Strains
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L. reuteri
JCM1112
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Type strain, synonymous to ATCC 23272, DSM 20016 and F275. Human isolate.
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Japanese Collection of Microorganisms (Riken, Japan)
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L. reuteri
ATCC PTA 6475
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Synonymous to MM4-1A. Finnish mother's milk isolate.
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Biogaia AB (Stockholm, Sweden)
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6475::pocR
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EmR, pocR insertion mutant derivative of L. reuteri ATCC PTA 6475
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This study
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Lc. lactis
NZ9000
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MG1363 pepN:nisRK, cloning host.
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NIZO culture collection (Ede, The Netherlands)
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L. delbrueckii
NIZO235
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L. delbrueckii subsp. lactis ATCC 7830. Vitamin B12 assay indicator strain.
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NIZO culture collection (Ede, The Netherlands)
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Plasmids
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pCR®2.1
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Used in routine cloning and to construct pJKS100
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Invitrogen (Carlsbad, CA)
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pLEM5
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L. reuteri replication origin used to construct pJKS100
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[28]
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pNZ7021
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CmR, pNZ8148 derivative with the nisin promoter replaced by the pepN promoter
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[23]
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pNZ7748
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CmR, pNZ7021 derivative harboring lreu_1750 downstream of the pepN promoter.
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This study
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pVE6007
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CmR, repA-positive temperature-sensitive derivative of pWV01
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[27]
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pORI28
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EmR, repA-negative derivative of pWV01
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[35]
|
pORIpocR
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EmR, pORI28 derivative harboring internal fragment of gene encoding putative PocR
|
This study
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pJKS100
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CmR, E. coli-L. reuteri shuttle vector
|
This study
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pJKS101
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CmR, pJKS100 derivative expressing 6475 pocR gene under control of its natural promoter
|
This study
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Primers
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5' - 3'
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Application
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P180
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AAAAGGTACC GTAGGCGAAATTCAAATGTACG
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Amplification of lreu_1750 and addition of Kpn I site
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P181
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GAATAAATAAGAGGCTGGGCAC
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Amplification of lreu_1750
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P182
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ATGAACTCTATTCAGGAATTG
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Control of pNZ7748
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LR0062F-BHI
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TGACGGATCC TAA CACAAGCATTACCGGAGCAATTG
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Amplification of internal fragment of putative pocR, addition of Bam HI site and translational stop codon
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LR0062R-ERI
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TGACGAATTC GCGTCTGATTCTATATGTGATTC
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Amplification of internal fragment of putative pocR and addition of Eco RI site
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LR0062 FL F
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CGCTTTATCCTCAATTTGTTACG
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Amplification of wild-type pocR gene and natural promoter
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LR0062 FL R
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GCTTTTACCATTGCATCAGCAG
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Amplification of wild-type pocR gene and natural promoter
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