Rank) named in the Materials and Methods. We identified four genes strongly up-regulated by iron limitation [9] and compared their expression between

drip-flow biofilm, three standard comparison data sets [15, 18, 20], and a positive control in which the bacterial culture was deliberately iron-limited (Figure 3C) [22]. All four genes were highly ranked in the iron-limited positive control. The expression rank of these four genes in the drip flow biofilm was consistently lower in comparison to the reference data sets. These data suggest that bacteria

in the drip-flow find more biofilm as grown in this study did not experience limitation for iron. The concentration of iron in the medium, added in the form see more of ferrous ammonium sulfate, was 1.5 μM. From the literature, we identified four genes that are induced by the presence of nitrate in the medium, either under aerobic or anaerobic conditions [25]. The expression rank of these genes is compared in Figure 3D. The rank for the drip-flow biofilm for all four genes was higher than the three standard comparison data sets and lower than a nitrate-amended positive control. The medium used to grow the biofilm did not contain added nitrate. Figure 3E

presents a comparison of gene rank for four growth phase responsive genes. Three genes associated with stationary phase, cspD, rmf, and rpoS, [26–29] were very highly ranked in both our drip flow biofilm and the comparison data set that was sampled in stationary phase. The fourth gene whose expression is associated with early exponential phase growth, fis, [26, 29] showed the inverse ranking. Phosphoribosylglycinamide formyltransferase The biofilm and stationary phase culture had similar ranks for the fis gene, while the two systems in which bacteria were rapidly growing had much higher ranks. These comparisons suggest that many of the cells in the biofilm exhibit stationary phase character. To further explore the potential relationship between transcript levels for these genes and growth state, we plotted gene rank for fis and rpoS as a function of specific growth rate, where a growth rate was reported or optical density versus time data permitted a quantitative estimation (not shown).