BACKGROUND
the physiological activities of bacteria growing in biofilms are difficult to divine, because these activities are diverse, change with time as the biofilm develops, and are subject to extreme micro scale spatial heterogeneity  <cit> .
it is also clear that the metabolism and activities of a particular biofilm will be shaped by the specific chemical and physical environment in which it grows.
these realities make it difficult to develop a consensus picture of the physiology of the biofilm state as there is so little overlap in the lists of genes differentially expressed between the planktonic and biofilm states of pseudomonas aeruginosa prepared by different experimenters  <cit> .
however, there are biofilm physiological traits, such as antimicrobial tolerance  <cit>  and reduced growth rate  <cit> , for which there is considerable consensus.
these robust phenotypes, with their functional and evolutionary importance, should have discernable biochemical and genetic bases.
we sought to understand these phenotypes with an unconventional interpretation of transcriptional profiling studies.
conventional interpretations of transcriptional profiling studies compare two paired data sets that differ in a single controlled variable .
in this study, we have obtained the transcriptome for a single biofilm specimen, ranked the transcripts based on the signal intensity to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and a number of published data sets.
for example, if we wish to discern whether the biofilm is responding to iron limitation, we first identify a set of genes that are up-regulated in response to iron deprivation .
the rank of each of these transcripts in the biofilm data set is then compared to transcript ranks for the same genes in data sets collected from both rapidly growing and deliberately iron-starved cultures.
in this way it becomes possible to evaluate physiological activities in the biofilm rather than just documenting differences between the biofilm and a reference state.
in the experiments reported here, rna was extracted from an entire, homogenized biofilm specimen.
an obvious concern with this approach is that it neglects the inherent biological heterogeneity of the biofilm  <cit> .
we would like to address this concern upfront with two points.
first, just because a population is heterogeneous does not mean that measurements of population averages are invalid.
population averages are very widely and informatively used in biology.
second, we suggest that even the concept of an average may not be appropriate in this case.
the current conceptual model of p. aeruginosa drip-flow biofilms is that they consist of two distinct populations: an aerobic, metabolically active upper layer and a lower, and larger, layer consisting of inactive cells containing very low levels of mrna  <cit> .
because the inactive cells contain so little rna, this majority is expected to be essentially invisible on the microarray.
from this perspective, the transcriptomes reported here may best be thought of as reflecting the properties of the transcriptionally-active subpopulation rather than the average behavior of the entire population.
these concepts are elaborated on in the results and discussion.
RESULTS
three day old drip flow biofilms of p. aeruginosa were characterized with respect to antibiotic tolerance, oxygen availability, and microscale patterns of protein synthetic activity.
these biofilms contained  <dig>  ±  <dig>  cfu cm- <dig>
reduced antibiotic susceptibility of biofilm bacteria
p. aeruginosa cells grown in biofilms were protected from killing by tobramycin and ciprofloxacin, in comparison to actively growing planktonic bacteria.
both antibiotics rapidly and effectively reduced viable cell numbers in an aerobic, planktonic culture.
after  <dig> h of treatment with  <dig> μg ml- <dig> tobramycin or  <dig>  μg ml- <dig> ciprofloxacin, planktonic log reductions measured were  <dig>  ±  <dig>   and  <dig>  ±  <dig>   for tobramycin and ciprofloxacin, respectively.
in contrast, neither antibiotic was very effective against biofilms of p. aeruginosa.
after  <dig> h exposure to antibiotic in continuously flowing medium, the log reductions in viable cell numbers were  <dig>  ±  <dig>   and  <dig>  ±  <dig>   for tobramycin and ciprofloxacin, respectively.
the log reductions measured for biofilm bacteria were 23% and 28% of the planktonic log reductions for the two antibiotics, respectively.
reduced killing of the biofilm in comparison to planktonic cells was statistically significant .
these data demonstrate that these drip-flow biofilms exhibit the antibiotic-tolerant phenotype that is considered a hallmark of the biofilm mode of growth.
when biofilm bacteria were dispersed prior to antibiotic exposure, they again became susceptible to the antibiotics.
log reductions measured for biofilm cells re-suspended into aerated medium and treated with tobramycin or ciprofloxacin for  <dig> h were  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
this degree of killing was the same as that measured for planktonic bacteria, indicating that susceptibility was rapidly and fully restored upon dispersal of cells from the biofilm.
low oxygen concentrations in biofilms
an oxygen microelectrode was used to demonstrate the presence of oxygen concentration gradients in this system .
the oxygen concentration in the flowing fluid above the biofilm was approximately  <dig> mg l- <dig>  oxygen concentration decreased to  <dig>  mg l- <dig> or less inside the biofilm.
a similar profile was measured in a duplicate experiment.
the oxygen concentrations shown in figure 1a may not define the lower bound of oxygen concentration inside the biofilm because the electrode was positioned only partway into the biofilm, to avoid electrode breakage.
the utilization of oxygen by bacteria is coupled to their simultaneous uptake and oxidation of a carbon source.
to investigate this coupling, the oxygen microelectrode was positioned at a depth part way into the biofilm where the oxygen concentration was less than  <dig>  mg l- <dig> .
the medium flowing over the biofilm was then changed from complete pbm to pbm lacking glucose and ammonium sulfate.
within a few minutes after switching to this starvation medium, the oxygen concentration in the biofilm abruptly rose to approximately  <dig> mg l- <dig>  when the complete medium containing glucose and the nitrogen source was restored, the oxygen concentration quickly dropped back to its previous low level.
upon switching once again to the starvation medium, the oxygen concentration again returned to the higher level.
restoring the complete medium again caused the oxygen concentration to fall.
the same behavior was observed in a duplicate experiment.
these experiments show that oxygen and glucose utilization are interdependent.
heterogeneous patterns of protein synthetic activity in biofilms
the induction of a gfp has been used to reveal regions of active protein synthesis in biofilms  <cit> .
when this technique was applied to p. aeruginosa biofilms grown in drip-flow reactors, a stratified pattern of activity was observed .
expression of gfp was localized in a band at the top of the biofilm adjacent to the source of nutrients and oxygen.
the dimension of the gfp-expressing zone averaged  <dig> ±  <dig> μm .
the average thickness of the entire biofilm was  <dig> ±  <dig> μm  .
while the predominant zone of activity was along the air interface , gfp fluorescence was occasionally observed in thin strata in the interior and even at the bottom of the biofilm .
the observation of fluorescent gfp at the bottom of the biofilm argues against the interpretation that these patterns are an artifact of incomplete iptg penetration.
in prior studies, the facile penetration of iptg throughout p. aeruginosa biofilms has been demonstrated  <cit> .
