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Journal of Bacteriology, July 2005, p. 5019-5022, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.5019-5022.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Gene Encoding the Glutamate Dehydrogenase in Lactococcus lactis Is Part of a Remnant Tn3 Transposon Carried by a Large Plasmid

Catherine Tanous,¹ Emilie Chambellon,¹ Anne-Marie Sepulchre,² and Mireille Yvon^1*

Institut National de la Recherche Agronomique, Unité de Biochimie et Structure des Protéines, 78352 Jouy-en-Josas, France,¹ DANISCO, ZA de Buxières, BP10, F-86220 Dangé-Saint-Romain, France²

Received 1 March 2005/ Accepted 18 April 2005

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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.

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Lactococcus lactis is a lactic acid bacterium widely used as a starter culture in the production of fermented dairy products. Although this bacterium is commonly found in milk, it is also naturally present in other environments.

Previously, we detected glutamate dehydrogenase (GDH) activity in some L. lactis strains isolated from plants and animals (21). This activity could be of interest in the dairy industry, since it stimulated conversion of amino acids to aroma compounds by providing, from glutamate, -ketoglutarate required for amino acid transamination that initiates the conversion. However, no gdh gene was identified in the two L. lactis genomes available (strain IL1403 [GenBank accession no. AE005176]) (4) and SK11 (Joint Genome Institute [http://genome.ornl.gov/microbial/lcre/]), suggesting that GDH activity in L. lactis is carried by a mobile genetic element.

Cloning and characterization of the gene responsible for GDH activity in L. lactis NCDO 1867 isolated from peas. A 1-kb fragment of the gdh gene of L. lactis NCDO 1867 was first amplified by PCR with degenerate oligonucleotides deduced from sequences of known bacterial GDHs (3F and 8R [Table 1]). This fragment was cloned into the cloning vector TOPO-XL (PCR cloning kit, Invitrogen) and sequenced. The rest of the gene and its flanking regions were then amplified by inverse PCR (Takara kit; Takara Shuzo Co., Japan) by using the oligonucleotides 24R/23R and 25F/26F (Table 1). Amplified fragments were purified (Qiaquick kit; QIAGEN) and sequenced. The gdh gene was then disrupted by a single crossing over with the pGhost9 vector containing a 965-bp internal fragment of gdh (generated with oligonucleotides 58F and 1023R [Table 1]) as described by Maguin et al. (12). The gene disruption totally suppressed the GDH activity of the strain, indicating that this gene is the only gene responsible for GDH activity in L. lactis. Indeed, the NAD- and NADP-dependent activities in cell extracts of the wild-type strain NCDO 1867 determined as previously described (8) were 1.4 ± 0.3 and 5.7 ± 0.7 nmol min^–1 mg protein^–1, respectively, while in the gdh mutant TIL487, they were both equal to zero (0 and 0.2 ± 0.1 nmol min^–1 mg protein^–1).

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TABLE 1. Oligonucleotides used in this study

The sequence surrounding gdh suggests that the gene is monocistronic (Fig. 1b). The gene encodes a 448-amino-acid protein, which is in agreement with the sizes of monomers of hexameric GDHs purified from other bacteria (1, 13). The protein sequence (Fig. 1c) exhibits the putative GDH active site, the NAD(P) coenzyme binding motif, and the three highly conserved domains characteristic of family I of hexameric GDHs (2). This classification was confirmed by the construction of a phylogenetic tree from the sequence alignments with known GDHs (Clustal W, version 1.8 [22]) (Fig. 2).

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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 (IRL) or at the right (IRR) 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).

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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.

As are other GDHs of family I, GDH of L. lactis is more active in glutamate biosynthesis (mean activity ± standard deviation, 103 ± 14 nmol min^–1 mg protein^–1) than in glutamate catabolism (14 ± 2 nmol min^–1 mg protein^–1) according to the activity determined by a spectrometric method (7). Consequently, in addition to its role in glutamate catabolism (21), GDH of L. lactis plays a major role in glutamate biosynthesis, as do other GDHs of family I. Indeed, while the wild-type strain was capable of growing (mean growth rate ± standard deviation, 0.46 ± 0.07 h^–1) after a long latency phase (11 h) in a chemically defined medium (20) without glutamate or glutamine, no growth was observed for the gdh mutant strain.

