title: 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 alpha-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.
authors.affiliation: Aquatic Animal Health Laboratory, Centre for Marine Studies, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Roslina A
authors.initials: RA
authors.lastname: Nawawi
authors.forename: Justice C F
authors.initials: JC
authors.lastname: Baiano
authors.forename: E Charlotte E
authors.initials: EC
authors.lastname: Kvennefors
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Barnes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D002170:Canada; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D007557:Israel; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010473:Perciformes / Q000382:microbiology*; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D013785:Thailand; D014481:United States
chemical_list: D004269:DNA, Bacterial; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase