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

Abstract

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 ␣-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.

Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

“…Strains were recovered from stock without defrosting and grown on Columbia agar base containing 5% defibrinated sheep blood (Oxoid, Australia) at 28°C for 24 to 48 h. Where isolates were obtained from farm cases through veterinarians, histopathology was performed by the veterinary laboratories using standard techniques. Identity of strains was confirmed by diagnostic PCR as previously described (20,27) and by sequencing the 16S rRNA gene (26,27).…”

Decreasing levels of the fish pathogen Streptococcus iniae following inoculation into the sludge digester of a zero-discharge recirculating aquaculture system (RAS)

“…PCR amplification was performed in a 50-µL reaction volume containing DNA template (80 ng) using the LOX 1/2 primer set, which specifically amplifies the gene encoding lactate oxidase, producing an 870-bp (Mata et al, 2004b) or 920-bp (Nawawi et al, 2009) …”

“…S. iniae has been reported to infect a large variety of cultured fish species under salinities ranging from fresh water to marine water (Salati, 2011;Nawawi et al, 2009). Several PCR-based assays have been developed for the detection of S. iniae (Berridge et al, 1998;Lau et al, 2004;Mata et al, 2004a,b;Zlotkin et al, 1998).…”

On the origin of Group B Streptococcus from disease outbreaks in wild marine fish in Australia

“…Lactate metabolism has previously been shown to be variable in fish pathogenic streptococci. In a case of Streptococcus iniae infection in barramundi (Lates calcarifer), molecular epidemiology revealed a novel lactate oxidase, which was confined to a single farm in Northern Territory with extreme tidal flow (Nawawi et al, 2009). The authors suggested an evolutionary advantage for these strains of S. iniae to have a more efficient enzyme under conditions where lactate concentration is high (Nawawi et al, 2009).…”

“…In a case of Streptococcus iniae infection in barramundi (Lates calcarifer), molecular epidemiology revealed a novel lactate oxidase, which was confined to a single farm in Northern Territory with extreme tidal flow (Nawawi et al, 2009). The authors suggested an evolutionary advantage for these strains of S. iniae to have a more efficient enzyme under conditions where lactate concentration is high (Nawawi et al, 2009). In common with S. iniae and other fish pathogenic steptococci, GBS are essentially systemic blood pathogens highly adapted to survive and proliferate within the circulatory system prior to colonisation of the central nervous system (CNS).…”