*the thickness of the area of gfp expression as well as the overall thickness of the biofilm was measured  <dig> times.
measurement of pseudomonas aeruginosa pao <dig> carrying plasmid pab <dig> containing an iptg-inducible gfp with and without iptg are compared with p. aeruginosa carrying plasmid pmf <dig> lacking gfp.
†the uncertainties indicated are standard deviations.
transcriptional profiling of biofilms - nutritional and growth status
the rna was extracted from 3-day old p. aeruginosa drip-flow reactor grown biofilms and subjected to global transcriptional profiling.
these microarray data have been deposited to gene expression omnibus  accession gse <dig>  we compared the expression of individual genes or groups of genes indicative of specific physiological activities by analyzing the rank of the selected transcript in the drip flow biofilm transcriptome across  <dig> different experiments and planktonic comparator transcriptomes  <cit>  listed in table  <dig> and additional file  <dig>
● 2% o <dig> - gold
●  <dig> % o <dig> - red
● od ~  <dig>  - white
additional file  <dig> contains a version of this table that includes colored symbols for visual identification of the symbols used in figures  <dig>   <dig>  and  <dig>
when grown on glucose, p. aeruginosa expresses an outer membrane protein, oprb, which is involved in the uptake of sugars  <cit> .
figure 3a compares the rank of the oprb  transcript in several data sets, including our drip-flow reactor biofilm.
this gene is highly expressed in the biofilm  and also highly expressed in one other transcriptome from a study  <cit>  in which the bacteria were grown on a glucose-minimal medium .
the rank of the pa <dig> transcript is lower in cells grown on minimal media supplemented with acetate or citrate, lower still on complex media such as lb or bhi, and lowest of all on a minimal amino acid medium.
the straightforward interpretation of this comparison is that the strong expression of oprb in the drip-flow biofilm implies the presence of glucose in the system.
since the medium used in this study contained glucose as the sole carbon and energy source, these results illustrate the face validity of our approach.
to further demonstrate the potential to diagnose metabolic activities from transcript ranks, we conducted a more comprehensive analysis of relationship between the presence or absence of glucose and the ranks of selected gene transcripts.
fifty eight samples were identified in which no glucose was present in the medium.
eleven samples were identified in which glucose was the sole or predominant carbon source.
differences in the ranks of pairs of genes, identified by inspection, were found to discriminate the glucose-present and glucose-absent data sets .
the drip-flow biofilm data group with the glucose-present comparators, as expected.
the six glucose-absent points that overlap with the glucose-present cluster are from a single investigation in which glycerol was the predominant carbon source.
the extensive commonality of pathways for catabolism of glucose and glycerol may explain this overlap.
alvarez-ortega and harwood  <cit>  identified genes induced under conditions of low oxygen concentration.
from their results, we identified a subset of seven genes that were particularly strongly induced by low oxygen and whose transcript rank increased monotonically with decreasing oxygen concentration.
figure 3b compares the rank for these seven genes between drip-flow biofilms in this study and the alvarez-ortega and harwood  <cit>  data.
the rankings of the transcripts for the biofilm were consistent with low oxygen concentrations for six of seven transcripts.
this comparison indicates that the biofilm experienced oxygen limitation.
a recent investigation reported  <dig> genes induced by transferring p. aeruginosa from aerobic to anaerobic conditions  <cit> .
thirty-five genes appearing on this list also appear in table  <dig>  a significant overlap .
this overlap reinforces the interpretation of an oxygen-limited physiology of the drip-flow biofilm population.
*average rank in drip flow biofilms  and three comparator microarray data sets  named in the materials and methods.
we identified four genes strongly up-regulated by iron limitation  <cit>  and compared their expression between drip-flow biofilm, three standard comparison data sets  <cit> , and a positive control in which the bacterial culture was deliberately iron-limited   <cit> .
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 biofilm as grown in this study did not experience limitation for iron.
the concentration of iron in the medium, added in the form of ferrous ammonium sulfate, was  <dig>  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  <cit> .
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.
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 .
though the data is somewhat noisy, it is clear that rpos gene rank decreases with increasing growth rate.
the rank of the fis gene is relatively constant above a specific growth rate of approximately  <dig>  h- <dig>  and decreases below this growth rate.
the difference in gene rank between rpos and fis increases with specific growth rate .
this analysis points to the possibility of inferring growth rate from transcriptomic data.
for example, in the drip-flow biofilm the difference in rpos and fis gene rank was - <dig> ±  <dig> .
from figure 3f, this difference corresponds to a specific growth rate of approximately  <dig>  h- <dig>  taking the results of figures 3e and 3f together, it appears as if bacteria in the biofilm were growing very slowly.
oxygen availability limits growth in biofilm
in this experimental system, two potentially limiting substrates for bacterial growth were glucose and oxygen.
the composition of the medium used ensured excess nitrogen, phosphorous, sulfur, and other elemental requirements.
for example, the molar ratio of ammonium to glucose carbon was  <dig> , which provided approximately ten-fold excess nitrogen.
there is no basis for anticipating that glucose was limiting in any part of the biofilms that were grown in this study.
this can best be appreciated by a simple calculation.
as derived by williamson and mccarty  <cit> , the metabolic substrate that will first be depleted in a biofilm can be determined by calculating the dimensionless quantity degsg/deo2so2ygo <dig>  this ratio is a measure of the relative diffusive fluxes of glucose and oxygen into the biofilm, where de denotes the effective diffusion coefficient of the respective substrate in the biofilm, s denotes the bulk fluid concentration of the respective substrate, and ygo <dig> is the stoichiometric coefficient relating the consumption of glucose and oxygen.
in the present case, we take the effective diffusion coefficients of oxygen and glucose to be  <dig>  × 10- <dig> cm <dig> s- <dig> and  <dig>  × 10- <dig> cm <dig> s- <dig>  respectively  <cit> .
the yield coefficient has been carefully measured, in biofilms of this bacterium, and is  <dig>  g glucose per g oxygen  <cit> .
