Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi (Lates calcarifer)

In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of α-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.Streptococcus iniae is a major pathogen of farmed fish, resulting in severe economic losses globally estimated at U.S. $150 million annually (27). S. iniae is essentially a blood pathogen, with infection resulting in a generalized septicemia and meningitis (1). During infection the pathogen avoids phagocytosis by means of an antiopsonic capsule (4, 18, 22) and by binding host serum components including immunoglobulins (3) and fibrinogen (2). Little is known, however, about the metabolism of S. iniae during infection although lactic acid bacteria may produce l-lactate from fermentation of glucose. In S. iniae a lactate oxidase gene, lctO, has been characterized previously (11). The product of the lctO gene in S. iniae is a flavin enzyme (l-lactate 2-monooxygenase, EC 1.13.12.4), which catalyzes the oxidation of lactate to pyruvate, coupled with reduction of O₂ to H₂O₂ (11). Lactate oxidase has been extensively characterized, both structurally and functionally, in the cold-water marine pathogen Aerococcus viridans (9, 30, 35, 36); thus, the catalytic activities of these enzymes are relatively well understood. In S. iniae, lactate can be utilized as an energy source through an aerobic but nonrespiratory mode of metabolism (11), a mechanism that is coupled to hydrogen peroxide production in Streptococcus pyogenes (26).Since the discovery of the lactate oxidase gene in S. iniae, its presence has been routinely used for PCR-based diagnosis, overcoming the lack of specificity of commercial biochemical diagnostic kits and other molecular methods. Confirmation of isolate identity as S. iniae by commercial bacterial identification kits is problematic because the biochemical profile is absent from databases supplied with the kits or because the databases are unable to identify atypical strains with confidence (25). Identification of isolates by molecular methods such as PCR is more reliable since isolates with atypical biochemical profiles can confidently be identified. PCR has been used to amplify sections of the 16S rRNA gene (37), the chaperonin HSP60 (12), and the 16S-23S rRNA gene intergenic spacer region (5) for identification of S. iniae. The development of the lactate oxidase gene (lctO) PCR assay by Mata et al. (21) reported that the primer pair LOX-1/LOX-2 could be used successfully to aid in the identification of S. iniae via the generation of a specific 870-bp product. Moreover, the LOX-1/LOX-2 primer pair overcame the problem of nonspecific amplification of Streptococcus difficilis that had previously been reported with the 16S rRNA gene primer pair described previously (21, 37).In Australia, S. iniae causes major economic loss in farmed barramundi (Lates calcarifer, Bloch) (1, 6). Barramundi, also known as Asian sea bass, are perciform euryhaline fish native to Australia and tropical southeast Asia. In Australia, barramundi have both cultural and commercial significance in terms of their iconic status among indigenous populations and the recent rapid growth of commercial farming. The value of intensive barramundi culture in Australia increased from Australian $15.5 million in 2004 to Australian $23.5 million in 2006 (34). There is also increasing farmed output of L. calcarifer in Malaysia, Indonesia, Taiwan, and Vietnam (33) and small to medium recirculating aquaculture ventures in the United States and United Kingdom using imported fingerlings.During routine diagnostic screening of S. iniae isolated throughout Australia from diseased barramundi, a novel variant of the lctO gene was found that resulted in amplicons of 920 bp following PCR using the LOX-1/LOX-2 primer pair. Isolates expressing the novel lactate oxidase gene were isolated only from a single site in the Northern Territory, Australia. In the present study, the novel lctO variant is investigated genetically and phenotypically in order to better understand how the larger gene product may have arisen from this single site.