BACKGROUND
the prokaryotic diversity of the extremely acidic waters of the río tinto  has been studied for many years and is well characterized  <cit> .
among the most abundant microorganisms are the gram-negative iron oxidizing bacteria leptospirillum ferrooxidans and acidithiobacillus ferrooxidans, both accounting for more than 70% of the prokaryotic population in the water column  <cit> .
these bacteria have special relevance for the biomining industry because they are used to extract metals through the bioleaching of sulfide ores, and may produce acidification of mine drainages  <cit> .
in addition, due to their very limited nutrient requirements and their association with iron and sulfur minerals, they are good models for the study of the origin, evolution and adaptation of life on earth and elsewhere, particularly mars  <cit> .
the acidophile prokaryotic communities play a critical ecological role because they are responsible for maintaining the low ph and, as a consequence, other physicochemical conditions of the ecosystem, like the elevated heavy metal concentration.
they are also critical for the existence of fully operative iron and sulfur cycles in the río tinto ecosystem  <cit> .
we previously reported the environmental transcriptomic fingerprint of l. ferrooxidans in its natural planktonic microbial community under high iron and sulfur content , as well as high oxidative stress  <cit> .
the cell density in the water column is relatively low , however, there are several types of macroscopic filaments  in the river or attached to solid substrates forming true biofilms with similar prokaryotic diversity as that found in the water column  <cit> .
many bacterial species live predominantly in biofilms in both natural and artificial environments  <cit> .
biofilms can be defined as matrix-enclosed bacterial populations  <cit> , dynamic structures in which transitions between the planktonic and biofilm modes of growth occur as a response to different environmental signals.
biofilms constitute habitats where microorganisms exhibit physiological heterogeneity and behavioral characteristics that makes them different of those with a free living lifestyle  <cit> .
it has been reported that ore bioleaching requires the formation of biofilms as the mode for bacterium-mineral interaction  <cit> .
extracellular polymeric substances from biofilms seem to mediate in the attachment of cells to the solid substrates  and the electrochemical reactions operating in biocorrosion and bioleaching processes  <cit> .
among the factors affecting the biofilm formation and maintenance is the so called quorum sensing, by which microorganisms communicate via the secretion of chemical signaling molecules like acyl homoserine lactone, principally used by gram-negative bacteria  <cit> .
up to two different acyl homoserine lactone production systems have been identified in a. ferrooxidans  <cit> , however very little is known about quorum sensing in leptospirillum spp.
metaproteomic  <cit>  and metagenomic  <cit>  studies in acidic biofilms dominated by leptospirillum ferriphilum reported the presence of the luxri-like genes, involved in quorum sensing through acyl homoserine lactone.
due to the important role of extremely acidophilic biofilms, both in microbial ecology and in industrial bioleaching, the aim of this work was to gain insights into the physiological differences between the planktonic  and sessile  modes of life through the analysis of the leptospirillum spp.
environmental transcriptomes.
RESULTS
physicochemical and microbiological characterization of sampling sites
biomass was collected from water and filamentous biofilms .
the sampling site was mainly characterized by a high metal  and salt  content .
however, differences in some parameters could be observed between the two sampling times.
in general, the  <dig> samples contained higher concentrations of iron, sulfate, al, cu, mg, mn or zn, while they had a lower amount of k+ and acetate  than the  <dig> ones .
significant concentrations of acetate in water were also determined in other campaigns:  <dig>  mm in june  <dig>  or  <dig>  mm in july  <dig>
other acetate measurements:  <dig>  mm on june  <dig>  or  <dig>  mm on july  <dig>
uld, under the limit of detection.
we estimated the microbial community composition by two color simultaneous hybridization of total rna  to a prokaryotic acidophile microarray  already tested and validated with this type of samples  <cit> .
the pam analysis  indicated the predominance of a. ferrooxidans  and leptospirillum spp.
