ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes

ABSTRACTTwo efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for “oligonucleotide-mediatedrecombineering followed byBxb1integrasetargeting”) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this “drag and drop” strategy by the construction of insertions or deletions in over 100 genes inMycobacteriumtuberculosisandM. smegmatis.IMPORTANCEWe sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use inM. smegmatisandM. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1attPsite into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries anattBsite and a drug selection marker, we show both formation of a chromosomalattPsite and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3′ mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.

Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination

ABSTRACT Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3′-to-5′ proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3′ ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDVr) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3′-to-5′ exonuclease domain of the viral polymerase. Viruses encoding a CDVr mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3′ ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3′-to-5′ exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD+ Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes

ABSTRACT To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD + background, we studied 31 strains with all combinations of null alleles of exonuclease I (Δxon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD Δxon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon+), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3′ DNA overhangs, giving protection against Xon+. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD + cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.

Genetic Requirements of Phage λ Red-Mediated Gene Replacement in Escherichia coli K-12

ABSTRACT Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing thered genes of bacteriophage λ in place ofrecBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac− chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced inruvAB and recQ strains. In this genetic background, mutations in recF, recO, andrecR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the ninregion of phage λ partially complemented both the viability and recombination defects of the recF, recO, andrecR mutants and the recombination defect ofruvC but not of ruvAB or recQmutants.

Roles of RuvC and RecG in Phage λ Red-Mediated Recombination

ABSTRACT The recombination properties of Escherichia colistrains expressing the red genes of bacteriophage λ and lacking recBCD function either by mutation or by expression of λ gam were examined. The substrates for recombination were nonreplicating λ chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+recG+ , ruvC recG +,ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome’s restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in arecG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flankinglac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher inrecG than in recG +. These observations favor models in which RecG tends to push invading 3′-ended strands back out of recombination intermediates.

Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in Hfr x F- crosses.

The formation of recombinants during conjugation between Hfr and F- strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located approximately 500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located > 300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange(s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec+ crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to approximately 1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.