Models of target-substrate structures.

<p>(A) When the segment of the target molecule being deleted is larger than the segment being inserted, the target must bend to permit target-substrate annealing. For short insertions, this reaction has higher recombination efficiency, suggesting that the genome more easily adopts a favorable structure. (B) When a recombineering substrate directs an insertion without concomitant deletion, the substrate must bend to achieve target-substrate annealing. The flexibility of the substrate is likely to influence the stability of recombineering heteroduplex intermediate. The green arrow indicates the direction of DNA replication fork movement.</p

Substrate and Target Sequence Length Influence RecTE_Psy Recombineering Efficiency in <em>Pseudomonas syringae</em>

<div><p>We are developing a new recombineering system to assist experimental manipulation of the <em>Pseudomonas syringae</em> genome. <em>P. syringae</em> is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified orthologs of the lambda Red <em>bet/exo</em> and Rac <em>recET</em> genes in <em>P. syringae</em> and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the properties of dsDNA substrates more closely to determine how they influence recombineering efficiency. We find that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTE_Psy mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.</p> </div

Deletion size affects RecTE_Psy-mediated recombination efficiency.

<p>(A) Recombination substrates were generated using PCR primers to amplify the <i>neo</i> gene with 80 nt homologies to the <i>pvsA</i> gene at the ends of the PCR product. The size of the deletion is determined by the distance between the <i>pvsA</i> flanking sequences as they exist on the <i>P. syringae</i> DC3000 genome. Homologous sequences are shown in pink. 500 ng of each substrate was used in electroporations. (B) Recombineering efficiency was determined for each substrate. Between 20 to 60 kanamycin resistant clones from each deletion length were analyzed using PCR to determine whether the <i>neo</i> gene had integrated and produced a deletion in the correct location. The percentage of kanamycin resistant clones with the correct deletion is indicated above each bar. The results are the average of four independent replicates, error bars indicate standard deviation.</p

The amount of dsDNA substrate influences RecTE_Psy recombination frequency.

<p>The amount of PCR product added for electroporations was varied in order to determine the effect of substrate concentration on recombination. (A) Recombineering substrates were generated using PCR. The PCR product had 1 kb flanks identical to target location (the PSPTO_1203 locus) at each end of the <i>neo</i> gene encoding kanamycin resistance. This substrate deletes 500 bp of the 543 bp PSPTO_1203 gene and inserts the 1.3 kb <i>neo</i> gene in its place. (B) Maximum recombination frequency was observed when 500 ng of PCR product was added to the electroporations with RecTE_Psy present. The results are the average of four independent replicates, error bars indicate standard deviation. No kanamycin resistant colonies were observed in the absence of the RecTE_Psy expression vector (data not shown).</p

Insertion length influences recombination efficiency.

<p>(A) The length of the region between the homologous flanking sequences was varied to evaluate whether its length affects recombination frequency. Substrates were designed to generate a 2 kb chromosomal deletion upon insertion of the length indicated. Homologous sequences are shown in pink. (B) Recombineering efficiency with substrates that insert different amounts of sequence. PCR was used to analyze the <i>pvsA</i> alleles of 8–18 kanamycin resistant clones for each deletion length to determine whether the <i>neo</i> gene had integrated and produced the deletion in the correct location. The percentage of kanamycin resistant clones with the correct deletion is indicated above each bar. The results are the average of three independent replicates, error bars indicate standard deviation.</p

Phosphorothioate bonds on the matching lagging strand provide a modest increase in recombineering.

<p>(A) Locations of the 5′ phosphorothioate bonds at the four terminal nucleotides are indicated as red spheres. Leading, Lagging and Both indicate the location of the 5′ phosphorothioate bonds on the respective strands of the substrates relative to the target location. (B) Recombination frequency for 80 nt flank protected substrates. 200 ng of each substrate was used in each electroporation. The results are the average of at least four independent replicates, error bars indicate standard deviation.</p

RecTE_Psy-mediated recombination frequency is sensitive to flank length.

<p>(A) Recombination substrates were generated by PCR using primers that flank the ΔPSPTO_1203::<i>neo</i> allele to produce substrates with homologies of indicated lengths. For example, each of the substrates is composed of the <i>neo</i> gene flanked by the indicated amount of genomic sequence. The substrate was then gel purified and 0.46 pmol of each substrate was used to electroporate cells containing the RecTE_Psy expression vector or empty vector (0.46 pmol corresponds to 200 ng of the 80 bp flanks substrate). (B) Recombination frequencies with these substrates in wild-type and <i>recA</i>⁻<i>P. syringae</i> pv. <i>tomato</i> DC3000 with pUCP24/recTE are shown. The results are the average of at least three independent replicates, error bars indicate standard deviation. No kanamycin resistant colonies were observed in control transformations of cells containing the pUCP24/61 empty vector (no RecTE_Psy). PCR was used to confirm that for each length tested the kanamycin resistance gene had integrated into the correct location.</p

Identification of a Twin-Arginine Translocation System in Pseudomonas syringae pv. tomato DC3000 and Its Contribution to Pathogenicity and Fitness

The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium