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Journal of Bacteriology, December 2005, p. 8462-8469, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8462-8469.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Requirements for Potassium Ion-Dependent Colony Spreading in Bacillus subtilis

Rebecca F. Kinsinger,1 Daniel B. Kearns,2,{dagger} Marina Hale,1 and Ray Fall1*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215,1 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, Massachusetts 021382

Received 26 August 2005/ Accepted 3 October 2005

Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K+) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K+ requirement, directed mutagenesis of the higher-affinity K+ transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K+-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K+-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K+ ions, as well as growth conditions that promote sliding motility.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215. Phone: (303) 492-7914. Fax: (303) 492-1149. E-mail: fall{at}colorado.edu.

{dagger} Present address: Department of Biology, Indiana University, Bloomington, Indiana 47405.

Journal of Bacteriology, December 2005, p. 8462-8469, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8462-8469.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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