with the bulk fluid concentration of glucose at  <dig> mg l- <dig> and the bulk fluid concentration of oxygen at  <dig> mg l- <dig>  the quantity given by the ratio above has a value of  <dig> .
this value being greater than  <dig> means that glucose is provided in excess and that oxygen is the limiting substrate.
this interpretation is consistent with the strong expression of oprb in biofilm specimens  and the analysis shown in figure 4a.
microelectrode measurements provided direct chemical evidence of reduced oxygen availability .
steep oxygen concentration gradients were measured in the vicinity of the biofilm, with parts of the biofilm experiencing oxygen concentrations of  <dig>  mg l- <dig> or less .
these measurements are concordant with the transcriptomic analysis of biofilm bacteria that provides direct biological evidence of oxygen limitation .
the following describes our physical understanding of the concentration gradients in this particular biofilm system.
in the aerobic layer, both oxygen and glucose are consumed.
once the oxygen has been depleted, utilization of glucose stops.
abundant glucose, approximately  <dig> mg l- <dig>  is predicted to be available at the bottom of the biofilms studied in this investigation.
we note that p. aeruginosa is unable to ferment glucose and no arginine was present, precluding fermentative growth  <cit> .
no alternative electron acceptor, such as nitrate, was added to the medium used in these studies.
therefore, growth by denitrification was also precluded.
the expression of genes associated with denitrification in the biofilm  may have been a response to oxygen limitation.
in summary, once oxygen was depleted in this system, one would predict that growth would cease.
biofilm harbors slowly-growing or non-growing bacteria
we hypothesize that oxygen limitation in p. aeruginosa drip-flow biofilms resulted in slow growth or lack of growth of many of the bacteria in the biofilm.
the expression of an inducible gfp was focused in a sharply demarcated band immediately adjacent to the oxygen source.
this band represented approximately 38% of the biofilm, indicating that as much as 62% of the biofilm could be anoxic and anabolically inactive.
because alternative fermentable substrates or electron acceptors were absent, oxygen limitation is expected to be sufficient to lead to arrested growth in anoxic regions of the biofilm.
this interpretation is qualitatively consistent with previous studies of oxygen availability and spatial patterns of physiological activity in some other p. aeruginosa biofilms  <cit> .
transcriptomic data show that the biofilm exhibited stationary phase character .
this is evident in the pronounced expression of rmf, a stationary-phase inhibitor of ribosome function  <cit> , cspd, a stationary-phase inhibitor of replication  <cit> , and rpos, a stationary-phase sigma factor <cit> .
in a previous investigation, we independently reported the elevated expression of rpos in p. aeruginosa biofilms  <cit> .
a gene associated with early exponential phase growth, fis, was expressed at relatively low levels, consistent with very slow growth.
our estimate of an average specific growth rate of  <dig>  h- <dig> is approximately ten percent of the specific growth rate of p. aeruginosa in this medium of  <dig>  h- <dig>  colony biofilms of a mucoid strain of p. aeruginosa had a reported specific growth rate that was two percent of the maximum specific growth rate in that system  <cit> .
here we consider two alternative conceptual models for growth and activity within the biofilm.
these models attempt to address the microscale heterogeneity that is obviously present and which the transcriptional analysis is incapable of resolving.
both of these conceptual models view the biofilm as having two layers of differing growth rates.
in the first model, an aerobic layer representing the upper 40% of the biofilm grows at  <dig>  h- <dig> while the bottom layer has a specific growth rate of zero.
the population average growth rate  would be  <dig>  h- <dig>  in the second model, an aerobic layer representing the upper 40% of the biofilm grows at  <dig>  h- <dig> while the bottom layer has a specific growth rate of zero.
the population average growth rate would be  <dig>  h- <dig>  we believe that the second model is the more realistic.
the transcriptome obtained in this study does not represent the average behavior of the biofilm.
it reflects rather the activities of the transcriptionally-active subpopulation, which is the aerobic upper layer.
localized gene expression measurements performed by microdissection and pcr show that the rpos transcript is more abundant in the upper layer of the biofilm compared to the middle or bottom layers  <cit> .
this confirms that the "active" cells in the biofilm are in fact in a stationary phase-like state and that the inactive cells are depleted of most mrna.
transcriptional profiling of biofilms - stress responses and quorum sensing
the same approach of comparing ranks of selected genes indicative of specific physiological activities was applied to examine oxidative stress, copper stress, efflux pump activities, and quorum sensing in drip-flow biofilms.
the expression levels, as quantified by transcript rank, of five genes associated with oxidative stress  <cit>  were not in general elevated in reference to the comparators .
the only possible exception, a putative glutathione peroxidase , is difficult to interpret clearly since this gene is also induced under copper stress .
thus we conclude that no unusual oxidative stress is occurring.
we noticed that several genes associated with copper stress, as reported by teitzel et al.
<cit> , were highly expressed in drip-flow biofilms .
the nominal copper concentration in pbm is  <dig>  μm, which is much less than the  <dig> mm teitzel et al.
used.
we identified another data set, that of love and co-workers  <cit> , in which an acetate minimal medium was supplemented with trace elements including cu at a final concentration of  <dig>  μm.
copper stress genes were highly expressed in this case as well.
we therefore suggest that micro molar concentrations of copper are sufficient to induce a copper stress response when p. aeruginosa is grown in minimal media.
efflux pumps were not up-regulated in p. aeruginosa biofilms in general .
the one instance of obvious high level expression, pa <dig>  is associated with copper stress  <cit> .
three different laboratories have published data on the set of genes regulated by homoserine lactone quorum sensing in p. aeruginosa  <cit> .
we selected a consensus subset of seven of these genes that are more highly expressed under conditions of active quorum sensing and compared the drip-flow biofilm transcriptome to the standard reference data sets .
the biofilm rank was relatively low for all but one of these genes, pa <dig> or rsal.
though rsal is itself quorum sensing activated, the rsal gene product is a negative regulator that represses many other quorum-sensing activated genes  <cit> .
thus the high level expression of rsal may be consistent with repression of many of the other genes shown in figure 5d.
these data show, surprisingly, that homoserine lactone quorum sensing is not active in these drip-flow biofilms.
to further demonstrate the potential for differences in transcript ranks to serve as indices of specific physiological activities, homoserine lactone quorum sensing was examined in a fashion analogous to that described above for glucose  and growth rate .
