Yu Zhiliang, Wang Ju, Lin Jianxun, Zhao Minyan, Qiu Juanping
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, China.
Department of Electrical Engineering, Columbia University, New York 10027, United States of America.
PLoS One. 2015 Mar 27;10(3):e0122741. doi: 10.1371/journal.pone.0122741. eCollection 2015.
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.
细菌L-氨基酸氧化酶（LAAO）被认为在海洋生态位中发挥着重要的生物学和生态学作用，因此越来越受到关注以了解其产生的调控机制。在本研究中，我们利用转座子诱变技术研究了海洋细菌假交替单胞菌属Rf-1中参与LAAO产生的基因。在筛选的4000多个突变体中，有15个突变体的LAAO活性发生了显著变化。在12个突变体中证实了所需的转座子插入，并鉴定了其中被破坏的基因及其相应功能。对LAAO活性和lao基因表达的分析表明，GntR家族转录调节因子、甲基化酶、非核糖体肽合成酶、TonB依赖性血红素受体家族、Na+/H+反向转运蛋白和相关的亚砷酸盐通透酶、N-乙酰转移酶GCN5、酮酸还原异构酶和SAM依赖性甲基转移酶及其编码基因可能在转录、转录后、翻译和/或翻译后水平上参与上调或下调途径。将nhaD和sdmT基因分别互补到相应的LAAO活性丧失的突变体中。任一基因的互补都能恢复LAAO活性和lao基因表达，证明它们在LAAO生物合成中的调控作用。本研究首次深入了解了假交替单胞菌属Rf-1中LAAO产生的分子调控机制，这对于更好地理解LAAO的生物学和生态学作用具有重要意义。