, both in water and in filaments along the two sampling times.
other groups of the microbial community with significant representation were the firmicutes, some alpha- and beta-proteobacteria, acidimicrobium spp., and some eukaryotes .
in addition, the pam analysis allowed us to estimate the relative proportion of the rna of each strain between the plankton and biofilm , which was considered for normalization purposes .
the composition of the free-living and the sessile prokaryotic communities was very similar in  <dig>  while it showed clear differences in  <dig>  when some groups like firmicutes , alpha and betaproteobacteria, and acidomicrobium predominated in plankton, while leptospirillum ferrodiazotrophum was only detected in the biofilm.
we assume that other leptospirillum spp.
present in the biofilm  may be contributing to the microarray hybridization, as deduced from comparative genome hybridization  of l. ferriphilum using the l. ferrooxidans microarray .
by comparing the genome sequences of l. ferrooxidans to that of l. rubarum and l. ferrodiazotrophum , we estimated a degree of sequence identity around 70-75%.
this level of sequence identity has, in fact, allowed us to perform transcriptome studies of l. ferriphilum using the l. ferrooxidans microarray  <cit> .
we hybridized at a temperature 5°c lower than that used for l. ferrooxidans transcriptome studies and the environmental transcriptome reported herein.
finally, the possible interference on the transcriptome studies by the other major component of the microbial community, the a. ferrooxidans group, was considered negligible following the cgh results .
comparing the leptospirillum spp.
environmental biofilm and planktonic transcriptomes
total amplified rna from plankton and biofilm was fluorescently labeled , mixed and set to hybridize with the genomic l. ferrooxidans dna microarray.
the signal intensities and the quality of the microarrays assayed with the samples collected in  <dig> were higher than those of  <dig>  probably due to a better quality of the extracted rna or to a higher metabolic activity in the  <dig> samples .
as expected, most of the probes on the microarray showed a log <dig> ratio  between - <dig> and + <dig> in the experiments with samples from the two campaigns .
more than  <dig> probes bearing the l. ferrooxidans 16s rrna gene showed strong reproducible signals and log <dig> ratios between - <dig>  and +  <dig>  from the  <dig> and  <dig> microarrays after normalization.
these consistent results suggested the absence of rna amplification bias in any of the samples, although we cannot exclude the possibility of some preference in the amplification of particular genes or regions.
we selected  <dig> different clone probes  having a significant intensity  in at least one of the microarrays for further analysis.
then, up to  <dig> probes were selected having a biofilm vs plankton ratio higher than  <dig> in both campaign experiments, suggesting that the up-regulation of these genes was tightly related to the biofilm architecture and organization processes, rather than to differences in the environmental conditions .
in previous works  <cit> , those spots showing special up or down-regulation were selected and the corresponding clones were sequenced from both ends.
we identified different open reading frames preferentially expressed in biofilm, others preferentially expressed in free living cells, and others with high expression levels in both sites .
many of them were assigned to already known genes and some corresponded to hypothetical conserved proteins of unknown function.
as expected, the genes whose transcription was induced in biofilm corresponded preferentially to several functional categories related to biofilm formation and maintenance or key steps of central metabolism:  cell envelope biogenesis and exopolysaccharide  synthesis;  quorum-sensing and chemotaxis ;  specific endopeptidase systems ;  two-component sensor kinase systems ;  cofactor biosynthesis ;  carbohydrate transport and metabolism;  pentose phosphate pathway;  deoxynucleotide biosynthesis; and  mixed acid fermentation .
on the other hand, the functional categories up-regulated in free living cells were related to:  ion transport, like po4= and k+;  amino acids  and fatty acids biosynthesis; and  tricarboxylic acid  cycle enzymes .
some of the more relevant up-regulated genes were also checked by rt-pcr and qrt-pcr using total amplified rna from biofilm and planktonic cells from the  <dig> campaign.
the preferential transcription detected by genome microarray experiments of mqsr, sira-like, poxb, acsa, pta, clpx, rpfg-like, acka and the unknown orf ygit-like , was confirmed by rt-pcr .
the qrt-pcr results further confirmed the induction of mqsr , sira-like  and poxb  in the biofilm, and pora  and pta  in planktonic cells.
effect of monosaccharides and small organic acids on the growth of l. ferrooxidans
the toxic effect of small organic acids  and other organic compounds on the growth of acidophile chemolithoautotrophs is well known  <cit> .
the presence of significant amounts of acetate  in the acidic water of the río tinto spring suggested that leptospirillum spp.
were able either to grow or at least to tolerate this relatively high concentrations.
additionally, the induction of genes like poxb, acka, or acsa also suggested that l. ferrooxidans and/or other leptospirillum spp.
are capable to produce acetate by mixed acid fermentation .
