Table 1. Transformation experiments and clone isolation.
| Cell Preparationa | |||||
|---|---|---|---|---|---|
| Donor DNA | Value | MIV-1 | MIV-2 | MIV-3 | Late-log |
| RR666 donor DNA | NovR/CFU | 6.40 × 10−4 | 9.20 × 10−4 | 2.40 × 10−3 | 1.33 × 10−5 |
| NalR/CFU | 9.50 × 10−4 | 4.00 × 10−3 | 2.50 × 10−3 | ND | |
| Congressionb | 14.5 | 8.7 | 7.0 | ND | |
| RR3131 donor DNA | NovR/CFU | 2.70 × 10−4 | 6.10 × 10−4 | 7.30 × 10−4 | 1.15 × 10−5 |
| NalR/CFU | 8.50 × 10−4 | 1.10 × 10−3 | 8.80 × 10−4 | ND | |
| Congressionb | 9.6 | 8.2 | 6.2 | ND | |
| Strains collected and sequencedc | NalR (36) | RR4001-12 | RR4033-44 | RR4065-76 | ND |
| NovR (44) | RR4013-24 | RR4045-56 | RR4077-88 | RR4117-24 | |
| None (16)d | RR4025-26 | ND | RR4101-10,13-16 | ND | |
CFU, colony-forming unit; ND , no data.
Three independent MIV cultures of the lab strain Rd (also known as KW20, strain RR722) were prepared and transformed with genomic DNA from either MAP7 (RR666) or a NovR NalR derivative of 86-028NP (RR3131) as donor [(DNA) ~1 genome/cell]. A fourth late-log culture (OD600 = 1.2) also was transformed. The transformation frequency of the donor NovR and NalR alleles were measured by standard plating assay.
Congression was measured as the observed:expected ratio of NovRNalR recombinants, or (NovRNalR/CFU) / (NovR/CFU * NalR/CFU).
Isolated colonies were propagated from each transformation with RR3131 donor DNA for genome sequencing. The antibiotic used for selection is indicated in column 2, with the total number of isolated clones indicated in parentheses. Strain numbers are indicated for each selected type (Table S1). In addition to the strains listed here, the donor and recipient genomes were sequenced in parallel as controls (RR722, RR666, and RR3131).
For viable CFUs isolated from MIV cultures (none), DNAs were pooled into pairs prior to library construction to reduce sequencing costs.