TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant phenotype(s) or genotype(s)aSource or reference
Strains
    E. coli BL21DE3Allows IPTG-inducible expressionNovagen
    E. coli TOP10Cloning hostInvitrogen
    L. lactis MG1363Plasmid-free derivative of NCDO7129
    L. lactis NZ9700Nisin producer14
    L. lactis NZ9000MG1363 pepN::nisRK, allows nisin-inducible expression14
    L. lactis NZ9000(ΔacmA)Derivative of NZ9000 lacking acmA4
    L. lactis PA1001Derivative of NZ9000 lacking acmA and htrAThis study
    Lactobacillus caseiWild typeATCC 393
    Lactobacillus sakeWild typeNCFB 2714
    Lactobacillus salivariusWild typeNCFB 2747
    Lactobacillus curvatusWild typeNCFB 2739
    Bacillus subtilisWild typeLaboratory collection
    Mycobacterium smegmatisWild typeATCC 700084
Plasmids
    pNG400Cmr, pNZ8048 derivative containing PnisA, usp45ss, and a transcription terminatorLaboratory collection
    pNG3041Cmr, pNZ8048 derivative containing PnisA, prtP preprosequence, and msa2-acmA PA (MSA2-PA)39
    pNG3042Cmr, pNZ8048 derivative containing PnisA, prtP preprosequence, and msa2-acmD PA (nucleotides 1286 to 2371)bThis study
    pPA1Cmr, pNZ8048 derivative containing c-myc, B. licheniformis α-amylase (amyL) under control of PnisA, and usp45ssThis study
    pPA3Cmr, pNZ8048 derivative containing c-myc, the acmA PA (nucleotides 835 to 1492)c under control of PnisA, and usp45ss (PA3)This study
    pPA5pPA3 derivative, B. licheniformis α-amylase (amyL) upstream of the acmA PA sequence (α-PA)This study
    pPA6 (1-6)pPA3 derivative containing one to six copies of first LysM repeat of acmA PA (nucleotides 819 to 1038)cThis study
    pPA95Cmr, pNZ8048 derivative containing c-myc, the E. coli mltD LysM repeats (nucleotides 927 to 1313)d under control of PnisA, and usp45ssThis study
    pPA165Cmr, pNZ8048 derivative containing c-myc, human LysM repeat erap140 (amino acids 116 to 158 [accession no. AAM27392]; in duplicate) under control of PnisA, and usp45ssThis study
    pPA171Cmr, pNZ8048 derivative containing P. berghei CSP B-cell epitope sequences upstream of the acmA PA sequence under control of PnisA and usp45ss; the epitopes PPPPNPND and NANDPAPP were duplicated and fused to the PA, [PPPPNPND]2-[NANDPAPP]2-PA (CSP[2×B]-PA)This study
    pPA193Apr, pET32c (Novagen) derivative producing CSP[2×B]-HisThis study
    pGBL1Emr, pGB14 derivative, β-lactamase secretion vector28
    pGBLREmr, pGBL1 derivative producing secreted β-lactamase fused to the AcmA PA (β-PA)4
    pINT-HtrAEmr, pORI280 derivative containing a 900-bp chromosomal DNA fragment 5′ of htrA and a 1,200-bp fragment 3′ of htrA; primers used to generate the chromosomal fragments were HtrA.1 (5′-CGGTCTCACTAGCGCATTTTCTAAATTTTATTTTTGCGG), HtrA.2 (5′-CCCCCCGCGGCCGCCCTTTCAATTATTACTCTTTGTAGC), HtrA.3 (5′-GGGGGGGCGGCCGCCATACTAAAGTCTGAAATTTTG), and HtrA.4 (5′-CGGTCTCAAATTACGTTTATATTTCTGTAAAGTCTATC)eThis study
    pTOPO-htrAApr Kmr, derivative of pCR4Blunt-TOPO (Invitrogen) containing the L. lactis htrA geneThis study
  • a PnisA, nisin-inducible promoter; usp45ss, signal sequence of usp45.

  • b See reference 3.

  • c See reference 5.

  • d See reference 8.

  • e See reference 18. Sequences homologous to the MG1363 chromosome sequence are underlined.