the eight quorum sensing positive samples plotted in figure 4b are planktonic cultures with optical densities greater than  <dig> .
the  <dig> quorum sensing negative samples in this figure are either from quorum sensing deficient mutants or planktonic cultures of very low optical density.
the drip-flow biofilm data points clearly do not group with quorum sensing positive benchmarks .
quorum sensing has been associated with biofilm development in p. aeruginosa by many investigators  <cit> , so our finding that this communication system is silent in three-day old drip-flow biofilms seems at odds with the literature.
this result is internally consistent, however, with the elevated expression of two negative regulators of quorum sensing, rsal  <cit>  and algr, another repressor of quorum sensing  <cit> .
the algr gene transcript ranked  <dig> in drip-flow biofilms and  <dig> in the same comparator data sets used to compile table  <dig>  we speculate that quorum sensing may have been active at an earlier stage of biofilm formation in the drip-flow reactor.
transcriptional profiling - biofilm extracellular matrix genes
extracellular polysaccharides and proteins are common constituents of the biofilm matrix.
there are four putative or known polysaccharide biosynthetic operons in p. aeruginosa  <cit> .
both pel and psl genes were expressed in the biofilm while alginate biosynthetic genes were not.
only the pel genes were up-regulated in biofilms compared to the three planktonic controls .
the low level of expression of algd in the drip-flow biofilm is consistent with prior reports that alginate is not an important constituent of pao <dig> biofilms  <cit> .
our transcriptomic data suggest that the pel and psl polysaccharides may be important constituents of the extracellular matrix of drip-flow biofilms while alginate is unimportant .
the rank of the cdra gene, a recently described adhesin that interacts with the psl polysaccharide  <cit> , was not much different in drip-flow biofilms and planktonic comparators.
genes associated with the elaboration of type iv pili were strongly expressed in drip-flow biofilms .
this has led us to speculate that these extracellular proteinaceous appendages contribute to the mechanical stability of the biofilm rather than motility, perhaps by binding to extracellular dna  <cit> .
transcriptional profiling - independent identification of upregulated genes in biofilms
all of the preceding analyses were predicated using a priori identification of a set of genes associated with discrete physiological conditions.
the comparison of transcript ranks can also be used to identify genes that are differentially regulated between the drip-flow biofilm data set and planktonic comparator data sets.
table  <dig> reports the  <dig> genes that ranked more highly in the drip-flow biofilm than in the comparator data set, by fold-changes in rank ranging from  <dig> to more than  <dig>  some of the salient features of this list are genes associated with oxygen limitation , copper stress , bacteriophage pf <dig> , denitrification , ethanol metabolism , and three genes involved in type iv fimbrial biogenesis.
seven of the genes listed in table  <dig>  appear on the consensus list of gene transcripts upregulated in p. aeruginosa biofilms reported by patell et al  <cit> .
biological basis of biofilm antibiotic tolerance
p. aeruginosa strain pao <dig> formed biofilms in the drip-flow reactor that were poorly killed by tobramycin or ciprofloxacin.
this result is concordant with many previous investigations of antibiotic susceptibility of p. aeruginosa biofilms developed in other in vitro systems  <cit> .
a plausible and long-standing explanation for reduced antibiotic susceptibility in biofilms is that nutrient limitation leads to slow growth or stationary phase existence for many of the cells in a biofilm, reducing their antimicrobial susceptibility  <cit> .
this mechanism is consistent with all of our data.
multiple lines of evidence support oxygen limitation and arrested growth in drip-flow biofilms: oxygen concentration gradients , expression of genes associated with oxygen limitation  and stationary phase existence , and stratified patterns of protein synthetic activity .
in a previous study using a different in vitro biofilm model, we reported that oxygen limitation could account for  <dig> percent or more of the protection from six antibiotics observed in p. aeruginosa colony biofilms  <cit> .
a recent report showed that ciprofloxacin and tetracycline preferentially killed the metabolically active subpopulation in p. aeruginosa biofilms  <cit> .
oxygen limitation is known to occur in vivo in cystic fibrosis patients  <cit> .
further, molecular biological evidence suggests that p. aeruginosa in the cystic fibrosis lung experiences anaerobic conditions  <cit> .
in an investigation of in situ growth rates of p. aeruginosa obtained from chronic lung infections, approximately 11% of cells were determined to be in a non-growing stationary-phase based on their ribosome content  <cit> .
the average specific growth rate of the growing bacterial cells was  <dig>  h- <dig>  this shows that a non-growing population may be relevant in vivo, though it suggests that the population of bacteria in the infected lung were overall more active than we describe here for drip-flow biofilms.
heterogeneity within the biofilm
here we remark on the "averaging" that occurs when the entire biofilm is mashed up and extracted rna is analyzed.
this method mixes together the rna from transcriptionally active cells in the aerobic upper layer of the biofilm with rna from inactive bacteria in the lower layers of the biofilm.
the result is not a simple average of the activities of the two layers because there is so much less mrna in the inactive bacteria.
indeed, the inactive bacteria may contribute little to the overall microarray signal.
for this reason, the transcriptome that has been examined in this work may best be thought of as representing the transcriptionally-active supopulation of bacteria rather than an average of the entire biofilm population.
a recently described laser capture microdissection technique provides a direct experimental approach for quantifying the amount of specific rna sequences in distinct regions of the biofilm  <cit> .
this method begins with cryoembedding an intact biofilm and preparing frozen cross sections.
small user-defined areas of the cross section can be physically removed and amplified by pcr to detect specific transcripts.
application of this approach to drip-flow p. aeruginosa biofilms has revealed that the upper layer of the biofilm is enriched in mrna compared to the lower layers  <cit> .
for example, in drip-flow biofilms the number of rna copies of the housekeeping gene acpp was approximately  <dig> times smaller at the bottom of the biofilm compared to the top  <cit> .
for the rhlr transcript the difference between top and bottom was approximately 30-fold  <cit> .
utility of ranked transcriptome analysis
conventional transcriptional profiling is applied to paired samples and allows for the discovery of genes that are differentially regulated between the two samples.
for example, comparing the transcriptomes of samples grown at two different temperatures or in the presence and absence of a signaling molecule leads directly to the identification of genes regulated by temperature or by the specific signal chemistry.
this is the usual usage of transcriptional profiling technology.
in this investigation, we sought to use transcriptional profiling to provide insight about the physiological activities of a single sample.
rather than chronicling the differences between two conditions , we wanted to ask and answer the question "what is the transcriptionally active biofilm cell doing?"
to do this, we ranked the transcriptome, which makes manifest the priorities of the cell, at least at the transcriptional level.
to interpret this ladder of genes, we independently identified from the literature sets of genes as markers of particular physiological activities and then compared the ranks of these genes to the ranks in several planktonic comparator transcriptomes.
as the public database of transcriptional data expands, this approach becomes more and more feasible and powerful.
our effort is a preliminary one that surely will benefit from many improvements.
