Identification of an atypical replicative genetic element in Rhodococcus jostii RHA1

Abstract

By exploring the use of plasmids to confer Rhodococcus jostii RHA1 the possibility of utilizing xylose to produce lipids we have observed that the plasmid used was not always maintained in the transformants as expected. Instead, we observed an illegitimate integration of the antibiotic resistance gene from the plasmid into the recombinant cells. Genome sequencing of the transformants has provided evidence that this illegitimate integration is not size-, site-or sequence-specific. But even more surprising, genome sequencing revealed the presence of an unexpected circular multicopy replicative element (75-80 kb) that appears to be excised from the chromosome as a consequence of the stress generated by the antibiotic used in the selection process. The excised fragment does not contain any of the typical features of genomic islands. These results provide evidence that the genome of this oleaginous strain is more plastic than initially anticipated and our findings open the option of developing new ways to genetically modify this strain by using illegitimate recombinant approaches. But even more remarkably, the discovery of this atypical replicative element raises new questions about the existence of novel mechanisms of evolution in bacteria.

Keywords: Rhodococcus; antibiotic resistance; illegitimate recombination; kanamycin; replicative genetic element.

Figure 1

Illegitimate recombination of plasmid pNVSxylABatf1 in the chromosome of R. jostii RHA1. (A) Schematic description of recombination observed in clone 1. (B) Schematic description of recombination observed in clone 2. Green arrows represent the genes located in the R. jostii chromosome that are deleted after recombination: (1) LuxR C-terminal-related transcriptional regulator; (2) acyl--CoA ligase family protein; (3) DUF3054 domain-containing protein; (4) TetR/AcrR family transcriptional regulator; (5) flippase-like domain-containing protein; (6) YceI family protein; (7) TetR/AcrR family transcriptional regulator; (8) Alpha/beta hydrolase. The blue arrows represent the genes located in plasmid pNVSxylABatf1 that are inserted after recombination. The orange triangles represent the recombination sites in the chromosome of R. jostii marked as I and II for clone 1 and as V and VI for clone 2, and in the plasmid pNVSxylABatf1 marked as III and IV for clone 1 and as VII and VIII for clone 2. Numbers in the sequences indicate the position at the R. jostii chromosome. The sequences surrounding the insertion sites both in the chromosome and in the plasmid are indicated.

Figure 2

Coverage of the reads sequenced by Illumina. (A) Reference genome of Rhodococcus jostii RHA1, showing alignment of the genomes of the two transformed microorganisms, with the 80 kb excised fragment clearly visible, indicated by a notable increase in read coverage. (B) Zoom of the genomic island region with high coverage in clones 1 and 2. The zoom also highlights the region with high coverage in the WT strain, which does not present the excised element.

Figure 3

Semiquantitative PCR of different R. jostii kanamycin resistant clones electroporated with pNVS plasmid. (A) PCR amplification performed with primers iGIforward and iGIreverse inside the mobile region. Lane 1. 1 kb DNA Ladder. Lanes 2, 3, 4, 5, 7, 8, 9. Km resistant clones; Lane 6. Wild type. Lane 10. Negative control. (B) PCR amplification performed with primers oGIforward and oGIreverse outside the mobile region. Lane 1. 100 bp DNA Ladder. Lanes 2. Wild type. Lane 3 Clone 1 (sequenced). Lane 4. Clone 2 (sequenced). PCR amplification performed with primers iGIforward and iGIreverse inside the mobile region. Lane 5. Wild type. Lane 6. Clone 1 (sequenced). Lane 7. Clone 2 (sequenced). Lane 8. 100 bp DNA Ladder.