Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation

Abstract

We report the cloning of lldA, a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH). Escherichia coli contains a single L-LDH gene (lldD) in the lld operon (previously lct). E. coli grown in complex media does not have L-LDH activity, but the activity is induced by growth in defined medium with L-lactate as the carbon source. In contrast, meningococci contain at least one L-LDH in addition to the lldA gene product. These enzymes are active in meningococci grown in complex media and are not dependent on growth in L-lactate. The predicted amino acid sequence of lldA is homologous to that of E. coli lldD and of other prokaryotic and eukaryotic flavin mononucleotide-containing enzymes that catalyze the oxidation of L-lactate and other small ␣-hydroxy acids. A mutant with a deletion in lldA was found to have reduced L-LDH activity. However, this mutant was able to grow on L-lactate, indicating that a second L-LDH must exist. Activity of the lldA enzyme was affected by growth conditions, being increased by growth on a defined medium with either L-lactate or pyruvate as the carbon source. For meningococci grown on a complex medium, activity of the lldA enzyme was increased by growth on plates or in well-aerated broth. A second L-lactate-oxidizing activity was seen in bacteria grown in poorly aerated broth. Neisseria gonorrhoeae contains a homolog of lldA. As for meningococci, mutation of the gonococcal lldA reduced L-LDH activity but did not affect growth on L-lactate.Neisseria meningitidis is able to grow well with either Dlactate or L-lactate as its principal carbon source (9). The D isomer of lactate is not produced by mammalian cells, but since it is produced from glucose by some lactic acid bacteria, it may be available to meningococci in the nasopharynx, the site of initial colonization. Lactate produced by the mammalian host is the L isomer and may be utilized by meningococci in the bloodstream or cerebrospinal fluid as well as in the nasopharynx. Study of the lactate-oxidizing enzymes and their regulation may provide insight into the physiology of meningococci at different stages of infection and the process of adaptation to various sites within the host.Several lactate-oxidizing enzymes of gram-negative bacteria have been characterized and shown to be membrane-associated flavoproteins that are components of the electron transport chain (18,19). The activity of these enzymes can be measured in intact bacteria and in some membrane fractions by lactate-dependent oxygen uptake or by lactate-dependent dye reduction. In general, these enzymes are isomer specific. However, in both Escherichia coli (29) and N. meningitidis (9), purified D-lactate dehydrogenase (D-LDH) has been found to have a low-affinity activity toward L-lactate in addition to a high-affinity D-lactate-oxidizing activity. We have previously described the purification of meningococcal D-LDH and the cloning of its gene, dld (9). Insertional inactivation of dld produced a mutant lacking D-LDH activity and unable to grow on D-lactate but wi...

Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization

“…The novel L-LDH (LldEFG) is present in Ͼ80 bacteria that do not contain the canonical LldD enzyme, including B. subtilis, for which we confirmed the role of yvfV-yvfW-yvbY genes in L-lactate oxidation. Although the simultaneous presence of lldEFG and lldD genes in Ϸ40 bacterial species including E. coli and Neisseria meningitidis is puzzling, the existence of residual L-LDH activity in ⌬lldD/⌬dld mutant of N. meningitidis (30) is consistent with the proposed L-LDH function of lldEFG operon (Table S1). Secondly, the lldEFG (NMB1436-38) operon of N. meningitidis was implicated in the increased resistance to H 2 O 2 (31).…”

A Widely Conserved Gene Cluster Required for Lactate Utilization inBacillus subtilisand Its Involvement in Biofilm Formation

“…For instance, orthologs of lldD were found in only two out of eight species of Streptococcus examined (22). Also, in at least one species, Neisseria meningitidis, which does contain an ortholog of the gene, a null mutation of lldD did not impair growth on L-lactate (18).…”

An NMR and enzyme study of the carbon metabolism of Neisseria meningitidis

“…This contradicts the work of Erwin & Gotschlich (1993), who showed that N. meningitidis was able to grow on lactate at least as well as on glucose. Growth on lactate requires the presence of LDH ; Erwin & Gotschlich (1996) demonstrated the presence of at least two -lactate-specific LDHs and one -lactatespecific LDH in N. meningitidis. This has recently been confirmed by the genome sequence (Tettelin et al, 2000).…”