EVALUATING PHENOTYPIC EXPRESSION OF SELECTED GENES ON PBASL58 MEGAPLASMID IN PSEUDOMONAS

Abstract

Horizontal gene transfer (HGT) is the exchange of genetic material between bacterial cells. Megaplasmids, which are extrachromosomal DNA over 350 kilobases in length, can be shared between bacteria in a population through HGT. The megaplasmid pBASL58, disc Leaf58 Pseudomonas first found on Arabidopsis overed in plants, contains genetic information that appears to be redundant with the bacterial chromosome (1). It is hypothesized that pBASL58 must confer fitness benefits that justify the energetic cost of pla smid retention. One such fitness benefit is a type 1F clustered, regularly interspaced, short palindromic repeat (CRISPR) system. Plasmid DNA that is transferred by HGT may be targeted by CRISPR cut DNA to prevent replication of associated proteins, which foreign material. pBASL58 encodes the only known type 1F CRISPR system contained entirely on a megaplasmid in Gram negative bacteria. Here, we evaluate the phenotypic expression of this CRISPR system as well as a tmRNA ribosome rescue system that is also e ncoded on pBASL58. Such ribosome rescue systems mark and dispose of peptides that have stalled ribosomes (2). For normal cell growth and activity, the nonfunctional peptide sequence must be degraded in order to free up the ribosome for translation of more mRNA. To evaluate expression of the CRISPR system, target plasmids were created based upon the target sequences encoded in the CRISPR loci. Triparental conjugations were used to test whether CRISPR associated (Cas) proteins would cut target DNA and thus pr event replication. The tmRNA ribosome rescue system’s phenotypic expression was evaluated by comparing deletion mutants of the corresponding sspB gene to wildtype strains in a motility assay. There is no evidence that the CRISPR system is actively being tr conditions. However, deletion of the transposon sspB anscribed and translated in laboratory gene reduces the growth of Pseudomonas over a 24hour period, possibly indicating a fitness benefit for retention of the pBASL58 megaplasmid in Pseudomonas.