Abstract
Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO.
Highlights
L-Amino acid oxidase (LAAO; EC 1.4.3.2) is usually a flavin adenine dinucleotide (FAD)containing homodimeric protein, which functions in stereospecific oxidative deamination of L-amino acids to the corresponding a-keto acids with release of NH4+ and H2O2 [1]
Altered LAAO activity was detected in 300 mutants,(data not shown) in which 15 mutants displayed significant altered or abolished LAAO activity (Fig. 1)
Pseudoalteromonas cells are advantageous in their contest for nutrients, space and colonization of surfaces, and are protected against predators grazing at surfaces [30, 31]
Summary
L-Amino acid oxidase (LAAO; EC 1.4.3.2) is usually a flavin adenine dinucleotide (FAD)containing homodimeric protein, which functions in stereospecific oxidative deamination of L-amino acids to the corresponding a-keto acids with release of NH4+ and H2O2 [1]. It plays important biological roles, such as apoptosis [2], cytotoxicity [3], edema [4], hemolysis, hemorrhage [5], inducing or inhibiting platelet aggregation [6], and parasite-killing and antimicrobial. The Genes Regulating LAAO Expression in Pseudoalteromonas sp.
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