Table 1 Bacterial strains, plasmids and oligonucleotides

Bacterial strains

EC10B

E. coli DH10B derivative, with repA integrated into the glgB gene. Kan^r.

[20]

NZ9000

Nisin responsive L. lactis MG1363 derivative, with nisRK integrated into the pepN gene.

[26]

EGD-e

L. monocytogenes 1/2a strain. Genome sequenced. Obtained from Werner Goebel.

[39]

EGD-eΔinlA

EGD-e with the E-cadherin interacting region of InlA deleted (amino acids 80 to 506)

[20]

EGD-eΔinlA::pIMK2inlA

EGD-e ΔinlA with InlA over expressed from the Phelp promoter integrated at tRNA^Arg locus, Kan^r

[20]

EGD-e InlA^m*

EGD-e with inlA residues S192N and Y369 S modified in the chromosome.

This study

EGD-e A

EGD-eΔinlA with inlA locus recreated containing SDM change N259Y in the chromosome.

This study

EGD-e B

EGD-eΔinlA with inlA locus recreated containing SDM change Q190L in the chromosome.

This study

EGD-e C

EGD-eΔinlA with inlA the locus recreated containing SDM changes S173I, L185F and L188F in the chromosome.

This study

EGD-e D

EGD-eΔinlA with inlA locus recreated containing SDM changes T164A, K301I and G303E in the chromosome.

This study

EGD-e InlA^m* ::pIMC3ery

EGD-e InlA^m* with the IPTG inducible expression of erythromycin integrated in the tRNA^ARG locus, Cm^r.

This study

EGD-e::pIMC3kan

EGD-e with the IPTG inducible expression of kanamycin integrated in the tRNA^ARG locus, Cm^r.

[18]

EGD-e A::pIMC3kan

EGD-e A with the IPTG inducible expression of kanamycin integrated in the tRNA^ARG locus, Cm^r

This study

EGD-e B::pIMC3kan

EGD-e B with the IPTG inducible expression of kanamycin integrated in the tRNA^ARG locus, Cm^r

This study

EGD-e C::pIMC3kan

EGD-e C with the IPTG inducible expression of kanamycin integrated in the tRNA^ARG locus, Cm^r

This study

EGD-e D::pIMC3kan

EGD-e D with the IPTG inducible expression of kanamycin integrated in the tRNA^ARG locus, Cm^r

This study

NZ9700

Nisin producer, progeny of NIZO B8 and MG1363 (Rif^r and Strp^r) conjugation.

[26]

Plasmids

pNZB

Nisin inducible plasmid with heterologous gene expressed from the nisA promoter. Bgl II site upstream of nisA removed.

This study

pNZBinlA^WT

Internalin A from EGD-e containing the entire gene including signal sequence. Cloned into Nco I/Pst I of pNZB.

This study

pNZBinlA^m*

Internalin A containing S192N and Y369 S in pNZB.

This study

pNZBinlA Bank-iii

Error Prone PCR with low level of mutation 0-4.5 nt per kb.

This study

pNZBinlA Bank-iv

Error Prone PCR with medium level of mutation 4.5-9 nt per kb.

This study

pNZBinlA Bank-v

Error Prone PCR with high level of mutation 9-16 nt per kb.

This study

pNZBinlA Bank-vi

Error Prone PCR with very high level of mutation 9-16 nt per kb.

This study

pORI280

RepA negative gene replacement vector, constitutive lacZ, 5.3 kb, Em^r.

[40]

pORI280inlA(SDM)

PCR amplified mutated inlA^m* into pORI280 as Nco I/Pst I fragment. Contains wild type inlA promoter.

This study

pORI280inlA(A)

PCR amplified mutated inlA (from bank v clone 6 containing N259Y) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter.

This study

pORI280inlA(B)

PCR amplified mutated inlA (from bank iii clone 3 containing Q190L) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter.

This study

pORI280inlA(C)

PCR amplified mutated inlA (from bank v clone 6 containing S173I, L185F, L188F) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter.

This study

pORI280inlA(D)

PCR amplified mutated inlA (from bank v clone 8 containing T164A, K301I, G303E) into pORI280 as Nco I/Pst I fragment. Contains Wt inlA promoter.

This study

pVE6007

Temperature-sensitive helper plasmid, supplies RepA in trans. Cm^r.

[41]

Name

Oligonucleotide sequence (5'-3') a

IM194 (inlA-F)

ATATCCATGG AAAAAAACGATATGTATGGTTG

IM188 (inlA-R)

TTTTCTGCAG TTATTTACTAGCACGTGCTTTTTTAG

IM345 (S192N SDM-F)

CAGGTTTAACTAGTCTACAGCAATTAAAT TTTGGTAATCAAGTGACAGATTTAAAACC

IM346 (S192N SDM-R)

GGTTTTAAATCTGTCACTTGATTACCAAAATTTAATTGCTGTAGACTAGTTAAACCTG

IM349 (Y369 S SDM-F)

CAAAGCTTCAAAGATTATTTTTCTCT AATAACAAGGTAAGTGACGTAAG

IM350 (Y369 S SDM-R)

CTTACGTCACTTACCTTGTTATTAGAGAAAAATAATCTTTGAAGCTTTG

IM490 (Chromosome-F)

ATATCCATGG AAAAGGAGTGTATATAGTG AGAAAAAAACGATATGTATGG

IM466 (Chromosome-R)

ATATCTGCAG CAAACGTTGCTGTATAGCTATTGG

IM467 (inlA out-F)

TATATAGGAAAAATGTGCTGGAACG

IM468 (inlA out-R)

TCCTTGATAGTCTACTGCTTGAGTCG

IM317 (inlA muta-F)

AAACAGATCT AGACCAAGTTACAACG

IM318 (inlA muta-R)

AATTCCA CTTCTTTGG TTGTTTCTTTGC