 tonA31 tsx-78 supE44
McrA- McrB
New England Biolabs
Rosetta (DE3)
F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE (Cmr)
Novagen
H26
DS70 pyrE2
(Allers et al, 2004)
GZ108
H26 ΔpanB
(Zhou et al, 2008)
HM1052
H26 ΔubaA
(Miranda et al, 2011)
Plasmidsb
pET24b
Cmr, Kmr, expression vector for E. coli (DE3) strains
Strain, plasmid
E. coli
Hfx. volcanii
(Humbard et al, 2009)
pJAM2001
Ampr Nvr; C-terminal StrepII expression vector for Hfx.
volcanii strains
Ampr Nvr; PAN-A/1-StrepII
pJAM1106
Ampr Nvr; Flag-His-SAMP1 in pJAM202c
This study
pJAM1119
Ampr Nvr; MoaE-StrepII
(Hepowit et al, 2012)
pJAM1796
Ampr Nvr; Flag-SAMP1-MoaE-M1A-StrepII
(Hepowit et al, 2012)
pJAM1131
Cmr, Kmr, Flag-His-SAMP1 in pET24b
This study
pJAM809
This study
a
Abbreviations: Ampr, ampicillin resistance; Nvr, novobiocin resistance ; Cmr, chloramphenicol
resistance; Kmr, kanamycin resistance; -StrepII, C-terminal StrepII tag fusion protein; His-, N-terminal
poly-His6 tag fusion protein; Flag-, N-terminal Flag tag fusion protein; Flag-His-, N-terminal tandem
affinity Flag and poly-His6 tagged protein.
b
To generate pJAM2001, PCR was used with primers 1/2 to isolate an Hfx. volcanii genomic fragment
carrying the panA/1 gene that was ligated into the NdeI to KpnI sites of plasmid pJAM809. To
generate pJAM1106, PCR was used with primers 5/6 to generate an Hfx. volcanii genomic fragment
carrying the samp1 gene that was ligated into the KpnI to BlpI sites of pJAM939. Plasmid pJAM1131
was derived by NdeI to BlpI ligation of the Flag-His6-SAMP1 coding region of pJAM1106 into plasmid
vector pET24b. PCRs were with Hfx. volcanii H26 or DS70 genomic DNA as the template using primers
listed in Table S2 and Phusion® High-Fidelity DNA Polymerase according to Manufacturer’s protocol
(New England Biolabs). DNA fragments were treated with Antarctic Phosphatase and/or extracted
from gels after separation by 0.8% (w/v) agarose gel electrophoresis in 1X TAE buffer with MinElute®
gel extraction kit (Qiagen) as needed. Plasmid vectors and digested PCR-amplified products were
ligated overnight at 16˚C with T4 DNA ligase (New England Biolabs). The fidelity of all plasmids was
confirmed by DNA sequencing (ICBR DNA Sequencing Core, University of Florida).
1
Table S2. List of primers used in this study.
Primer
no.
1
Primer name
Oligonucletide Sequence (5’ to 3’)a
HVPanA Nde1 FW
5’-CGGCATcatatgATGACCGATACT-3’
2
HVPanA Kpn1 RV
5’-ATggtacccGCGAACGCGC-3’
3
Hvo_2619 NdeI up
5’-CTGCCCTcatatgGAGTGGAAGCTGT-3’
4
Hvo_2619 BlpI down
5’-TTAATgctcagcTCACCCACCGGC-3’
5
HVO_2619 KpnI-His Up
5’-AAggtaccCACCACCACCACCACCACGAGTGG
AAGCTGTTC-3’
6
HVO_2619 BlpI down
5’-TTAATgctcagcCTAGCCGCCGCTGACCGG-3’
a
Lowercase letters indicate restriction enzyme site. Italicized uppercase letters indicate random bases
introduced into primer to enhance cleavage of PCR product by restriction enzyme.
2
Table S3. Modified guanidine-HCl buffered conditions used for denaturing and renaturing the halophilic
proteins by far western blottinga.
Buffer Reagents/
Conditions
8 M Guanidine-HCl (mL)
Glycerol (mL)
NaCl (g)
500 mM HEPES (mL)
0.5 M EDTA (mL)
10% Tween-20 (mL)
Milk powder (g)
1M DTT (µL)
ddH2O (mL)
Total volume (mL)
Time/Temperature
1
150 (6M)
20
1.16 (0.1 M)
8
0.4
2
4
200
21.4
200
30 min/RT
Far Western Blotting Steps
2
3
4
74.4 (3 M)
24.8 (1 M)
2.48 (0.1 M)
20
20
20
1.16 (0.1 M)
1.16 (0.1 M)
2.32 (0.2 M)
8
8
8
0.4
0.4
0.4
2
2
2
4
4
4
200
200
200
97
146.6
168.92
200
200
200
30 min/RT
30 min/RT
30 min/4°C
a
5
0 (0 M)
20
24 (2 M)
8
0.4
2
4
200
171.4
200
overnight/4°C
Protocol was adapted from the far western blotting method of (Wu et al, 2007) for the halophilic (saltloving) proteins of this study. All solutions were freshly prepared prior to use. Membrane-bound proteins
were denatured in step 1 and renatured in a stepwise fashion (steps 2 to 5) resulting in a decrease in the
concentration of guanidine-HCl from 6 to 0 M and increase the NaCl concentration from 0.1 to 2 M. The
final buffer of step 5 was supplemented with 2M NaCl to enhance renaturation of the halophilic proteins.
3
Supplemental References
Allers T, Ngo HP, Mevarech M, Lloyd RG (2004) Development of additional selectable markers for the
halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol 70:
943-953
Hepowit NL, Uthandi S, Miranda HV, Toniutti M, Prunetti L, Olivarez O, De Vera IM, Fanucci GE, Chen
S, Maupin-Furlow JA (2012) Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves
ubiquitin-like small archaeal modifier proteins (SAMPs) from protein-conjugates. Mol Microbiol 86:
971-987
Humbard M, Zhou G, Maupin-Furlow J (2009) The N-terminal penultimate residue of 20S proteasome
alpha 1 influences its N-alpha acetylation and protein levels as well as growth rate and stress
responses of Haloferax volcanii. JOURNAL OF BACTERIOLOGY 191: 3794-3803
Miranda HV, Nembhard N, Su D, Hepowit N, Krause DJ, Pritz JR, Phillips C, Söll D, Maupin-Furlow JA
(2011) E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur
transfer in archaea. Proc Natl Acad Sci U S A 108: 4417-4422
Wu Y, Li Q, Chen XZ (2007) Detecting protein-protein interactions by Far western blotting. Nat Protoc
2: 3278-3284
Zhou G, Kowalczyk D, Humbard M, Rohatgi S, Maupin-Furlow J (2008) Proteasomal components
required for cell growth and stress responses in the haloarchaeon Haloferax volcanii. J Bacteriol 190:
8096-8105
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