Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production

Abstract

Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 microg ml(-1) for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.

FIG. 1.

Southern blot hybridization of EcoRI (lanes 1 and 2)- or KpnI plus SacI (lanes 3 and 4)-digested chromosomal DNA of E. faecalis JH2-2 (lanes 1 and 3) and an E. faecalis tdc mutant (lanes 2 and 4) with the F4S1-F3S2 DNA fragment as a probe. Migration of DNA fragments of the molecular size marker Smartladder (Eurogentec) is indicated on the left.

FIG. 2.

Amino acid sequence comparison of E. faecalis Tdc (Tdc Ef) with the probable glutamate decarboxylases from V. cholerae (Gad Vc [17]) and Methanothermobacter thermautotrophicus (Gad Mt [43]); decarboxylases from Pyrococcus abyssi (Dec Pa; accession number NP 126894), Pyrococcus horikoshii (Dec Ph [25]), and Methanococcus jannaschii (Dec Mj [7]); and l-2,4-diaminobutyrate decarboxylases from Bacillus halodurans (Ddc Bh [48]) and from Haemophilus influenzae (Ddc Hi [15]). Amino acid residues that are shared by at least four proteins are shaded. An underlined number in a sequence indicates the number of amino acids omitted at that position to save space. The boxes represent the VHVDAAY motif and the pyridoxal phosphate attachment site.

FIG. 3.

Growth and tyramine production of E. faecalis JH2-2 and the 16G10 mutant in Maijala broth. Squares, JH2-2; circles, 16G10 mutant; solid symbols, log of UFC numbers; open symbols, tyramine concentration in supernatant.

FIG. 4.

Transcriptional analysis. (A) Northern blot hybridization of RNA isolated from exponentially growing E. faecalis JH2-2 cells in Maijala broth. Hybridization was performed with the F2S1-F2S2 single-stranded labeled probe. The size of the transcript was estimated by comparing the band mobility with those of standards in an RNA ladder (0.56 to 9.4 kb) (Amersham International). (B) RT-PCR assays conducted on mRNA isolated from exponentially growing E. faecalis JH2-2 cells in Maijala broth. RTs were performed with oligonucleotides F2S2 for lanes 1 and 2, F5S2 for lanes 3 to 6, and F8S2 for lanes 7 to 10. To ensure the absence of genomic DNA, negative controls were performed without reverse transcriptase (even lanes). PCRs were performed with primers F2S1 and F2S2 (lanes 1 and 2), F3S1 and F5S2 (lanes 3 and 4), F5S1 and F5S2 (lanes 5 and 6), F3S1 and F8S2 (lanes 7 and 8), or F6S1 and F8S2 (lanes 9 and 10). Lanes M contain DNA fragments of the molecular weight marker Smartladder (Eurogentec).