acetate may be produced from the hexoses and pentoses in the biofilm matrix to render pyruvate via the embden-meyerhof-parnas pathway and then to produce acetyl phosphate  and acetate through reactions catalyzed by poxb and acka, as it was described in lactobacillus plantarum  <cit> .
to check whether the presence of hexoses and pentoses as well as organic acids were toxic, we cultivated our l. ferrooxidans strain under different concentrations of glucose, arabinose, pyruvate and acetate.
the results showed a concentration-dependent growth on glucose and arabinose , as monitored by following the iron oxidation and cell counting .
no growth was detected in media containing pyruvate  or acetate .
discussion
transcriptomic fingerprints in extremely acidophilic biofilms
the analysis of the environmental transcriptomes of the leptospirillum spp.
in planktonic and biofilm natural communities resulted in the detection of several genes preferentially up-regulated in the biofilm.
most of them are related to functional categories and mechanisms known to be involved in biofilm formation and maintenance in other bacteria.
in addition, genes involved in mixed acid fermentation were also induced in the biofilm.
some of these genes and their functional categories are discussed below and in additional file.
genes involved in biofilm production and maintenance
several small size genes encoding hypothetical proteins were highly up-regulated in the streamers .
a sira-like gene that may be part of a two-component regulatory system was induced in the biofilm.
in several species, sira/bara orthologues are required for virulence gene expression, exoenzyme and antibiotic production, motility, and biofilm formation  <cit> .
a bdla-like gene is highly expressed both in biofilm and plankton.
this gene is involved in regulating chemotaxis and biofilm dispersion in pseudomonas aeruginosa  <cit> .
the authors proposed a model by which the bdla protein acts as a sensor of environmental cues and controls both biofilm formation and biofilm dispersion/detachment by modulating the enzymatic activity of c-di-gmp-forming and hydrolyzing ggdef/eal diguanylate cyclase containing domains.
similarly, an rpfg-rpfc two-component system is being expressed in both lifestyles, with a slight preference for biofilm.
the rpfgc operon is involved in the positive regulation of the synthesis of extracellular enzymes and polysaccharides  <cit> , in the regulation of the formation and dispersal of biofilms, and is required for full virulence in xanthomonas campestris  <cit> .
several proteins containing ggdef or ggdef/eal domains are being expressed , some of them preferentially in the biofilm, and others in both lifestyles.
genes for quorum sensing and motility
we identified a putative mqsr /mqsa  -like operon  <cit>  and both genes were highly up-regulated in the biofilm .
the mqsr-like protein shares 60% identity to the e. coli homologue, while mqsa  has 45% identity to that of e. coli .
mqsr  is directly associated with biofilm development and is linked to the development of persister cells  <cit> .
in addition, and relevant for the streamer biofilms, deletion of mqsr leads to a reduction in biomass, substratum coverage, and changes the biofilm architecture when cultured in flow cell devices.
mqsr transcriptionally regulates expression of genes necessary for motility, like flia or mota.
very recently, kim et al.
<cit>  have reported that mqsr and mqsa  are in fact a toxin/antitoxin  pair which regulates additional loci besides its own.
the system may be additionally regulated through the degradation of mqsa by clpx/p and lon proteases  <cit> .
the genes encoding these proteases are also up-regulated in the rio tinto biofilm .
in our study, the l. ferrooxidans mqsr-like protein might be the responsible for more than two-fold upregulation of a chemotaxis and motility operon consisting of flia-chey-chea-orf-mcp-chew-mota-motb, where flia is a sigma 28-like encoding gene .
a flagellar motor switch flim-like gene, located downstream of a methyl-accepting chemotaxis gene bdla  is also induced in biofilm streamers.
on the contrary, a putative operon encoding a sigma- <dig> like factor followed by flgb-flgc-flie-flifg-orf is preferentially expressed in plankton, which may indicate different peculiarities in motility between both lifestyles: "swimming motility" in plankton and "swarming motility" in biofilm.
a bioinformatic search of mqsr-like genes in public leptospirillum spp.
genome sequences  was unsuccessful, so it seems to be only present in our l. ferrooxidans strain.
genes for the synthesis of cell wall structures
among the highest up-regulated genes was lnt, encoding an apolipoprotein n-acyltransferase.
this enzyme is involved in lipoprotein biosynthesis by transferring the acyl group to the apolipoproteins.
lipoproteins are integral membrane proteins that bind to specific enzymes or transport proteins across the cell membrane.
the lnt protein is essential in e. coli, and its depletion causes mislocalization of outer membrane lipoproteins  <cit> .
lipoproteins are involved in functions like cell wall synthesis, secretion, or flagellar assembly and motility in salmonella sp.
