TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain or plasmid | Description or sequencea | Source or reference |
|---|---|---|
| Strains | ||
| F. tularensis LVS | F. tularensis subsp. holarctica live vaccine strain | ATCC 29684 |
| E. coli DH5α | endA1 recA1 | Clontech |
| Plasmids | ||
| pRIT5 | Apr in E. coli, Cmr in Gram-positive organisms, pC194 origin of replication | Pharmacia (29) |
| pTE | pRIT5 vector in which the protein A gene was deleted; E. coli-F. tularensis shuttle vector | This study |
| pWH1012 | Souce of the gfp+ gene | 37 |
| pTRAP | Promoterless gfp+ gene from pWH1012; inserted as an EcoRI-PstI fragment in pTE | This work |
| pASC-1 | E. coli-Bacillus expression vector, carrying the B. anthracis pagA gene | 8 |
| pKK214 | Tetr in E. coli and F. tularensis; p15A origin of replication in E. coli; oriFT in F. tularensis; promoterless cat gene | 18 |
| pKK202 | Tetr in E. coli and F. tularensis; p15A origin of replication in E. coli; oriFT in F. tularensis | 30 |
| pTRAP (groEL-gfp) | pTRAP containing the groEL promoter upstream from the gfp gene | This work |
| pKK (bfr-cat) | pKK214 containing the bfr promoter upstream from the cat gene | This work |
| Primers | ||
| GFP-F | gtcgatcatcgaattctctagagatcttacaataaggagtacgtatATGGCTAGCAAAGGAGAAG | |
| GFP-R | tgaccatagcctgcagtggtaccccgggTTATTTGTAGAGCTCATCCATGC | |
| GFP-seq | TTTGTGCCCATTAACATCACC | |
| CAT-seqF | CCAAAACGATCTCAAGAAGATC | |
| CAT-seqR | GATGCCATTGGGATATATCAACGG | |
| PE P2Ab | GACGAATGTTCATAACAATCTTACTCC | |
| PE P2B | GCACGACGAACTAATACTCTATCTTG | |
| PE P3A | CCACAGATACCTAAAATATGAATATG | |
| PE P3B | CTAATACTGCTAAAGAACCCATAAAAG | |
| PE P39A | GCTCAACTATTATATGGTTAACTCTAG | |
| PE P39B | CTTCTGGCTTAAGATCTTCTTC | |
| PE PbfrA | CTTGTTTATTTTCTAATTTAAGTTCC | |
| PE PbfrB | CGAGTTCTAAGATTTTATTTAATTG | |
| PE P18A | GTTTGATTTTTTTCATTGTATTGC | |
| PE P18B | CTAACTAGGGTAAGTGTAGCTAATG | |
| CAT-F | ATGGACAACTTCTTCGCCCC | |
| CAT-R | CAAACGGCATGATGAACCTG | |
| Tet-F | CTAACAATGCGCTCATCGTCA | |
| Tet-R | CCGGCAGTACCGGCATAAC | |
| Det-P2F | TGGTAATGCTCAAGAGAAACCTAG | |
| Det-P2R | CTTGTTTTGCAGCCATTTTTAATTTC | |
| Det-P39F | CTACAGATATGACTGATAAACTAACTG | |
| Det-P39R | TTCTTCTTTAACGCCTAATTGCTC | |
| Det-P29F | CTAGATAAGATTGAAAATTAAACTC | |
| Det-P29R | CTGTTAACATAAATTTACTCC | |
| Det-PbfrF | GCATAATATCATTTTTATTAAAATATC | |
| Det-PbfrR | TGTTTATTTTCTAATTTAAGTTC | |
| groEL-F | GCCAAAAAACagatctCCTATTGTATGGATTAGTCGAGC | |
| groEL-R | CGAATGTTCtacgtaATCTTACTCCTTTG | |
| bfr-F | caatactgcatctagaGATCCATACCCATGATGGTTAC | |
| bfr-R | cataagaattcctgcagGATCAATAATTTCTTGTTTATTTTC |
a
The region of homology to the coding sequence is in uppercase letters. The restriction sites, used for cloning of the corresponding PCR fragments, are underlined.
b
PE primers were used for primer extension analyses of the various promoters as indicated by their numbers.