Theresa Zwiener1, Frank Mickoleit1, Marina Dziuba1,2, Christian Rückert3, Tobias Busche3, Jörn Kalinowski3, Damien Faivre4,5, René Uebe1, Dirk Schüler6. 1. Department of Microbiology, University of Bayreuth, Bayreuth, Germany. 2. Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia. 3. Center for Biotechnology, University of Bielefeld, Bielefeld, Germany. 4. Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. 5. Aix-Marseille Université, CEA, CNRS, BIAM 13108, Saint Paul lez Durance, France. 6. Department of Microbiology, University of Bayreuth, Bayreuth, Germany. dirk.schueler@uni-bayreuth.de.
Abstract
BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.
BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.
Authors: Anna Pollithy; Tina Romer; Claus Lang; Frank D Müller; Jonas Helma; Heinrich Leonhardt; Ulrich Rothbauer; Dirk Schüler Journal: Appl Environ Microbiol Date: 2011-07-15 Impact factor: 4.792