The location of gdh on a large plasmid was demonstrated by the transfer of the gdh gene by electroporation in a plasmid-free strain. Indeed, the plasmid DNA of the gdh-negative mutant strain TIL487, which contains the pGhost9 (Ery^r) inserted in the gdh gene, was used to transform the plasmid-free strain MG1363. The transformants (Ery^r) were verified for the presence of the gdh gene disrupted with pGhost9 by PCR and for the presence of a plasmid of about 70 kb. The plasmid location of gdh in L. lactis is very original, since gdh has always been found in the chromosomes of bacteria, particularly in Lactobacillus plantarum strains, streptococci, and Listeria innocua strains in which gdh genes share the highest identities with the L. lactis gene.

gdh is part of a remnant transposon of the Tn3 family. The study of the genetic environment of gdh revealed that it is part of a structure clearly related to a complex transposon of the Tn3 family (Fig. 1a). The putative transposon (6.5 kb) is delimited by two imperfect inverted repeats of 28 bp with a GGGG terminal sequence characteristic of Tn3 transposons (19). It also includes a resolvase of 197 amino acids (ORF5) that contains the invariant serine in the N-terminal part and the C-terminal DNA binding domain characteristic of Tn3 resolvase (19) and is similar to the resolvase of Tn552 of Staphylococcus aureus (80% identity) (15). Moreover, we identified a putative resolution site sharing 70% identity with site I of Tn3 transposons (10, 15, 18). The putative transposon also encodes two proteins of 105 and 705 amino acids highly similar to known cadmium resistance proteins. In particular, they are 100% identical to functional CadA and CadC found in other L. lactis plasmids (AF243383 and U78967) (11, 14), in a Listeria innocua plasmid (NC_003383), and in the chromosomes of Listeria monocytogenes (EAL08508.1) and Streptococcus thermophilus (AJ315964) (16). They also exhibited 70 and 54% identities with CadA and CadC carried by the Tn3 transposon in L. monocytogenes (L28104) (9). Finally, ORF2 of the putative transposon encodes a transposase of 200 amino acids which is different from and much shorter than the 1,000-amino-acid transposases (TnpA) usually carried by Tn3 transposons (3). This transposase shared 98% identity with IS1216, widespread in composite transposons of gram-positive bacteria. This suggests that the original Tn3 transposase has been deleted and IS1216 inserted and that the putative transposon is not functional. Indeed, we have no evidence that this element transposes. Firstly, we did not identify direct repeats, which are normally the result of Tn3 transposon insertion, at each end (3, 19). Secondly, attempts to transpose by mating procedures resulted in the transfer of the whole plasmid (data not shown). Due to its location on a mobilizable or conjugative plasmid, the initial Tn3 transposon may have evolved into an immobilized element.

The characteristics of the remnant transposon suggest that it originates from a bacterium other than Lactococcus sp. Indeed, transposons of the Tn3 family, as well as the gdh gene, have never been reported in Lactococcus sp., while they are frequently found in other gram-positive bacteria. The percentages of identity of cadA/cadC and gdh genes with homologous genes of other bacteria suggest that this transposon could originate from Streptococcus strains, which are often found in the same biotopes (16, 17).

Interestingly, this remnant transposon carries, in addition to heavy metal resistance genes which are usually carried by Tn3 transposons, a functional gdh gene. Generally, auxiliary genes of transposons confer a selective advantage to the host. The gdh gene confers to the strains the ability to biosynthesize glutamate from -ketoglutarate and ammonia and, therefore, the ability to assimilate ammonia (2). This capacity could be an advantage in certain environments, such as vegetal environments which contain high levels of ammonia and are poor in glutamate and other free amino acids. However, L. lactis GDH seems also to be partly involved in cadmium resistance, since the gdh mutant is more sensitive to CdCl₂ than the wild-type strain (Table 2). This impact of GDH on cadmium resistance may be related to a better elimination of ammonia generated by cell protein degradation resulting from cell damages, as suggested for GDH in tomato plants under cadmium stress (5).

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TABLE 2. Percentages of growth inhibition of L. lactis NCDO 1867 and the gdh mutant TIL487 in the presence of CdCl2a

Nucleotide sequence accession number. The nucleotide sequence reported in this paper appears in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AY849557.

 
This work was funded by a Eureka research grant (E!2536). We are also grateful to Danisco and the Fromageries Bel for their financial support.

We would like to thank Véronique Monnet and Philippe Horvath for their critical reading of the manuscript.

 
* Corresponding author. Mailing address: INRA, Unité de Biochimie et Structure des Protéines, 78352 Jouy-en-Josas, France. Phone: 33 1 34 65 21 59. Fax: 33 1 34 65 21 63. E-mail: mireille.yvon{at}jouy.inra.fr.

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Journal of Bacteriology, July 2005, p. 5019-5022, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.5019-5022.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Tanous, C., Chambellon, E., Yvon, M. (2007). Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity. Microbiology 153: 1664-1675 [Abstract] [Full Text]  
• Tanous, C., Chambellon, E., Le Bars, D., Delespaul, G., Yvon, M. (2006). Glutamate Dehydrogenase Activity Can Be Transmitted Naturally to Lactococcus lactis Strains To Stimulate Amino Acid Conversion to Aroma Compounds. Appl. Environ. Microbiol. 72: 1402-1409 [Abstract] [Full Text]  

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