Proteomic approach for characterization of hop-inducible proteins in Lactobacillus brevis

Abstract

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-alpha-acids, are described as ionophores which exchange H+ for cellular divalent cations, e.g., Mn2+, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of Lactobacillus brevis TMW 1.465 and hop adaptation in its variant L. brevis TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in L. brevis TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.

FIG. 1.

2D electrophoretic analysis of Coomassie blue-stained total protein from cells of L. brevis TMW 1.465A grown under hop stress. HI, proteins detected under hop stress conditions only; AI, proteins detected in acid-stressed cells; HO, proteins overexpressed in hop-stressed cells; HR, proteins repressed in hop-stressed cells.

FIG. 2.

Induction factors of proteins overexpressed under hop and acid stress conditions. The normalized volume of each protein spot (HO Protein nr.) was calculated using the Image Master2D Elite software. The induction factors were calculated by dividing each stress-regulated protein spot volume by the corresponding spot volume of the reference condition. Shown are the means of two independent experiments.

FIG. 3.

Protein HI10 expression of L. brevis TMW 1.465 under reference conditions (A) or acid stress (B) or of L. brevis TMW 1.465A under hop stress conditions (C).

FIG. 4.

Mn²⁺-stimulated enzymes in intermediate carbon metabolism are highlighted. Hop-regulated enzymes are boldfaced. Pgk, phosphoglycerate kinase; Gpm, phosphoglyceromutase; Eno, enolase; Pyk, pyruvate kinase; Ppc, phosphoenolpyruvate carboxylase; Mdh, malate dehydrogenase; Ldh, lactate dehydrogenase; Als, acetolactate synthetase; Ald, acetolactate decarboxylase; ButA, acetoin dehydrogenase. Adapted from reference .