In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members

Abstract

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of anAcinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and themeta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied inP. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from thePu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pmpromoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction ofPm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

The Pseudomonas quinolone signal (PQS), and its precursor HHQ, modulate interspecies and interkingdom behaviour

“…Biofilms were grown at room-temperature in flow chambers with individual channel dimensions of 1 Â 4 Â 40 mm. The flow system was assembled and prepared as described previously by Moller et al (1998). Before inoculation, cells were pre-exposed to 10 mM HHQ or methanol control for 12 h, with a starting OD 600 nm of 0.05, shaking at 30 1C in TSB.…”

Chromosomally located gene fusions constructed in Acinetobacter sp. ADP1 for the detection of salicylate

“…Many studies have utilized recombinant methods where reporter genes are fused to the promoters of degradation genes (Applegate et al ., 1998;Willardson et al ., 1998;Stiner and Halverson, 2002). This approach facilitates the identification of user-defined compounds for a variety of matrices, such as biofilms (Moller et al ., 1998), contaminated water, plant and soil research (Belkin, 2003). King and colleagues (1990) constructed the first naphthalene and salicyclate responsive biosensor using a plasmid-based luxCDABE gene fusion derived from the NAH7 plasmid of Pseudomonas fluorescens .…”

Biofilm production by clinical strains ofAcinetobacter baumanniiisolated from patients hospitalized in two tertiary care hospitals

“…Furthermore, biofilm formation by Acinetobacter spp. may be influenced by its interaction within a biofilm with other bacterial species (Moller et al, 1998). A gene cup (chaperone-usher pathways) has been reported in Pseudomonas aeruginosa as an important factor in biofilm formation (Vallet et al, 2004).…”