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Molecular Analysis of Glutamate Decarboxylases in Enterococcus avium.

ABSTRACT: Enterococcus avium (E. avium) is a common bacterium inhabiting the intestines of humans and other animals. Most strains of this species can produce gamma-aminobutyric acid (GABA) via the glutamate decarboxylase (GAD) system, but the presence and genetic organization of their GAD systems are poorly characterized. In this study, our bioinformatics analyses showed that the GAD system in E. avium strains was generally encoded by three gadB genes (gadB1, gadB2, and gadB3), together with an antiporter gene (gadC) and regulator gene (gadR), and these genes are organized in a cluster. This finding contrasts with that for other lactic acid bacteria. E. avium SDMCC050406, a GABA producer isolated from human feces, was employed to investigate the contribution of the three gadB genes to GABA biosynthesis. The results showed that the relative expression level of gadB3 was higher than those of gadB1 and gadB2 in the exponential growth and stationary phases, and this was accompanied by the synchronous transcription of gadC. After heterologous expression of the three gadB genes in Escherichia coli BL21 (DE3), the K _m value of the purified GAD3 was 4.26 ± 0.48 mM, a value lower than those of the purified GAD1 and GAD2. Moreover, gadB3 gene inactivation caused decreased GABA production, accompanied by a reduction in resistance to acid stress. These results indicated that gadB3 plays a crucial role in GABA biosynthesis and this property endowed the strain with acid tolerance. Our findings provided insights into how E. avium strains survive the acidic environments of fermented foods and throughout transit through the stomach and gut while maintaining cell viability.

SUBMITTER: Gu X

PROVIDER: S-EPMC8461050 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.

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Molecular Analysis of Glutamate Decarboxylases in Enterococcus avium.

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