|
Strains
|
|
E. coli
|
| TOP10 |
Competent E. coli for plasmid transformation |
Invitrogen |
| DC10B |
Competent E. coli for direct plasmid transformation into clinical isolates of S. aureus
|
(46) |
| BL21 (DE3) |
E. coli for expression of recombinant proteins with IPTG |
Promega |
|
S. aureus
|
|
|
| ISP479C |
8325-4 derivative, with SaeSL allele |
(47) |
| CYL316 |
RN4220(pYL112Δ19), L54 int gene, |
(34) |
| RN4220 |
Restriction-deficient S. aureus strain |
(48) |
| RN4220-30 |
RN4220 saeP::kanA
|
This work |
| RN4220-217 |
RN4220 rny::ermC
|
(11) |
| Newman |
Wild-type |
(49) |
| Newman-29 |
Newman, sae::kanA
|
(20) |
| Newman-30 |
Newman saeP::kanA
|
This study |
| Newman-217 |
Newman rny::ermC
|
(11) |
| Newman-217-30 |
Newman rny::ermC, saeP::kanA
|
This work |
| PR01 |
ΔpyrFE mutant of the clinical strain SA564RD |
(39) |
| PR01-01 |
PR01 with the RNase J1 gene deleted |
(39) |
|
Plasmids
|
|
For SaeP mutant construction
|
| pMAD |
Vector for allelic replacement |
(33) |
| pBT2 |
Cloning vector |
(31) |
| pCWsae30 |
pMAD with cloned saeP::kanA
|
This study |
|
For PCR template
|
| pCWsae19 |
pCR2.1-Topo with saePQRS with stop codon in saeP
|
(T. Geiger, unpublished) |
|
Integrative plasmids
|
| pCG188 |
pCL84 with truncated gfp gene cassette from pc183 |
(11) |
| pCG212 |
pCG188 with sae region from P1 to upstream of P3 |
(11) |
| pCG213 |
pCG188 with sae region from P1 to upstream of P3 with deletion of 13 bp in the CS |
This work |
| pCG218 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the left stem loop (terminator) |
This work |
| pCG219 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the right stem loop |
This work |
| pCG223 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the entire region between the two stem loops |
This work |
| pCG379 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the two stem loops |
This work |
| pCG301 |
pCG188 with sae region between the 2 stem loops under the native P1 promoter |
This work |
| pCG392 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the sequence downstream of CS (Rrs) |
This work |
| pCG394 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the CS and alternative CS (aCS) |
This work |
| pCG484 |
pCG188 with sae region from P1 to upstream of P3 with deletion of the Rrs counterpart |
This work |
| pCG589 |
pCG212 with sae region with mutated CS (C instead of T) |
This work |
|
Replicative plasmids
|
| pCG246 |
E. coli/Staphylococcus shuttle vector, pCN47 derivative with cat cassette |
(50) |
| pCG599 |
pCG246 with sae-gfp region from plasmid pCG212 |
This work |
| pCG600 |
pCG246 with sae-gfp region from plasmid pCG484 |
This work |
| pCG601 |
pCG246 with sae-gfp region from plasmid pCG392 |
This work |
| pCG616 |
pCG246 with sae-gfp region from plasmid pCG589 |
This work |
| pCG618 |
pCG599 with sae region with point mutation which affect Rrs secondary structure: CC in +3 and +8 instead of AA, CC in +14 and +15 instead of TT (the position is indicated as distance to the cleavage site) |
This work |
| pCG620 |
pCG246 with sae-gfp region from plasmid pCG223 |
This work |
|
For complementation
|
| pCG296 |
pCG246 with rny for complementation |
(11) |
| pCG322 |
pCG246 with rny without active site for complementation |
This work |
| pCG596 |
pCG246 with rny carrying mutations in the active site (H367A D368A) |
This work |
|
For protein purification
|
| pET15b |
Protein expression vector, IPTG inducible |
Novagen |
| pCG249 |
pET15b with rny (without transmembrane domain) |
This work |
