Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi

Abstract

Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

Fig. 1

Recombination event mediated by ΦC31 integrase between attP and attB. The short vertical line in the middle of the attP and attB sites indicates the core region (TT) where recombination occurs. The arrow indicates the position of primers used in sequencing analysis and PCR amplification.

Fig. 2

Sequences of the attachment sites for ΦC31 integrase in Rhodococcus equi and closely related species. The core sequence is TT, in bold at the center of each attB. Conserved nucleotide bases in each attB site flanking the core sequence are also in bold.

Fig. 3

Stability of integrated pSET152 and the episomal plasmid pMV261-hyg. Rhodococcus equi pSET152-integrated strain int1 and 103+hyg were cultured in BHI without the addition of antibiotics for up to 100 generations. At approximately the 20th, 40th, 60th, 80th, and 100th generation, the samples of cultures were diluted and plated on BHI plates with or without antibiotics and the number of colonies arising was counted. Bacterial numbers obtained from the BHI plates without antibiotic were used as controls to calculate the percentage of control CFU arising from growth on plates with antibiotic supplementation. This experiment is representative of three independent experiments.

Fig. 4

ΦC31 integrase-based integration does not alter intracellular replication. The intracellular growth of various Rhodococcus equi strains in RAW264 macrophages was determined by lysis of infected macrophages and subsequent plating of lysates. Macrophages were infected with wild-type R. equi 103+, the pSET152-integrated strain (int1), a vapA mutant and a vapA mutant complemented with wild-type vapA (strain vapA-/vapA). At various time points post infection, macrophages were lysed and the lysate was serially diluted and plated. The number of CFU was determined after 48 h of incubation at 37 °C. Each time point represents the mean ± SD for three replicate lysates.