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. i...

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Bibliographic Details
Published in:	Applied and Environmental Microbiology Vol. 75; no. 9; pp. 2908 - 2919
Main Authors:	Nawawi, Roslina A, Baiano, Justice C.F, Kvennefors, E. Charlotte E, Barnes, Andrew C
Format:	Journal Article
Language:	English
Published:	Washington, DC American Society for Microbiology 01-05-2009 American Society for Microbiology (ASM)
Subjects:	Amino Acid Sequence Animals Australia Biological and medical sciences Biotechnology Canada Catalytic oxidation DNA, Bacterial - chemistry DNA, Bacterial - genetics E coli Enzymes Escherichia coli Escherichia coli - genetics Evolution, Molecular Evolutionary and Genomic Microbiology Fundamental and applied biological sciences. Psychology Israel Lates calcarifer Metabolism Methods. Procedures. Technologies Microbiology Mixed Function Oxygenases - genetics Mixed Function Oxygenases - metabolism Models, Molecular Molecular Sequence Data Molecular weight Mutagenesis, Insertional Perciformes - microbiology Protein engineering Protein Structure, Tertiary Repetitive Sequences, Amino Acid Sequence Alignment Sequence Analysis, DNA Streptococcus - enzymology Streptococcus - genetics Streptococcus - isolation & purification Streptococcus iniae Thailand United States Canada Israel Australia Thailand United States USA ISW, Indian Ocean, Mascarene Is., Reunion Directed molecular evolution Streptococcaceae Lactate 2-monooxygenase Vertebrata Lates calcarifer Streptococcus Enzyme Pisces Bacteria Micrococcales Isolate Oxidoreductases
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Corresponding author. Mailing address: Aquatic Animal Health Laboratory, Centre for Marine Studies, The University of Queensland, Brisbane, Queensland 4072, Australia. Phone: 61 7 3346 9416. Fax: 61 7 3365 4755. E-mail: a.barnes@uq.edu.au
ISSN:	0099-2240 1098-5336 1098-6596
DOI:	10.1128/AEM.02147-08