ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes

Plasmids constructed for ORBIT. (a) Construct pKM444 expresses the Che9c phage RecT annealase and the Bxb1 phage integrase, both driven from the Ptet promoter. A similar construct (pKM461) additionally contains the sacRB counter-selectable marker for curing the plasmid following gene modification. (b) One of the ORBIT payload plasmids (pKM446) used for integration into the chromosomal attP site created by an oligonucleotide. In this case, the plasmid payload contains a Flag-DAS+4 inducible degradation tag that is ultimately going to be fused to the 3′ end of the target gene. Cam^r, chloramphenicol resistance. (c) Three genes in M. smegmatis were targeted for C-terminal tagging. Following the ORBIT protocol for each target gene, the total numbers of colonies obtained (from multiple trials) ranged between 20 to 200 CFU/transformation. Electroporations performed with payload plasmid only (no targeting oligonucleotides) produced, on average, 5-fold-lower total numbers of colonies. The number of correct recombinants (of 4 candidates tested) for each target gene is shown. (d) PCR analysis of the 5′ junctions of each candidate tested. (e) Primer positions for verification by PCR of the recombinants are shown. Blue arrows, 5′ junction; brown arrows, 3′ junction. In each case where a 5′ junction was verified, the 3′ junction was also verified (not shown). The 5′ junctions were confirmed by DNA sequencing.

Knockdown phenotypes of ORBIT-generated DAS+4-tagged strains. The growth phenotypes of the Flag-DAS+4 tagged strains constructed as described in the Fig. 2 legend were analyzed after transformation of an SspB-expressing plasmid. (a) The recA–Flag-DAS+4 strain was transformed with an SspB-producing plasmid, pGMCgS-TetON-18 (streptomycin resistance [Strep^r]), under the control of the reverse TetR repressor. In this scenario, RecA is degraded in the absence of anhydrotetracycline (ATc). Ten-fold serial dilutions of the cells grown overnight without ATc were spotted on LB-streptomycin plates, either with or without ATc, and the cells were exposed to 20 J/m² of UV. Preferential UV sensitivity of the recA–Flag-DAS+4 strain in the absence of ATc was determined by comparing the levels of growth seen with and without the inducer. (b) The divIVA–Flag-DAS+4 strain was transformed with an SspB-producing plasmid, pGMCgS-TetOFF-18 (Strep^r), under the control of the wild-type TetR repressor. In this case, DivIVA is expected to be depleted in the presence of ATc. Preferential growth of the DivIVA–Flag-DAS+4 strain on LB plates in the presence of ATc was determined by comparing the levels of growth seen with and without the inducer. (c) The experiment was performed as described for panel B, except that leuB was the target and the cells were plated on 7H10-AD plates.

ORBIT-promoted gene alteration. The ORBIT process is initiated at the replication fork. An oligomer containing a single-stranded version of the Bxb1 attP site (top pictures, red lines) is coelectroporated with an attB-containing nonreplicating plasmid into a mycobacterial host cell expressing both RecT annealase and Bxb1 integrase. RecT promotes annealing of the oligonucleotide to the lagging strand template. Following DNA replication through this region, an attP site is formed in the chromosome (middle pictures). In the same outgrowth period, Bxb1 integrase promotes site-specific insertion of the plasmid into the chromosome (attB × attP). (Left side) The oligonucleotide is designed such that attP is inserted just before the stop codon. The integration event fuses the GFP tag in frame to the 3′ end of the target gene (with an attL site in frame between them); the recombinant is selected for by Hyg^r. (Right side) The oligonucleotide is designed such that attP replaces the target gene and the plasmid integration event allows hygromycin resistance to be used to select for the knockout.

Parameters of the ORBIT process. (A) The amount of target homology flanking the attP site in an oligonucleotide designed to create a polA–Flag-DAS+4 fusion in M. smegmatis was examined as a function of recombinant formation (Hyg^r). One microgram of each oligonucleotide was electroporated with 200 ng of pKM446. The frequency of targeting is expressed as the percentage of Hyg^r transformants following integration of pKM446 relative to a transformation control (20 ng of gentamicin resistance [Gen^r] plasmid pKM390). Experiments were performed in triplicate; standard errors are shown. Gent, gentamicin. (B) Colony counts were determined after electroporation of 1 μg of an oligonucleotide with 70-base flanks (designed to create a polA–Flag-DAS+4 fusion) with various amounts of pKM446. CFU counts per milliliter were determined following overnight growth of the electroporation mixtures in 2 ml LB. Experiments were performed in triplicate; standard errors are shown.

ORBIT-generated insertions and deletions in M. tuberculosis and M. smegmatis. Gene deletions and modifications were performed at a variety of positions in the chromosomes of both M. tuberculosis (a) and M. smegmatis (b) using ORBIT. In most cases, the oligonucleotides contained an attP site flanked by 70 bases of target homology. Insertions (C-terminal DAS+4 or His-Flag tags) are shown in red, deletions are shown in blue, and GFP tags are shown in green. Descriptions of all the modifications performed by ORBIT are provided in Table 1 (for M. smegmatis) and Table 2 (for M. tuberculosis).

ORBIT-generated GFP fusions. M. smegmatis cells containing GFP-tagged target genes were grown in 7H9-AD-Tween 80 to an optical density of 0.8. One microliter of the culture was spotted on an agarose pad for microscopy. Each bacterial strain was imaged using differential interference contrast (DIC) and GFP channels, as described in Material and Methods.