DNA binding kinetics of two response regulators, PlnC and PlnD, from the bacteriocin regulon of Lactobacillus plantarum C11

Bacteriocin production in the lactic acid bacterium Lactobacillus plantarum C11 is regulated through a quorum sensing based pathway involving two highly homologous response regulators (59% identity and 76% similarity), PlnC as a transcriptional activator and PlnD as a repressor. Previous in vitro studies have shown that both regulators bind, as homodimers, to the same DNA regulatory repeats to exert their regulatory functions. As the genes for these two proteins are located on the same auto-regulatory operon, hence being co-expressed upon gene activation, it is plausible that their opposite functions must somehow be differentially regulated, either in terms of timing and/or binding kinetics, so that their activities do not impair each other in an uncontrolled manner. To understand the nature behind this potential differentiation, we have studied the binding kinetics of the two regulators on five target promoters (P plnA , P plnM , P plnJ , P plnE and P plnG ) from the bacteriocin regulon of L. plantarum C11. By using surface plasmon resonance spectroscopy we obtained parameters such as association rates, dissociation rates and dissociation constants, showing that the two regulators indeed differ greatly from each other in terms of cooperative binding and binding strength to the different promoters. For instance, cooperativity is very strong for PlnC binding to the promoter of the regulatory operon (P plnA ), but not to the promoter of the transport operon (P plnG ), while the opposite is seen for PlnD binding to these two promoters. The estimated affinity constants indicate that PlnC can bind to P plnA to activate transcription of the key regulatory operon plnABCD without much interference from PlnD, and that the repressive function of PlnD might act through a different mechanism than repression of the regulatory operon. We have characterised the DNA binding kinetics of the two regulators PlnC and PlnD from the bacteriocin locus in L. plantarum C11. Our data show that PlnC and PlnD, despite their strong homology to each other, differ greatly from each other in terms of binding affinity and cooperativity to the different promoters of the pln regulon.