CONCLUSIONS
the physiological activities of mature p. aeruginosa biofilms were elucidated by integrating existing knowledge of gene functions and transcriptional responses, a public database of transcriptomic data, a whole-biofilm transcriptome, and other chemical and biological assay results.
the biofilm was found to be limited for oxygen, growing slowly, and exhibiting stationary phase character.

 the physiological activities of bacteria growing in biofilms are difficult to divine, because these activities are diverse, change with time as the biofilm develops, and are subject to extreme micro scale spatial heterogeneity. it is also clear that the metabolism and activities of a particular biofilm will be shaped by the specific chemical and physical environment in which it grows. these realities make it difficult to develop a consensus picture of the physiology of the biofilm state as there is so little overlap in the lists of genes differentially expressed between the planktonic and biofilm states of pseudomonas aeruginosa prepared by different experimenters. however, there are biofilm physiological traits, such as antimicrobial tolerance and reduced growth rate, for which there is considerable consensus. these robust phenotypes, with their functional and evolutionary importance, should have discernable biochemical and genetic bases. we sought to understand these phenotypes with an unconventional interpretation of transcriptional profiling studies. conventional interpretations of transcriptional profiling studies compare two paired data sets that differ in a single controlled variable. in this study, we have obtained the transcriptome for a single biofilm specimen, ranked the transcripts based on the signal intensity to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and a number of published data sets. for example, if we wish to discern whether the biofilm is responding to iron limitation, we first identify a set of genes that are up-regulated in response to iron deprivation. the rank of each of these transcripts in the biofilm data set is then compared to transcript ranks for the same genes in data sets collected from both rapidly growing and deliberately iron-starved cultures. in this way it becomes possible to evaluate physiological activities in the biofilm rather than just documenting differences between the biofilm and a reference state. in the experiments reported here, rna was extracted from an entire, homogenized biofilm specimen. an obvious concern with this approach is that it neglects the inherent biological heterogeneity of the biofilm. we would like to address this concern upfront with two points. first, just because a population is heterogeneous does not mean that measurements of population averages are invalid. population averages are very widely and informatively used in biology. second, we suggest that even the concept of an average may not be appropriate in this case. the current conceptual model of p. aeruginosa drip-flow biofilms is that they consist of two distinct populations: an aerobic, metabolically active upper layer and a lower, and larger, layer consisting of inactive cells containing very low levels of mrna. because the inactive cells contain so little rna, this majority is expected to be essentially invisible on the microarray. from this perspective, the transcriptomes reported here may best be thought of as reflecting the properties of the transcriptionally-active subpopulation rather than the average behavior of the entire population. these concepts are elaborated on in the results and discussion.  three day old drip flow biofilms of p. aeruginosa were characterized with respect to antibiotic tolerance, oxygen availability, and microscale patterns of protein synthetic activity. these biofilms contained ± cfu cm- reduced antibiotic susceptibility of biofilm bacteria p. aeruginosa cells grown in biofilms were protected from killing by tobramycin and ciprofloxacin, in comparison to actively growing planktonic bacteria. both antibiotics rapidly and effectively reduced viable cell numbers in an aerobic, planktonic culture. after h of treatment with μg ml- tobramycin or μg ml- ciprofloxacin, planktonic log reductions measured were ± and ± for tobramycin and ciprofloxacin, respectively. in contrast, neither antibiotic was very effective against biofilms of p. aeruginosa. after h exposure to antibiotic in continuously flowing medium, the log reductions in viable cell numbers were ± and ± for tobramycin and ciprofloxacin, respectively. the log reductions measured for biofilm bacteria were 23% and 28% of the planktonic log reductions for the two antibiotics, respectively. reduced killing of the biofilm in comparison to planktonic cells was statistically significant. these data demonstrate that these drip-flow biofilms exhibit the antibiotic-tolerant phenotype that is considered a hallmark of the biofilm mode of growth. when biofilm bacteria were dispersed prior to antibiotic exposure, they again became susceptible to the antibiotics. log reductions measured for biofilm cells re-suspended into aerated medium and treated with tobramycin or ciprofloxacin for h were ± and ±, respectively. this degree of killing was the same as that measured for planktonic bacteria, indicating that susceptibility was rapidly and fully restored upon dispersal of cells from the biofilm. low oxygen concentrations in biofilms an oxygen microelectrode was used to demonstrate the presence of oxygen concentration gradients in this system. the oxygen concentration in the flowing fluid above the biofilm was approximately mg l- oxygen concentration decreased to mg l- or less inside the biofilm. a similar profile was measured in a duplicate experiment. the oxygen concentrations shown in figure 1a may not define the lower bound of oxygen concentration inside the biofilm because the electrode was positioned only partway into the biofilm, to avoid electrode breakage. the utilization of oxygen by bacteria is coupled to their simultaneous uptake and oxidation of a carbon source. to investigate this coupling, the oxygen microelectrode was positioned at a depth part way into the biofilm where the oxygen concentration was less than mg l-. the medium flowing over the biofilm was then changed from complete pbm to pbm lacking glucose and ammonium sulfate. within a few minutes after switching to this starvation medium, the oxygen concentration in the biofilm abruptly rose to approximately mg l- when the complete medium containing glucose and the nitrogen source was restored, the oxygen concentration quickly dropped back to its previous low level. upon switching once again to the starvation medium, the oxygen concentration again returned to the higher level. restoring the complete medium again caused the oxygen concentration to fall. the same behavior was observed in a duplicate experiment. these experiments show that oxygen and glucose utilization are interdependent. heterogeneous patterns of protein synthetic activity in biofilms the induction of a gfp has been used to reveal regions of active protein synthesis in biofilms. when this technique was applied to p. aeruginosa biofilms grown in drip-flow reactors, a stratified pattern of activity was observed. expression of gfp was localized in a band at the top of the biofilm adjacent to the source of nutrients and oxygen. the dimension of the gfp-expressing zone averaged ± μm. the average thickness of the entire biofilm was ± μm. while the predominant zone of activity was along the air interface, gfp fluorescence was occasionally observed in thin strata in the interior and even at the bottom of the biofilm. the observation of fluorescent gfp at the bottom of the biofilm argues against the interpretation that these patterns are an artifact of incomplete iptg penetration. in prior studies, the facile penetration of iptg throughout p. aeruginosa biofilms has been demonstrated. *the thickness