Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

Abstract

We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32°C but not 43°C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene.

A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains

“…The site-directed disruption of the hsdR genes in these strains was performed using the targetron system (mobile group II introns) adapted for S. aureus (26) through introduction of the targetron plasmid by phage transduction. As expected, the resulting UAMS-1 hsdR − mutant strain could be transformed with plasmid DNA (pCN33, pCN38, and pNL9162) (26,27), albeit at low frequency ( Table 1). The transformation efficiency with plasmid DNA prepared from RN4220 into SA564 was similar in the mutant and parent strains.…”

Methicillin-resistant Staphylococcus aureus (MRSA) Pyruvate Kinase as a Target for Bis-indole Alkaloids with Antibacterial Activities

“…Controls showed no products if the initial reverse transcription step was omitted indicating that the amplified bands in the experimental samples were derived from cellular RNA. Disruption of hsa, a previously confirmed essential gene in S. aureus was performed as a control (36).…”

DEAD-Box RNA Helicases in Gram-Positive RNA Decay

“…In the laboratory, we have used the targetron system (Yao et al, 2006) and for making clean deletions, a highly powerful selection/antiselection system that takes advantage of the sensitivity of bacteria to 5-FOA (Redder and Linder, 2012).…”