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Appl Environ Microbiol, April 1998, p. 1526-1531, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

Ching-Tsan Huang,1,dagger Karen D. Xu,1,2 Gordon A. McFeters,1,2 and Philip S. Stewart1,3,*

Center for Biofilm Engineering,1 Department of Microbiology,2 and Department of Chemical Engineering,3 Montana State University-Bozeman, Bozeman, Montana 59717-3980

Received 18 September 1997/Accepted 23 January 1998

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 µmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.

* Corresponding author. Mailing address: Center for Biofilm Engineering, 366 EPS Building, Montana State University, P.O. Box 173980, Bozeman, MT 59719-3980. Phone: (406) 994-2890. Fax: (406) 994-6098. E-mail: phil_s{at}erc.montana.edu.

dagger Present address: Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, Republic of China.




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