of the area of gfp expression as well as the overall thickness of the biofilm was measured times. measurement of pseudomonas aeruginosa pao carrying plasmid pab containing an iptg-inducible gfp with and without iptg are compared with p. aeruginosa carrying plasmid pmf lacking gfp. †the uncertainties indicated are standard deviations. transcriptional profiling of biofilms - nutritional and growth status the rna was extracted from 3-day old p. aeruginosa drip-flow reactor grown biofilms and subjected to global transcriptional profiling. these microarray data have been deposited to gene expression omnibus accession gse we compared the expression of individual genes or groups of genes indicative of specific physiological activities by analyzing the rank of the selected transcript in the drip flow biofilm transcriptome across different experiments and planktonic comparator transcriptomes listed in table and additional file ● 2% o - gold ● % o - red ● od ~ - white additional file contains a version of this table that includes colored symbols for visual identification of the symbols used in figures and when grown on glucose, p. aeruginosa expresses an outer membrane protein, oprb, which is involved in the uptake of sugars. figure 3a compares the rank of the oprb transcript in several data sets, including our drip-flow reactor biofilm. this gene is highly expressed in the biofilm and also highly expressed in one other transcriptome from a study in which the bacteria were grown on a glucose-minimal medium. the rank of the pa transcript is lower in cells grown on minimal media supplemented with acetate or citrate, lower still on complex media such as lb or bhi, and lowest of all on a minimal amino acid medium. the straightforward interpretation of this comparison is that the strong expression of oprb in the drip-flow biofilm implies the presence of glucose in the system. since the medium used in this study contained glucose as the sole carbon and energy source, these results illustrate the face validity of our approach. to further demonstrate the potential to diagnose metabolic activities from transcript ranks, we conducted a more comprehensive analysis of relationship between the presence or absence of glucose and the ranks of selected gene transcripts. fifty eight samples were identified in which no glucose was present in the medium. eleven samples were identified in which glucose was the sole or predominant carbon source. differences in the ranks of pairs of genes, identified by inspection, were found to discriminate the glucose-present and glucose-absent data sets. the drip-flow biofilm data group with the glucose-present comparators, as expected. the six glucose-absent points that overlap with the glucose-present cluster are from a single investigation in which glycerol was the predominant carbon source. the extensive commonality of pathways for catabolism of glucose and glycerol may explain this overlap. alvarez-ortega and harwood identified genes induced under conditions of low oxygen concentration. from their results, we identified a subset of seven genes that were particularly strongly induced by low oxygen and whose transcript rank increased monotonically with decreasing oxygen concentration. figure 3b compares the rank for these seven genes between drip-flow biofilms in this study and the alvarez-ortega and harwood data. the rankings of the transcripts for the biofilm were consistent with low oxygen concentrations for six of seven transcripts. this comparison indicates that the biofilm experienced oxygen limitation. a recent investigation reported genes induced by transferring p. aeruginosa from aerobic to anaerobic conditions. thirty-five genes appearing on this list also appear in table a significant overlap. this overlap reinforces the interpretation of an oxygen-limited physiology of the drip-flow biofilm population. *average rank in drip flow biofilms and three comparator microarray data sets named in the materials and methods. we identified four genes strongly up-regulated by iron limitation and compared their expression between drip-flow biofilm, three standard comparison data sets, and a positive control in which the bacterial culture was deliberately iron-limited. 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 biofilm as grown in this study did not experience limitation for iron. the concentration of iron in the medium, added in the form of ferrous ammonium sulfate, was 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. 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. 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. though the data is somewhat noisy, it is clear that rpos gene rank decreases with increasing growth rate. the rank of the fis gene is relatively constant above a specific growth rate of approximately h- and decreases below this growth rate. the difference in gene rank between rpos and fis increases with specific growth rate. this analysis points to the possibility of inferring growth rate from transcriptomic data. for example, in the drip-flow biofilm the difference in rpos and fis gene rank was - ±. from figure 3f, this difference corresponds to a specific growth rate of approximately h- taking the results of figures 3e and 3f together, it appears as if bacteria in the biofilm were growing very slowly. oxygen availability limits growth in biofilm in this experimental system, two potentially limiting substrates for bacterial growth were glucose and oxygen. the composition of the medium used ensured excess nitrogen, phosphorous, sulfur, and other elemental requirements. for example, the molar ratio of ammonium to glucose carbon was, which provided approximately ten-fold excess nitrogen. there is no basis for anticipating that glucose was limiting in any part of the biofilms that were grown in this study. this can best be appreciated by a simple calculation. as derived by williamson and mccarty, the metabolic substrate that will first be depleted in a biofilm can be determined by calculating the dimensionless quantity degsg/deo2so2ygo this ratio is a measure of the relative diffusive fluxes of glucose and oxygen into the biofilm, where de denotes the effective diffusion coefficient of the respective substrate in the biofilm, s denotes the bulk fluid concentration of the respective substrate, and ygo is the stoichiometric coefficient relating the consumption of glucose and oxygen. in the present case, we take the effective diffusion coefficients of oxygen and glucose to be × 10- cm s- and × 10- cm s- respectively. the yield coefficient has been carefully measured, in biofilms of this bacterium, and is g glucose per g oxygen. with the bulk fluid concentration of glucose at mg l- and the bulk fluid concentration of oxygen at mg l- the quantity given by the ratio above has a value of. this value being greater than means that glucose is provided in excess and that oxygen is the limiting substrate. this interpretation is consistent with the strong expression of oprb in biofilm specimens and the analysis shown in figure 4a. microelectrode measurements provided direct chemical evidence of reduced oxygen availability. steep oxygen concentration gradients were measured in the vicinity of the biofilm, with parts of the biofilm experiencing oxygen concentrations of mg l- or less. these measurements are concordant with the transcriptomic analysis of biofilm bacteria that provides direct biological evidence of oxygen limitation. the following describes our physical understanding of the concentration gradients in this particular biofilm system. in the aerobic layer, both oxygen and glucose are consumed. once the oxygen has been depleted, utilization of glucose stops. abundant glucose, approximately mg l- is predicted to be available at the bottom of the biofilms studied in this investigation. we note that p. aeruginosa is unable to ferment glucose and no arginine was present, precluding fermentative