Introduction
Material and methods
Bacterial strains and growth conditions
Strain or plasmid
|
Relevant genotype or characteristics
|
Source or reference
|
---|---|---|
Plasmids
| ||
pUC18 | Ampr, lacZ', pMB9 replicon | [9] |
pUC18: ESA_02131 | pUC18 harboring ESA_02131 gene under control of the native promoter | [7] |
pUC18: ESA_01706 | pUC18 harboring ESA_01706 gene under control the native promoter | [7] |
pUC18: ESA_01706CTE | pUC18 harboring chimeric ESA_01706 with fused C-terminal extension (ESA_02131) under control of the native promoter | This work |
Strains
| ||
Cronobacter sakazakii BAA-894 | C.sakazakii strain isolated from powdered formula associated with neonatal intensive care unit | [10] |
Escherichia coli DH5α | Intermediate cloning host.supE44 ΔlacU169(80lacZΔM15)R17 recA1 endA1 gyrA96 thi-1 relA1 | Invitrogen |
MKH13 | MC4100Δ(putPA)101Δ(proP)2Δ(proU) | [11] |
MKH13 pUC18:ESA_02131+ | Host strain harbouring pUC18: ESA_02131 plasmid. Ampr | [7] |
MKH13 pUC18:ESA_01706+ | Host strain harbouring pUC18: ESA_01706 plasmid. Ampr | [7] |
MKH13 pUC18:ESA_01706CTE | Host strain harbouring pUC18: ESA_01706CTE plasmid. Ampr | This work |
Creation of the chimeric ProPc protein
Primer name
|
Primer sequence (5' to 3')
|
Length
|
Characteristics
| |
---|---|---|---|---|
ESA_02131 | F | CATCGGCCGACAGGCCAGTCAATGAATGATGC | 32 | EagI cut site |
R | CATTCTAGAGAGTACAACGGAATGCGGGG | 29 | XbaI cut site | |
ESA_01706 | F | CATTCTAGAGTCGGGCGGCTCTTTATCTGG | 30 | XbaI cut site |
R | CATGGATCCTTGACCAGATGACGCAGTCTTTC | 32 | BamHI cut site | |
Chimeric-01706 | F | AATAAGCTTGTGGCTTTTTATGCCGGGCTGC | 31 | HindIII cut site |
R | CAGGCCAGTAATCAGCGCCGCGCCCATGAC | 30 | 3' SOEing overhang | |
Chimeric-02131CTE | F | CGCGGCGCTGATTACTGGCCTGACGATGAAAG | 32 | 5' SOEing overhang |
R | AATGGATCCTTACTCGTTAATACGAGGATGCTGG | 34 | BamHI cut site | |
pUC18 MCS Check | F | CATTAGCTCACTCATTAGGCACC | 23 | pUC18 insert check |
R | CATTGTAAAACGACGGCCAGTG | 22 | pUC18 insert check |
Osmotolerance assay
Results
C. sakazakii ProP structures
Chimeric protein (ProPc) expression in E. coli MKH13
Assessment of osmotolerance
Protein expressed
|
Gene locus tags
|
Name
|
5%
|
6%
|
7%
|
8%
|
9%
|
10%
| ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Max. OD
|
Growth rate (hr
−1
)
|
Max. OD
|
Growth rate (hr
−1
)
|
Max. OD
|
Growth rate (hr
−1
)
|
Max. OD
|
Growth rate (hr
−1
)
|
Max. OD
|
Growth rate (hr
−1
)
|
Max. OD
|
Growth rate (hr
−1
)
| |||
Native | ESA_02131 | ProP1 | 0.326 | 0.009 | 0.144 | 0.005 | 0.152 | 0.004 | 0.177 | 0.004 | 0.181 | 0.004 | 0.204 | 0.004 |
Native | ESA_01706 | ProP2 | 0.111 | 0.003 | 0.096 | 0.002 | 0.083 | 0.002 | 0.074 | 0.002 | 0.127 | 0.002 | 0.171 | 0.002 |
Chimeric | ESA_02131 ESA_01706 | ProPc | 0.189 | 0.004 | 0.130 | 0.003 | 0.113 | 0.003 | 0.117 | 0.003 | 0.124 | 0.003 | 0.141 | 0.003 |