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Journal of Bacteriology, November 1998, p. 5896-5905, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Targeted Mutagenesis of
54 Activator
Proteins in Myxococcus xanthus
Lisa
Gorski and
Dale
Kaiser*
Department of Biochemistry and Department of
Developmental Biology, Stanford University School of Medicine,
Stanford, California 94305-5329
Received 19 December 1997/Accepted 9 September 1998
Myxococcus xanthus DNA segments related to the highly
conserved central sequence of
54 activator proteins have
been investigated. A genetic technique designed to inactivate a gene
that encodes such an activator by inserting a plasmid-borne internal
fragment of the putative gene has been tested. When the internal
fragment inserted by homologous recombination into the corresponding
chromosomal locus, the expected duplication of the gene was observed by
Southern hybridization. The single restriction fragment characteristic
of each segment was replaced in the insertion strains by two
hybridizing fragments, and one of these fragments hybridized with the
kanamycin resistance gene of the plasmid vector. The combined molecular
weights of the two fragments from the insertion strains were equal to
the molecular weight of the original fragment plus the expected
molecular weight contributed by the vector. In the duplication, one
copy is expected to have an N-terminal deletion and the other copy is
expected to have a C-terminal deletion. In most cases, the net result
should be loss of activator function. If an activator is essential for
vegetative growth, then it should not be possible to obtain the
insertion strain by plasmid integration. Indeed, integrants for three
of the segments were not obtained in repeated trials; however, a
plausible explanation for these results other than lethality can be
offered. Of the seven insertions validated by Southern hybridization,
four strains exhibited defects in the development of fruiting bodies.
One of these failed to develop in submerged culture, though it
developed normally on agar. The other three showed arrested development
of fruiting bodies, each at a morphologically different stage of
aggregation. One of the mutants may be defective in the reception
pathway of A-signal.
*
Corresponding author. Mailing address: Department of
Biochemistry and Department of Developmental Biology, Stanford
University School of Medicine, Stanford, CA 94305-5329. Phone: (650)
723-6165. Fax: (650) 725-7739. E-mail:
Luttman{at}cmgm.stanford.edu.
Journal of Bacteriology, November 1998, p. 5896-5905, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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