Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis

We isolated a strain from hop-resistant Lactobacillus brevis ABBC45, which had lost a plasmid (pRH45) harboring a putative hop resistance gene, horA. The hop resistance level of this horA-deficient strain, named ABBC45^C, was initially low but gradually induced by repeated growth in media containing progressively increasing levels of hop compounds. Although the hop resistance level was substantially lower than that of the hop-adapted wild type strain, hop-adapted ABBC45^C (ABBC45^CR) was still capable of growing in beer, suggesting ABBC45 possesses at least two hop resistance mechanisms. Hop resistance acquired by ABBC45^CR gradually diminished to the pre-adapted level, when the strain was grown repeatedly in the absence of hop compounds. ABBC45^CR was found to be cross-resistant to several structurally unrelated drugs, including ethidium bromide, daunomycin and nisin. In addition, ABBC45^CR was shown to extrude ethidium in an energy-dependent manner, while ABBC45^C did not show such activity. This indicates that the efflux pump was induced by adaptation to hop compounds. The efflux activity of ethidium was reduced by the addition of hop compounds, suggesting hop compounds are also the substrate of the efflux pump. It was also shown that the efflux activity was completely dissipated with the abolition of proton motive force (PMF). These results, taken together, suggest the hop resistance mechanism of ABBC45^C is mediated by PMF-dependent multidrug efflux pump.