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

Abstract

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.