Article Figures & Data
Figures
- FIG. 1.
Genetic region containing tatABC of S. meliloti. A schematic diagram showing the relative positions of the relevant primers used to construct and verify constructs in the tat region. Numbered arrows correspond to the primers listed in Table 2. The plasmid above corresponds to pBP121, whereas the plasmid below corresponds to pBP79. Details of the construction and verification are in the text. The arrow above the Plac promoter indicates the direction of transcription nptII, neomycin phosphotransferase cassette.
- FIG. 2.
Stability of pBP79 containing the tatABC operon. Presence of pBP79 was measured by the proportion of Tc-resistant colonies found at each subculture. Error bars represent standard deviations of three independent experiments. Where not shown, error bars are smaller than the symbol. ▴, SRmA947; ⧫, SRmA938.
- FIG. 3.
Effect of tatABC mutation on cell morphology and viability in S. meliloti. (A and C) Rm1021. (B and D) SRmA947. Panels A and B show DIC images of S. meliloti cells. Panels C and D show cells that were stained using a BacLight LIVE/DEAD bacterial viability kit (Molecular Probes, Invitrogen) and viewed using a fluorescent microscope. Live cells fluoresce green, whereas dead cells fluoresce red. (E) Percentage of live cells in cultures of Rm1021, SRmA938, and SRmA947, stained using a BacLight LIVE/DEAD kit and quantitated using fluorescence. Data are presented as the mean of three independent replicates. Values in parentheses represent standard deviations.
Tables
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Relevant characteristic(s)a Reference or source Strains S. meliloti Rm1021 SU47 str-21 Smr 35 Rm2011 SU47 str-3 Smr 11 Rm5000 SU47 rif-5 Rifr 19 SRmA363 Rm1021 expR+ 38 SRmA938 Rm1021 pKnock-Gm containing 5′ nptII::3′ flanking tatABC This work SRmA946 Rm1021 pRK7813tatABC::pKnock-Gm This work SRmA947 Rm1021 tatABCΔ/pRK7813tatABC This work S. medicae SmD100 WSM419 Smr This work SmD104 SmD100(pBP92) This work SmD107 SmD107(pBP93) This work E. coli DH5α λ− φlacZDM15 D(lacZYA-argF)U169 recA1 endA hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 24 S17-1 recA derivative of MM294A with integrated RP4-2 (Tc::Mu::Km::Tn7) 58 MM294A pro-82 thi-1 hsdR17 supE44 20 MT607 MM294A recA56 20 MT616 MT607(pRK600) 20 Plasmids pEX18Tc Broad-host-range gene replacement vector; sacB Tcr 26 pBlueScript II SK Cloning vector; ColE1 oriV Apr Stratagene pRK600 pRK2013 npt::Tn9 Cmr 20 pRK7813 RK2 derivative carrying pUC9 polylinker and lambda cos site; Tcr 27 pKnock-Gm Suicide vector for insertional mutagenesis; R6K ori RP4 oriT Gmr 2 pPH1JI IncP plasmid; Gmr 7 pBP59 sacB from pEX18Tc inserted into pRK7813 This work pBP79 pBP59 with tatABC cloned into polylinker This work pBP91 pRK7813 tatC::nptII; oriented in the same direction as Plac This work pBP92 pRK7813 tatC::nptII; oriented in the opposite direction as Plac This work pBP103 pKnock-Gm containing upstream 5′ tatABC fragment This work pBP105 pBP103containing 3′ downstream tatABC fragment This work pBP121 pBP105 containing nptII cassette This work pMM4 tatC fragment cloned into pBS This work pMM13 Kanr fragment from pMM22 cloned into pMM4 This work pMM22 Kanr fragment cloned as a SmaI fragment into pBlueScript This work ↵a Cm, chloramphenicol; Gm, gentamicin; Kan, kanamycin; Nm, neomycin; Ap, ampicillin; Sm, streptomycin.
- TABLE 2.
Primers used in this work
No. Primer name Sequence (5′ → 3′) 1 sacB Fwd ATATCCCGGGCCATGGCCATCACATATACCTGCCGTTC 2 sacB Rvs ATATCCCGGGCCATGGTTATTTGTTAACTGTTAATTGTCCTTG 3 tatABC Fwd ATATGAATTCTGCGCGAGACGCGCGCGA 4 tatABC Rvs ATATAAGCTTCTCCTTCGACGCAGCATTGCG 5 5′ upstream tat Fwd ATATGCGGCCGCGGTTTCAGATGCGCGTAACG 6 5′ upstream tat Rvs ATATCCCGGGGTATGGCCAGCGTGTCGGC 7 3′ downstream tat Fwd ATATCCCGGGACCGCGGAGTTCTTGCGCC 8 3′ downstream tat Rvs ATATGGTACCCGACATTTGCGCTTTCGTCCG 9 tatC Fwd ATATGAATTCCGCCGGCTGCGAATAAG 10 tatC Rvs ATATGAATTCGCTGCATGACATCGGAG 11 tat confirmation Fwd CACCGAAGCCTGAAGAAGAC 12 Tat confirmation Rvs GCTCCTTCGAAGTCCATCAG 13 nptII out Left TTCGGAATCGTTTTCCGGGAG 14 nptII out Right TTAGCAGCCCTTGCGCCCTG - TABLE 3.
Frequency of single-crossover recombinants is dependent upon the orientation of Placa
Construct Orientation relative to Plac No. of recombinants S. meliloti S. medicae pBP91 Same direction 1.8 × 10−4 1.7 × 10−4 pBP92 Opposite direction <10−8 <10−8 ↵a Plasmid pBP91 or pBP92 were conjugated from E. coli into either S. meliloti or S. medicae and recombinants were selected on the basis of antibiotic resistance. Data presented are the mean of one experiment with three independent replicates where the standard deviation is less than 10%. The experiment was also replicated on three separate occasions with comparable data.
