Heterologous expression of thuricin CD immunity genes in Listeria monocytogenes

Abstract

Bacteriocins are ribosomally synthesized peptides that can have a narrow or broad spectrum of antimicrobial activity. Bacteriocin producers typically possess dedicated immunity systems that often consist of an ATP-binding cassette (ABC) transporter system and/or a dedicated immunity protein. Here we investigated the genes responsible for immunity to thuricin CD, a narrow-spectrum two-peptide sactibiotic produced by Bacillus thuringiensis DPC6431. Heterologous expression of putative thuricin CD immunity determinants allowed us to identify and investigate the relative importance of the individual genes and gene products that contribute to thuricin CD immunity. We established that TrnF and TrnG are the individual components of an ABC transporter system that provides immunity to thuricin CD. We also identified a hitherto overlooked open reading frame located upstream of trnF predicted to encode a 79-amino-acid transmembrane protein. We designated this newly discovered gene trnI and established that TrnI alone can provide protection against thuricin CD.

FIG 1

Organization of the thuricin CD gene cluster (A), TrnI topology prediction (B) and TrnG topology prediction (C). (A) Immunity genes are shaded in gray. trnI (formerly orf11) is a small immunity gene encoding a transmembrane immunity protein. trnFG encode a fully functional ABC transporter system. trnβ and trnα are the two structural genes (shaded in black). trnC and trnD are the two posttranslational modification genes and encode S-adenosylmethionine proteins (shaded in white). trnE encodes a C-terminal processing peptidase with a minor role in thuricin CD immunity (shaded in gray). orf12 (dashed outline), located between trnI and trnF, is unlikely to be a gene. (B) Hydrophobicity plot and membrane topology prediction of TrnI on the basis of TMHMM (23). Red line, transmembrane helix; blue line, beta preference; gray line, modified hydrophobic-moment index; violet boxes, predicted transmembrane helix positions. (C) Hydrophobicity plot and membrane topology prediction of TrnG based on TMHMM (23). Red line, transmembrane helix; blue line, beta preference; gray line, modified hydrophobic-moment index; violet boxes, predicted transmembrane helix positions.

FIG 2

Kill curve analysis. Survival and growth of wild-type L. monocytogenes 33013 and strains expressing trnFG, trnIF, trnE, trnG, trnI (formerly orf11), trnF, and trnIFG when challenged with a sublethal level (2.2 μM) of thuricin CD. The growth of L. monocytogenes 33013 in the absence of thuricin CD (No TCD) is also shown.