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Tes in aluminum chambers (28). Chambers were filled with 5 ml of 0 TSB
Tes in aluminum chambers (28). Chambers have been filled with 5 ml of 0 TSB and 250 l of an overnight get E-982 culture and incubated for 24 h without medium flow to let attachment. Reactors were then set at a 0angle, and TSB was dripped over the plate at 50 ml h. Biofilms were harvested into 0 ml of PBS and homogenized, and colony morphology was scored. ,000 colonies were examined at every single time point in Fig. b and at five days for Fig. 2 b and d . The amount of generations that take place during development in drip flow reactors is difficult to specifically identify, since cells are continuously lost in the effluent. Having said that, even if the number of lost cells is assumed to exceed the quantity in the biofilm by 00fold, 7 generations would have occurred in the course of biofilm development. Variant colonies had been developed in related abundance in drip flow reactors (28), tube reactors (29), and following five days of biofilm development in 96well microtiter dishes with day-to-day media alterations. Variants appeared at low numbers inside the rotating disk reactor (30) and in continuous culture flow cells (30). In some experiments, PA0 was tagged having a selectable marker (tetracycline resistance) around the chromosome (by using miniCTX). Variants made by the tagged strain contained this marker, confirming that variants weren’t contaminants. Flow cell experiments have been performed as previously described (30). The rotating disk reactor (30) was utilised for generating biofilmsBoles et al.MICROBIOLOGYFig. 2. Function of recA in biofilminduced diversity. (a) Micrographs of colonies created by 5dayold wildtype and recA biofilms. (b) Proportion of bacteria with variant colony morphology arising from biofilms right after 5 days of development. Biofilms were grown with isogenic wildtype, recA , recA complemented, and dinP strains. Information are suggests of 3 experiments; error bars show SEM. (c) Variance in swimming distance induced by biofilm growth. The swimming capability of bacteria from typical colonies from biofilms was compared using the capability of bacteria from the inoculum. The biofilminduced variation essential recA. Information will be the variance of 50 randomly picked wildtype and recA colonies. (d) Generation of auxotrophs by biofilms. Information are means of four experiments. Error bars show SEM. (e) Generation of strains overproducing pyomelanin by biofilms. Agar plates show pyomelaninoverproducing colonies from wildtype but not from recA biofilms. Information in the graph are the imply of 4 experiments; error bars show SEM.wrinkly colonies switched morphotypes after overnight passaging. A prime candidate for mediating such variation is RecA, which can create genetic changes by recombination (three) and by inducing errorprone DNA polymerases as part of the bacterial strain response (SOS response) (32). Inactivation of recA considerably decreased biofilminduced colony variation, and this defect was complemented by chromosomally inserted recA (Fig. 2 a and b). In contrast, recA mutation had no influence on the low number of variants made by prolonged planktonic growth, suggesting that these variants arise by a distinct mechanism (data not shown). Mutation of dinP, the only errorprone polymerase gene so far identified in P. aeruginosa (33), didn’t reduce biofilmassociated variation, suggesting that recA acts by a recombination mechanism (Fig. 2b).6632 pnas.org cgi doi 0.073 pnas.The involvement of RecA, which could mediate genetic PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24566461 transform anywhere inside the chromosome, led us to hypothesize that biofilmgenerated diversity could extend to other func.

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