growth. no alternative electron acceptor, such as nitrate, was added to the medium used in these studies. therefore, growth by denitrification was also precluded. the expression of genes associated with denitrification in the biofilm may have been a response to oxygen limitation. in summary, once oxygen was depleted in this system, one would predict that growth would cease. biofilm harbors slowly-growing or non-growing bacteria we hypothesize that oxygen limitation in p. aeruginosa drip-flow biofilms resulted in slow growth or lack of growth of many of the bacteria in the biofilm. the expression of an inducible gfp was focused in a sharply demarcated band immediately adjacent to the oxygen source. this band represented approximately 38% of the biofilm, indicating that as much as 62% of the biofilm could be anoxic and anabolically inactive. because alternative fermentable substrates or electron acceptors were absent, oxygen limitation is expected to be sufficient to lead to arrested growth in anoxic regions of the biofilm. this interpretation is qualitatively consistent with previous studies of oxygen availability and spatial patterns of physiological activity in some other p. aeruginosa biofilms. transcriptomic data show that the biofilm exhibited stationary phase character. this is evident in the pronounced expression of rmf, a stationary-phase inhibitor of ribosome function, cspd, a stationary-phase inhibitor of replication, and rpos, a stationary-phase sigma factor. in a previous investigation, we independently reported the elevated expression of rpos in p. aeruginosa biofilms. a gene associated with early exponential phase growth, fis, was expressed at relatively low levels, consistent with very slow growth. our estimate of an average specific growth rate of h- is approximately ten percent of the specific growth rate of p. aeruginosa in this medium of h- colony biofilms of a mucoid strain of p. aeruginosa had a reported specific growth rate that was two percent of the maximum specific growth rate in that system. here we consider two alternative conceptual models for growth and activity within the biofilm. these models attempt to address the microscale heterogeneity that is obviously present and which the transcriptional analysis is incapable of resolving. both of these conceptual models view the biofilm as having two layers of differing growth rates. in the first model, an aerobic layer representing the upper 40% of the biofilm grows at h- while the bottom layer has a specific growth rate of zero. the population average growth rate would be h- in the second model, an aerobic layer representing the upper 40% of the biofilm grows at h- while the bottom layer has a specific growth rate of zero. the population average growth rate would be h- we believe that the second model is the more realistic. the transcriptome obtained in this study does not represent the average behavior of the biofilm. it reflects rather the activities of the transcriptionally-active subpopulation, which is the aerobic upper layer. localized gene expression measurements performed by microdissection and pcr show that the rpos transcript is more abundant in the upper layer of the biofilm compared to the middle or bottom layers. this confirms that the "active" cells in the biofilm are in fact in a stationary phase-like state and that the inactive cells are depleted of most mrna. transcriptional profiling of biofilms - stress responses and quorum sensing the same approach of comparing ranks of selected genes indicative of specific physiological activities was applied to examine oxidative stress, copper stress, efflux pump activities, and quorum sensing in drip-flow biofilms. the expression levels, as quantified by transcript rank, of five genes associated with oxidative stress were not in general elevated in reference to the comparators. the only possible exception, a putative glutathione peroxidase, is difficult to interpret clearly since this gene is also induced under copper stress. thus we conclude that no unusual oxidative stress is occurring. we noticed that several genes associated with copper stress, as reported by teitzel et al., were highly expressed in drip-flow biofilms. the nominal copper concentration in pbm is μm, which is much less than the mm teitzel et al. used. we identified another data set, that of love and co-workers, in which an acetate minimal medium was supplemented with trace elements including cu at a final concentration of μm. copper stress genes were highly expressed in this case as well. we therefore suggest that micro molar concentrations of copper are sufficient to induce a copper stress response when p. aeruginosa is grown in minimal media. efflux pumps were not up-regulated in p. aeruginosa biofilms in general. the one instance of obvious high level expression, pa is associated with copper stress. three different laboratories have published data on the set of genes regulated by homoserine lactone quorum sensing in p. aeruginosa. we selected a consensus subset of seven of these genes that are more highly expressed under conditions of active quorum sensing and compared the drip-flow biofilm transcriptome to the standard reference data sets. the biofilm rank was relatively low for all but one of these genes, pa or rsal. though rsal is itself quorum sensing activated, the rsal gene product is a negative regulator that represses many other quorum-sensing activated genes. thus the high level expression of rsal may be consistent with repression of many of the other genes shown in figure 5d. these data show, surprisingly, that homoserine lactone quorum sensing is not active in these drip-flow biofilms. to further demonstrate the potential for differences in transcript ranks to serve as indices of specific physiological activities, homoserine lactone quorum sensing was examined in a fashion analogous to that described above for glucose and growth rate. the eight quorum sensing positive samples plotted in figure 4b are planktonic cultures with optical densities greater than. the quorum sensing negative samples in this figure are either from quorum sensing deficient mutants or planktonic cultures of very low optical density. the drip-flow biofilm data points clearly do not group with quorum sensing positive benchmarks. quorum sensing has been associated with biofilm development in p. aeruginosa by many investigators, so our finding that this communication system is silent in three-day old drip-flow biofilms seems at odds with the literature. this result is internally consistent, however, with the elevated expression of two negative regulators of quorum sensing, rsal and algr, another repressor of quorum sensing. the algr gene transcript ranked in drip-flow biofilms and in the same comparator data sets used to compile table we speculate that quorum sensing may have been active at an earlier stage of biofilm formation in the drip-flow reactor. transcriptional profiling - biofilm extracellular matrix genes extracellular polysaccharides and proteins are common constituents of the biofilm matrix. there are four putative or known polysaccharide biosynthetic operons in p. aeruginosa. both pel and psl genes were expressed in the biofilm while alginate biosynthetic genes were not. only the pel genes were up-regulated in biofilms compared to the three planktonic controls. the low level of expression of algd in the drip-flow biofilm is consistent with prior reports that alginate is not an important constituent of pao biofilms. our transcriptomic data suggest that the pel and psl polysaccharides may be important constituents of the extracellular matrix of drip-flow biofilms while alginate is unimportant. the rank of the cdra gene, a recently described adhesin that interacts with the psl polysaccharide, was not much different in drip-flow biofilms and planktonic comparators. genes associated with the elaboration of type iv pili were strongly