Table 1.
Strains and plasmids used in this study
| Strain or plasmid | Characteristica | Source or reference |
|---|---|---|
| Strains | ||
| Escherichia coli | ||
| BL21(λDE3) | F−ompT hsdSB(rB− mB−)gal dcm (DE3) | Novagen |
| DH5α | General cloning strain | Invitrogen |
| Streptococcus gordonii | ||
| M99 | Parental strain | 33 |
| PS846 | M99 ΔgspB::pEVP3; expressing no GspB | 9 |
| PS840 | M99 ΔgspB::cat ΔgtfA::spc; expressing no GspB or GtfA Cmr Spcr | This study |
| PS2865 | PS846 pMSP3545-gspB736FLAG; expressing glycosylated GspB736FLAG | This study |
| PS2866 | PS840 pMSP3545-gspB736FLAG; expressing nonglycosylated GspB736FLAG | This study |
| PS961 | M99 gspB736FLAG::pVA891; expressing glycosylated GspB736FLAG | 5 |
| PS1179 | M99 gspB736FLAG::pVA891, ΔgtfA::spc; expressing nonglycosylated GspB736FLAG | 5 |
| PS2336 | M99 expressing gspB joined domains SP, AST, and BR (codons 1 to 140 and 213 to 598) and a 3×FLAG tag in the C terminus | This study |
| PS1570 | PS846::pBMalE31; expressing GspBflag::MalE31 | 8 |
| PS2413 | PS1570 Δasp2::spc | This study |
| PS2414 | PS1570 Δasp3::spc | This study |
| Plasmids | ||
| pACYCDuet | E. coli expression vector; Cmr | Novagen |
| pETDuet | E. coli expression vector; Ampr | Novagen |
| pGEX-3x | E. coli GST fusion vector; Ampr | GE Healthcare |
| pET28c | E. coli expression vector; Kanr | Novagen |
| pET28b | E. coli expression vector; Kanr | Novagen |
| pMSP3545 | Cloning vector containing replication origin for Gram-positive bacteria and nisin-inducible promoter; Ermr | 11 |
| pMal-c2x | Vector expressing mature MalE (without a signal peptide) | New England Biolabs |
| pVA891-gspB736FLAG | pVA891+gspB736FLAG | 9 |
| pACYCDuet-gspB736FLAG | Vector expressing GspB736FLAG | This study |
| pETDuet-gspB736FLAG | Vector expressing GspB736FLAG | This study |
| pET28c-asp1 | Vector expressing H6Asp1 | This study |
| pET28c-GSTasp1 | pET28C+GSTasp1 | This study |
| pACYCDuet-GSTasp1 | pACYCDuet + GSTasp1 | This study |
| pJSC401asp2 | pJSC401+asp2 | 28 |
| pETDuet-HAasp2 | Vector expressing HAAsp2 carrying silent mutations at codons 346 and 363 | This study |
| pACYCDuet-GSTasp3 | Vector expressing GSTAsp3 | This study |
| pET28c-asp3 | Vector expressing H6Asp3 | This study |
| pET28c-GSTasp3 | pET28c+GSTasp3 | This study |
| pACYCDuet-GSTasp1-gspB736FLAG | Vector coexpressing GSTAsp1 and GspB736FLAG | This study |
| pETDuet-H6asp3-HAasp2 | Vector coexpressing H6Asp3 and HAAsp2 | This study |
| pET28c-asp2 | Vector expressing H6Asp2 | This study |
| pGEX-3x-sp | Vector expressing GST-SP | This study |
| pGEX-3x-ast | Vector expressing GST-AST | This study |
| pGEX-3x-srr1 | Vector expressing GST-SRR1 | This study |
| pGEX-3x-br | Vector expressing GST-BR | This study |
| pGEX-3x-srr2′ | Vector expressing the N terminus of GST-SRR2 | This study |
| pGEX-3x-gspB736 | Vector expressing GST-GspB736 | This study |
| pACYCDuet-sp.ast.s1FLAG | Vector expressing codons 1 to 214 of GspB736FLAG | This study |
| pACYCDuet-sp.astFLAG | Vector expressing codons 1 to 140 of GspB736FLAG | This study |
| pMSP3545-gspB736FLAG | Vector expressing GspB736FLAG | This study |
| pMalE31 | Vector expressing mature MalE with mutated codons 32 to 33 | This study |
| pB117 M | pVA891-gspB736FLAG with codons 118 to 736 replaced by malE31 | 8 |
| pACYCDuet-gspB1-117::malE31 | Vector expressing gspB736FLAG with codons 118 to 736 replaced by malE31 | This study |
| pET28b-sp.ast.br | pET28b+gspB joined domains (codons 1 to 140 and 213 to 598) | This study |
| pVA891-gspB736FLAG.H96L | pVA891+gspB736FLAG with an alteration of the indicated codon | 8 |
| pVA891-[sp.ast.br]FLAG | pVA891+gspB joined domains (codons 1 to 140 and 213 to 598) and a 3×FLAG tag in the C terminus | This study |
| pORF2K | pS326 carrying upstream and downstream fragments of asp2; Spcr | 37 |
| pORF3K | pS326 carrying upstream and downstream fragments of asp3; Spcr | 37 |
| pBMalE31 | pB736flagR with codons 146 to 560 replaced by malE31 | 8 |
a
Cmr, chloramphenicol resistance; Ermr, erythromycin resistance; Ampr, ampicillin resistance; Spcr, spectinomycin resistance; Kanr, kanamycin resistance.