Analysis of Extracellular Alginate Lyase (alyA) Expression and its Regulatory Region in a Marine Bacterial Strain, Pseudoalteromonas atlantica AR06, Using a gfp Gene Reporter System

Ryoji Matsushima, Ryuichi Watanabe, Masataka Tsuda, Toshiyuki Suzuki

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Extracellular alginate lyase (alyA) has an important role in the use of alginate in the marine bacterial strain Pseudoalteromonas atlantica AR06. Green fluorescent protein (GFP) is a convenient, useful tool for visualization of gene expression, and here we introduced the gfp gene at the end of alyA by homologous recombination in AR06. The gfp gene was expressed as a polycistronic transcript in reporter strain ARAG (AR06 alyA-gfp). The analysis of GFP fluorescence of ARAG in minimal medium containing a carbon source (sucrose, mannitol, glucose, or glucuronate) revealed that the alyA was induced by alginate and regulated by carbon catabolite repression by the coexistence of each carbon substrate (sucrose, mannitol, and glucose). The transcription start site of alyA was identified 192 bp upstream from translation start site of alyA and the 10-bp sequence was a palindrome in which the transcription start site [G] was at the end. Thirteen kinds of plasmids were constructed for promoter analysis of alyA, which contains between 215 and 618 bp of upstream sequence from the translation start and gfp. Longer sequences than 339 bp were capable to increase the GFP fluorescence responding to alginate in the homologous recombination alyA deletion mutant strain ARAΔ (AR06 ΔalyA). The 339-bp upstream sequence was proved sufficiency for reproduction of the catabolite repression of alyA. These results indicate that (a) transcription of alyA is probably regulated by substrate recognition systems driven by multiple factors and (b) regulation of alyA requires a cis element on the upstream region of alyA.

    Original languageEnglish
    Pages (from-to)349-356
    Number of pages8
    JournalMarine Biotechnology
    Volume15
    Issue number3
    DOIs
    Publication statusPublished - 2013 Jan 1

    Keywords

    • Alginate lyase
    • Catabolite repression
    • Extracellular enzyme
    • Pseudoalteromonas atlantica
    • Regulation of expression
    • gfp reporter gene

    ASJC Scopus subject areas

    • Biotechnology
    • Aquatic Science

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