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Applied and Environmental Microbiology, October 2004, p. 6188-6196, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6188-6196.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Stratified Growth in Pseudomonas aeruginosa Biofilms

Erin Werner,1 Frank Roe,1 Amandine Bugnicourt,1 Michael J. Franklin,2 Arne Heydorn,3 Søren Molin,3 Betsey Pitts,1 and Philip S. Stewart4*

Center for Biofilm Engineering,1 Department of Chemical Engineering,4 Department of Microbiology, Montana State University—Bozeman, Bozeman, Montana,2 BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark3

Received 19 November 2003/ Accepted 27 May 2004

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 µm wide in colony biofilms and 30 µm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 µm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.

* Corresponding author. Mailing address: Department of Chemical Engineering, Montana State University—Bozeman, Bozeman, MT 59717-3980. Phone: (406) 994-2890. Fax: (406) 994-6098. E-mail: phil_s{at}erc.montana.edu.

Applied and Environmental Microbiology, October 2004, p. 6188-6196, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6188-6196.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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