Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions
Description and/or genotype | Source or reference | |
---|---|---|
Strains | ||
E. coli K113 | BL21(DE3)/pLysS; expression host, CmR | Novagen |
Methanococcus maripaludis Mm900 | Δhpt | [7] |
M. maripaludis ∆flaK | Mm900∆flaK | [35] |
M. maripaludis ∆eppA | Mm900∆eppA | [11] |
M. maripaludis ∆flaK ∆eppA | Mm900∆flaK ∆eppA | [11] |
M. maripaludis ∆aglB | Mm900∆aglB | [34] |
M. maripaludis ∆flaK ∆aglB | Mm900∆flaK ∆aglB | This study |
M. maripaludis ∆eppA ∆aglB | Mm900∆eppA ∆aglB | This study |
Plasmids | ||
pKJ574 | pCRPrtNeo with inframe deletion of aglB | [34] |
pKJ697 | pCRPrtNeo with inframe deletion of eppA | [11] |
pET23a+ | T7 promoter-based expression vector, Ampr | Novagen |
pKJ900 | pET23a+ with epdE Nde1-Xho1 fragment C-terminal histagged | This study |
pWLG40 | hmv promoter-lacZ fusion plus Purr cassette; Ampr | [30] |
pKJ880 | pWLG40 with epdE complement | [12] |
pKJ1072 | pWLG40 with epdE C-terminal histagged complement | This study |
pKJ1079 | pWLG40 with epdE (+3 Gly) C-terminal histagged complement | This study |
pKJ1107 | pWLG40 with epdE C-terminal FLAG complement | This study |
pKJ1108 | pWLG40 with epdE (+3 Gly) C-terminal FLAG complement | This study |
pHW40 | nif promoter-lacZ fusion plus Purr cassette; Ampr | [30] |
pKJ1169 | pHW40 with epdD C-terminal FLAG complement | This study |
pKJ1226 | pHW40 with epdE C-terminal FLAG complement | This study |
pKJ1216 | pHW40 with +1 Ala +3 Gly epdE C-terminal FLAG complement | This study |
pKJ711 | pHW40 with epdE | [13] |
2.2. M. maripaludis Mutant Generation
Primers | Primer Sequence 5’ to 3’ | Restriction site |
---|---|---|
1685+3_sdm_For Nsi1 | CCAATGCATGAAATTTTTAGAAAAACTAACATCAAAAAAAGGTC AAATAGGAATGGAACTCGG * | Nsi1 |
1685+1+3_sdm_For Nsi1 | CCAATGCATGAAATTTTTAGAAAAACTAACATCAAAAAAAGGTG CAATAGGAATGGAACTCGG * | Nsi1 |
1685_Mlu_Rev | AGCACGCGTTTAATCCGTAATATTTGACATTTGTGAGG | Mlu1 |
1685_Nsi1_For | CCAATGCATGAAATTTTTAGAAAAACTAACATC | Nsi1 |
1685_histag_Mlu_Rev | CGCGACGCGTTTAGTGATGGTGGTGATGGTGATCCGTAATA TTTGACATTTGTGAGG | Mlu1 |
1685_FLAG_Mlu_Rev | CGACGCGTTTATTTGTCATCGTCATCTTTGTAATCATCCGTA ATATTTGACATTTGTGAG | Mlu1 |
1685_For_exp | GGAATTCCATATGATGAAATTTTTAGAAAAACTAACATC | NdeI |
1685-Rev_exp | CCGCTCGAGATCCGTAATATTTGACATTTGTGAG | XhoI |
1283_Nsi1_For | CCAATGCATGTCTGTTGCTTTAAAGAAGTTTTTTTCGAAACG | Nsi1 |
1283_FLAG_Mlu_Rev | GCTACGCGTTTATTTGTCATCGTCATCTTTGTAATCACTAACTT CACTTAAAGCATCTAT | Mlu1 |
FlaK_seq_For | AATATCTGGCGGATACAGG | - |
FlaK_seq_Rev | TTCAAAGCCAATAGATACTGC | - |
EppA_seq_For | CTGGAGCTGTATGAAATGCAAC | - |
EppA_seq_Rev | GGATGACCTGGGATAATGCAGG | - |
AglB_seq_For | CATAACCATATTTGTAATTAAC | - |
AglB_seq_Rev | CTCAATAGCCATAAAATCACC | - |
2.3. Complementation Experiments
2.4. Expression of Epitope-Tagged Versions of EpdD and EpdE in Various M. maripaludis Strains
2.5. Overexpression and Purification of His-Tagged EpdE
2.6. Western Blot Analysis
3. Results and Discussion
3.1. The +3 Position Alone Does Not Distinguish Archaellins from Pilins as FlaK Substrates
3.2. Posttranslational Modifications of Native EpdE in Various Mutant Strains
3.3. Signal Peptide Removal Is a Prerequisite for N-glycosylation of Pilins but Not Archaellins
4. Conclusions
Acknowledgments
Author Contributions
Supplementary Materials
Conflicts of Interest
References
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Nair, D.B.; Jarrell, K.F. Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis. Life 2015, 5, 85-101. https://doi.org/10.3390/life5010085
Nair DB, Jarrell KF. Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis. Life. 2015; 5(1):85-101. https://doi.org/10.3390/life5010085
Chicago/Turabian StyleNair, Divya B., and Ken F. Jarrell. 2015. "Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis" Life 5, no. 1: 85-101. https://doi.org/10.3390/life5010085
APA StyleNair, D. B., & Jarrell, K. F. (2015). Pilin Processing Follows a Different Temporal Route than That of Archaellins in Methanococcus maripaludis. Life, 5(1), 85-101. https://doi.org/10.3390/life5010085