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Integration into the Phage Attachment Site, attB, Impairs Multicellular Differentiation in Stigmatella aurantiaca

Georgia Institute of Technology, School of Biology, 310 Ferst Drive, Atlanta, Georgia 30332-0230,¹ National Research Council of Canada, Institute for Biological Sciences, M54, 1200 Montreal Road, Ottawa, Ontario, Canada K1W 1G7,² ZMBH, Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany³

Received 1 August 2005/ Accepted 23 November 2005

Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA^Asp and tRNA^Val, respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t_Mx8 terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t_Mx8 terminator sequence results in reduced levels of mature tRNA^Asp and tRNA^Val and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.