Article Figures & Data
Figures
- FIG. 1.
Physical map of the mariner-based transposon delivery vectors pMC38 and pMC39. The vectors comprise the Escherichia coli p15A low-copy-number replication origin (5), the RP4 ori for conjugative transfer (20), the pE194ts gram-positive temperature-sensitive replication origin (11, 25), a gram-negative chloramphenicol resistance gene (cat) (5), a gram-positive noninducible erythromycin resistance gene (ermC) (25) flanked by the 29-bp ITR of the Himar1 mariner (15), a gram-positive kanamycin resistance gene (kan) (12) as a screening marker for loss of the plasmid, and the Himar1 mariner transposase gene (tpase) (16). The indicated restriction sites represent those used for cloning, but they are not necessarily unique. Gm−, gram negative.
- FIG. 2.
Locations of 77 sequenced Himar1 insertions in the chromosome of L. monocytogenes. The bars located outside the circle indicate transposons that were oriented in the same direction as the positive strand, whereas the bars located inside the circle indicate transposons that were oriented in the opposite direction. Each quarter of the chromosome is identified with the approximate base pair, except for bp 1, which is identified by an arrow.
Tables
- TABLE 1.
Bacterial strains and vectors used in this study
Strain or vector Genotype or relevant feature Reference or source 10403S L. monocytogenes serotype 1/2a 3 CU1065 B. subtilis W168 trpC2 attSPβ J. D. Helmann DH5α E. coli supE44 ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Lab stock pCR2.1-TOPO Cloning vector from Invitrogen pDG780 Contains a gram-positive kan resistance cassette 12 pKSV7 Shuttle vector with gram-positive pE194ts ori 22 pLTV3 Tn917 delivery vector 4 pMC1 Gram-positive cat gene from pPL2 cloned into pCR2.1-TOPO This study pMC3 Gram-positive cat gene from pMC1 cloned into pMMOrf This study pMC14 Gram-positive kan gene from pDG780 cloned into pPL2 This study pMC25 mariner delivery vector with L. monocytogenes promoter PP60 This study pMC30 mariner delivery vector with L. monocytogenes promoter PactA This study pMC38 mariner delivery vector with B. subtilis promoter PmrgA This study pMC39 mariner delivery vector with B. subtilis promoter PkatA This study pMMOrf Contains 5′ and 3′ ITR from Himar1 15 pNF1100 Contains a copy of Himar1 derived from pMEnt-neo N. E. Freitag; 16 pPL2 Site-specific shuttle integration vector 17 pPL3e pPL2 derivative with gram-positive ermC gene 10 - TABLE 2.
Oligonucleotide primers used in this study
No. of primer Sequence 5′→3′a Characteristic (reference) 155 TTGGATCCCGGAGACGGTCACA BamHI 156 CGCATCTGTGCGGTATTTCA 188 ATCCGCATGCTGCAAGGCGATTAAGT SphI 194 CGGGTACCATCACACGCAAAAAGGA KpnI 195 CGGGTACCTAAATTCAAAATCTATC KpnI 205 GGTATAGCATATGAATCGCATCCGATTGCAG NdeI 206 TGTCAGACATATGGGCACACGAAAAACAAGT NdeI 207 GGCCACGCGTCGACTAGTACNNNNNNNNNNGTAAT ARB1B (8) 208 GGCCACGCGTCGACTAGTAC ARB2 (8) 234 GCGGATCCAGAGGAGTTTTATGAATATGGAAAAAAAGGAATTTCGTGTTT BamHI and RBS 247 GCGGTACCTATCATCAATACTATA KpnI 248 CAGGATCCGTGATCTGTTGACTTAAT BamHI 249 GCGGTACCTTTTCTTTGATGCTGA KpnI 250 GCGGATCCAATTTATAAGAACATAAT BamHI 254 CGTGGAATACGGGTTTGCTAAAAG 255 CAGTACAATCTGCTCTGATGCCGCATAGTT 256 TAGTTAAGCCAGCCCCGACACCCGCCAACA 257 CTTACAGACAAGCTGTGACCGTCT 269 GCTCTGATAAATATGAACATGATGAGTGAT 270 TGTGAAATACCGCACAGATGCGAAGGGCGA 271 GGGAATCATTTGAAGGTTGGTACT ↵ a Restriction sites are underlined, and the ribosome binding site is italicized.
- TABLE 3.
Comparison of transposon delivery vectors
Delivery vector Size of vector Size of transposon (bp) Total no. of mutants (CFU/ml ± SD)a % ± SD of CFUs bearing vector pLTV3(4) 22.1 kb 14,861 (9.80 ± 2.00) × 104 >50b pMC38 8,172 bp 1,395 (1.73 ± 1.07) × 106 2.4 ± 1.7 pMC39 8,291 bp 1,395 (1.08 ± 0.76) × 106 0.85 ± 0.01