expressed in drip-flow biofilms. this has led us to speculate that these extracellular proteinaceous appendages contribute to the mechanical stability of the biofilm rather than motility, perhaps by binding to extracellular dna. transcriptional profiling - independent identification of upregulated genes in biofilms all of the preceding analyses were predicated using a priori identification of a set of genes associated with discrete physiological conditions. the comparison of transcript ranks can also be used to identify genes that are differentially regulated between the drip-flow biofilm data set and planktonic comparator data sets. table reports the genes that ranked more highly in the drip-flow biofilm than in the comparator data set, by fold-changes in rank ranging from to more than some of the salient features of this list are genes associated with oxygen limitation, copper stress, bacteriophage pf, denitrification, ethanol metabolism, and three genes involved in type iv fimbrial biogenesis. seven of the genes listed in table appear on the consensus list of gene transcripts upregulated in p. aeruginosa biofilms reported by patell et al. biological basis of biofilm antibiotic tolerance p. aeruginosa strain pao formed biofilms in the drip-flow reactor that were poorly killed by tobramycin or ciprofloxacin. this result is concordant with many previous investigations of antibiotic susceptibility of p. aeruginosa biofilms developed in other in vitro systems. a plausible and long-standing explanation for reduced antibiotic susceptibility in biofilms is that nutrient limitation leads to slow growth or stationary phase existence for many of the cells in a biofilm, reducing their antimicrobial susceptibility. this mechanism is consistent with all of our data. multiple lines of evidence support oxygen limitation and arrested growth in drip-flow biofilms: oxygen concentration gradients, expression of genes associated with oxygen limitation and stationary phase existence, and stratified patterns of protein synthetic activity. in a previous study using a different in vitro biofilm model, we reported that oxygen limitation could account for percent or more of the protection from six antibiotics observed in p. aeruginosa colony biofilms. a recent report showed that ciprofloxacin and tetracycline preferentially killed the metabolically active subpopulation in p. aeruginosa biofilms. oxygen limitation is known to occur in vivo in cystic fibrosis patients. further, molecular biological evidence suggests that p. aeruginosa in the cystic fibrosis lung experiences anaerobic conditions. in an investigation of in situ growth rates of p. aeruginosa obtained from chronic lung infections, approximately 11% of cells were determined to be in a non-growing stationary-phase based on their ribosome content. the average specific growth rate of the growing bacterial cells was h- this shows that a non-growing population may be relevant in vivo, though it suggests that the population of bacteria in the infected lung were overall more active than we describe here for drip-flow biofilms. heterogeneity within the biofilm here we remark on the "averaging" that occurs when the entire biofilm is mashed up and extracted rna is analyzed. this method mixes together the rna from transcriptionally active cells in the aerobic upper layer of the biofilm with rna from inactive bacteria in the lower layers of the biofilm. the result is not a simple average of the activities of the two layers because there is so much less mrna in the inactive bacteria. indeed, the inactive bacteria may contribute little to the overall microarray signal. for this reason, the transcriptome that has been examined in this work may best be thought of as representing the transcriptionally-active supopulation of bacteria rather than an average of the entire biofilm population. a recently described laser capture microdissection technique provides a direct experimental approach for quantifying the amount of specific rna sequences in distinct regions of the biofilm. this method begins with cryoembedding an intact biofilm and preparing frozen cross sections. small user-defined areas of the cross section can be physically removed and amplified by pcr to detect specific transcripts. application of this approach to drip-flow p. aeruginosa biofilms has revealed that the upper layer of the biofilm is enriched in mrna compared to the lower layers. for example, in drip-flow biofilms the number of rna copies of the housekeeping gene acpp was approximately times smaller at the bottom of the biofilm compared to the top. for the rhlr transcript the difference between top and bottom was approximately 30-fold. utility of ranked transcriptome analysis conventional transcriptional profiling is applied to paired samples and allows for the discovery of genes that are differentially regulated between the two samples. for example, comparing the transcriptomes of samples grown at two different temperatures or in the presence and absence of a signaling molecule leads directly to the identification of genes regulated by temperature or by the specific signal chemistry. this is the usual usage of transcriptional profiling technology. in this investigation, we sought to use transcriptional profiling to provide insight about the physiological activities of a single sample. rather than chronicling the differences between two conditions, we wanted to ask and answer the question "what is the transcriptionally active biofilm cell doing?" to do this, we ranked the transcriptome, which makes manifest the priorities of the cell, at least at the transcriptional level. to interpret this ladder of genes, we independently identified from the literature sets of genes as markers of particular physiological activities and then compared the ranks of these genes to the ranks in several planktonic comparator transcriptomes. as the public database of transcriptional data expands, this approach becomes more and more feasible and powerful. our effort is a preliminary one that surely will benefit from many improvements.  the physiological activities of mature p. aeruginosa biofilms were elucidated by integrating existing knowledge of gene functions and transcriptional responses, a public database of transcriptomic data, a whole-biofilm transcriptome, and other chemical and biological assay results. the biofilm was found to be limited for oxygen, growing slowly, and exhibiting stationary phase character. 
by comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. transcriptome analysis was applied to characterize the physiological activities of pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. in contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared ranki ngs for a priori identified physiological marker genes between the biofilm and published data sets. biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. these biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be h- genes associated with elaboration of type iv pili were strongly expressed in the biofilm. the biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps.
 transcriptome analysis was applied to characterize the physiological activities of pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. in contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared ranki ngs for a priori identified physiological marker genes between the biofilm and published data sets.  biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. similar rankings were obtained from data sets published in the geo database http://www.ncbi.nlm.nih.gov/geo. by comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. these biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. genes associated with elaboration of type iv pili were strongly expressed in the biofilm. the biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be h-  collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity. 