Natural diversity of lactococci in γ-aminobutyric acid (GABA) production and genetic and phenotypic determinants

Abstract

Background: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M).

Results: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the P_gadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the P_gadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production.

Conclusions: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.

Keywords: GAD system; Lactococcus cremoris; Lactococcus lactis; γ-aminobutyric acid.

Fig. 1

Schematic representation of genetic organization of the GAD genes (i.e. regulator gadR, glutamate/GABA antiporter gadC, glutamate decarboxylase gadB, and two promoters P_gadR and P_gadCB) in lactococci: ∆ (blue) deletions observed in all L. cremoris species relative to L. lactis, ∆ (red) deletion or ▼ (red) insertion only observed in L. cremoris species with the cremoris phenotype, ▼ (grey) insertion site for the IS981 element in L. lactis S642. (not drawn to scale)

Fig. 2

Phylogenetic tree based on GAD system gene sequences. The tree was generated with the software MEGAX using the maximum likelihood method and the Tamura 3-parameter model with 1,000 bootstraps. The numbers at the branches indicate supporting bootstrap values. The scale bar indicates 0.01 substitution per nucleotide position

Fig. 3

GABA production (mM) of 132 lactococci (▲ L. lactis, ♦ L. cremoris phenotype lactis, ● L. cremoris phenotype cremoris) after 24 h of growth on glucose-YE medium supplemented with 34 mM L-glutamic acid without NaCl (A) and with 0.3 M NaCl (B). Values represent the mean of duplicate experiments. The strains mentioned in the text are MG1363, a L. cremoris strain with a lactis phenotype ♦, and NCDO2118, NCDO2727, S642 and EIP3I, which are L. lactis strains▲

Fig. 4

GABA production (mM) after 24 h of growth on glucose-YE medium supplemented with 34 mM L-glutamic acid for five strains without NaCl (filled symbol) and with 0.3 M NaCl (empty symbol) compared to specific GAD activity (mmol/min/g protein) (A) and specific GAD activity compared to gene expression fold change (FC) of gadC ∆▲ and gadB ○● (B)