Table S1. List of strains and plasmids used in this study.
Strain, plasmid / Phenotype, genotype and/or descriptiona / Ref/SourceE.coli
TOP10 / F–mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1araD139 Δ(araleu)7697 galUgalKrpsL(Strr) endA1 nupG / Invitrogen
GM2163 / dam-13::Tn9 dcm6 hsdR2 leuB6 his-4 thi-1 ara-14 lacY1 galK2galT22 xyl-5 mtl-1 rpsL136 tonA31 tsx-78 supE44 McrA- McrB / New England Biolabs
Rosetta (DE3) / F- ompThsdSB(rB- mB-) gal dcm (DE3) pRARE (Cmr) / Novagen
Hfx. volcanii
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
pJAM809 / AmprNvr; C-terminal StrepII expression vector for Hfx. volcaniistrains / (Humbard et al, 2009)
pJAM2001 / Ampr Nvr; PAN-A/1-StrepII / This study
pJAM1106 / AmprNvr; Flag-His-SAMP1 in pJAM202c / This study
pJAM1119 / AmprNvr; MoaE-StrepII / (Hepowit et al, 2012)
pJAM1796 / AmprNvr; Flag-SAMP1-MoaE-M1A-StrepII / (Hepowit et al, 2012)
pJAM1131 / Cmr, Kmr, Flag-His-SAMP1 in pET24b / This study
aAbbreviations: 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.
bTo 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 anHfx. 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).
Table S2. List of primers used in this study.
Primer no. / Primer name / Oligonucletide Sequence (5’ to 3’)a1 / HVPanA Nde1 FW / 5’-CGGCATcatatgATGACCGATACT-3’
2 / HVPanA Kpn1 RV / 5’-ATggtacccGCGAACGCGC-3’
3
4 / Hvo_2619 NdeI up
Hvo_2619 BlpI down / 5’-CTGCCCTcatatgGAGTGGAAGCTGT-3’
5’-TTAATgctcagcTCACCCACCGGC-3’
5
6 / HVO_2619 KpnI-His Up
HVO_2619 BlpI down / 5’-AAggtaccCACCACCACCACCACCACGAGTGG
AAGCTGTTC-3’
5’-TTAATgctcagcCTAGCCGCCGCTGACCGG-3’
aLowercase letters indicate restriction enzyme site. Italicized uppercase letters indicate random bases introduced into primer to enhance cleavage of PCR product by restriction enzyme.
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Table S3. Modified guanidine-HCl buffered conditionsused for denaturing and renaturingthe halophilic proteins byfar western blottinga.
Buffer Reagents/ Conditions / Far Western Blotting Steps1 / 2 / 3 / 4 / 5
8 M Guanidine-HCl (mL) / 150 (6M) / 74.4 (3 M) / 24.8 (1 M) / 2.48 (0.1 M) / 0 (0 M)
Glycerol (mL) / 20 / 20 / 20 / 20 / 20
NaCl (g) / 1.16 (0.1 M) / 1.16 (0.1 M) / 1.16 (0.1 M) / 2.32 (0.2 M) / 24 (2 M)
500 mM HEPES (mL) / 8 / 8 / 8 / 8 / 8
0.5 M EDTA (mL) / 0.4 / 0.4 / 0.4 / 0.4 / 0.4
10% Tween-20 (mL) / 2 / 2 / 2 / 2 / 2
Milk powder (g) / 4 / 4 / 4 / 4 / 4
1M DTT (µL) / 200 / 200 / 200 / 200 / 200
ddH2O (mL) / 21.4 / 97 / 146.6 / 168.92 / 171.4
Total volume (mL) / 200 / 200 / 200 / 200 / 200
Time/Temperature / 30 min/RT / 30 min/RT / 30 min/RT / 30 min/4°C / overnight/4°C
aProtocol was adapted from the far western blotting method of(Wu et al, 2007) for the halophilic (salt-loving) 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 theNaClconcentration from 0.1 to 2 M. The final buffer of step 5 was supplemented with 2M NaClto enhancerenaturation of the halophilic proteins.
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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 Microbiol70: 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 Microbiol86: 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 BACTERIOLOGY191: 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 A108: 4417-4422
Wu Y, Li Q, Chen XZ (2007) Detecting protein-protein interactions by Far western blotting. Nat Protoc2: 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 Bacteriol190: 8096-8105
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