The gene encoding the glutamate dehydrogenase in Lactococcus lactis is part of a remnant Tn3 transposon carried by a large plasmid

Abstract

The gene responsible for the uncommon glutamate dehydrogenase (GDH) activity of Lactococcus lactis was identified and characterized. It encodes a GDH of family I that is mainly active in glutamate biosynthesis, is carried by a large plasmid, and is included, with functional cadmium resistance genes, in a remnant Tn3-like transposon.

FIG. 1.

(a) Schematic representation of the transposon of L. lactis NCDO 1867 and its flanking regions (7,380 bp). Open reading frames 1 through 5 show high levels of identity with gdh, transposase IS1216, cadmium resistance gene cadA, cadmium resistance gene cadC, and resolvase tnpR, respectively. The resolution site (res site) is represented by a triangle. Inverted repeats at the left (IR_L) or at the right (IR_R) of the transposon are indicated by hachured triangles. The G+C contents of the transposon and flanking regions are indicated at the top of the figure. (b) Partial nucleotide sequence of the fragment containing the gdh gene of L. lactis NCDO 1867. In the upstream region, the two −10 extended consensus sequences are boxed and the ribosome binding site sequence is in a grey-tinted box. The ATG start codon and TAA stop codon are in bold, with the coding region designated by dots. Arrows show the potential terminator. (c) The deduced amino acid sequence of gdh shows conserved hexameric family I GDH regions (in bold and in grey-tinted boxes), the GDH active site (in bold and italics), and the NAD or NADP binding site (in bold and underlined).

FIG. 2.

Phylogenetic tree of sequenced GDH. The alignment and phylogenetic analysis was performed with CLUSTAL W (version 1.8) (22). Analysis of 1,000 computer-generated trees by bootstrapping (6) indicates the reliability of each branch point. All values of >100 are shown. The tree is arbitrarily rooted. GenBank accession numbers for the sequences are reported in parentheses. B. halodurans, Bacillus halodurans; C. acetobutylicum, Clostridium acetobutylicum; S. agalactiae, Streptococcus agalactiae; S. mutans, Streptococcus mutans; C. glutamicum, Corynebacterium glutamicum; Lb. plantarum, Lactobacillus plantarum; S. pneumoniae, Streptococcus pneumoniae; S. suis, Streptococcus suis; E. faecalis, Enterococcus faecalis; P. gingivalis; Porphyromonas gingivalis; N. meningitidis, Neisseria meningitidis; C. symbiosum, Clostridium symbiosum; C. difficile, Clostridium difficile; P. asaccharolyticus, Peptostreptococcus asaccharolyticus; B. subtilis, Bacillus subtilis.