<cit> .
thermosensitive lnt mutants in salmonella sp.
are non-flagellate at 42°c.
however, it has not been so far associated to biofilm formation or maintenance.
genes encoding key enzymes for the peptidoglycan synthesis are also up-regulated in biofilm, like murb, involved in the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan  <cit> .
additionally, a slt-like gene, encoding a soluble lytic murein transglycosylase for recycling of muropeptides during cell elongation and/or cell division is also up-regulated.
several putative glycosyl transferases involved in eps and lipoplysaccharide biosynthesis as well as cell wall biogenesis are being expressed both in biofilm and planktonic cells , some of them with slight preference for biofilm, like an o antigen polymerase and a putative lipoplysaccharide biosynthesis protein, and others with more than two fold induction in streamer biofilm .
genes involved in mixed acid fermentation
some genes involved in mixed acid fermentation showed up-regulation patterns as determined by microarray hybridization, rt-pcr and q-rt-pcr analysis .
this is the case for the genes poxb, pta and acka encoding a pyruvate oxidase, phosphotransacetylase and acetate kinase enzymes, respectively.
the pta gene is up-regulated in planktonic cells, while poxb and acka are preferentially expressed in biofilm.
these enzymes are critical for the control of the acetate, acetyl-phosphate  and acetyl-coa levels .
wolfe et al.
<cit>  showed evidence for an important role of acp as a global signal during the first steps of biofilm formation.
the pta and acka mutants showed aberrant biofilms and affected the expression of other genes involved in flagella, pili and fimbriae synthesis, stress proteins or colanic acid biosynthesis.
very recently, gueriri et al.
<cit>  reported that acp is an efficient phosphodonor for the response regulator degu in listeria monocytogenes, and that a double pta-acka mutant unable to synthesize acp was strongly affected in chemotaxis, motility and biofilm formation.
the high induction of poxb in the biofilm , may be the responsible for the significant amounts of acetate measured in the water stream .
no poxb genes have been identified in other leptospirillum genomes sequenced so far.
the l. ferrooxidans poxb protein  is also very similar to other putative poxb proteins from the gammaproteobacteria and firmicutes groups, like lactobacillus plantarum .
the location of poxb and mqsr close to each other, next to transposase coding genes and at the end of a contig , suggests that both genes may have been acquired by a horizontal gene transfer event from another microorganism.
the oxygen concentration drops drastically in the water column, so most of the submerged streamers are microaerophilic.
under these conditions, the primary fermenters  in the biofilm community can use the structural components of the biofilm  to produce pyruvate via the embden-meyerhof-parnas pathway.
then, the same or other bacteria  may oxidize and phosphorilate pyruvate by poxb  to acetylphosphate  which, in turn, can be transformed to acetate by acka .
a recent work reported the high hexose content in the extremely acidophilic biofilms from río tinto  <cit> , which indicates that sugars from the same biofilm can be a primary source of fermentable material.
it is known that acetic acid is toxic to chemolithoautotrophs like a. ferrooxidans  <cit>  and l. ferrooxidans , however it can also be assimilated, like in a. caldus  <cit> , or consumed by heterotrophic iron reducer microorganisms present in the community .
a recent work by nancucheo and johnson  <cit>  reported the presence of glycolic acid as an exudate in actively growing cultures of three chemolithotrophic acidophiles .
the glycolic acid showed similar toxicity as acetic acid with  <dig> strains of acidophiles screened , while only members of the firmicutes group  were capable to metabolize it.
similarly, the acetic acid we detected in the river's water can be consumed by sulfobacillus spp.
or other firmicutes .
we did not detect glycolic acid nor acetic acid production in the culture media of l. ferrooxidans even when it was cultivated in the presence of  <dig> % glucose or arabinose.
this result, together with the environmental transcriptomic studies shown herein suggest that only sessile l. ferrooxidans cells in the biofilm are capable to produce acetate.
the transcriptomic fingerprint of l. ferrooxidans planktonic cells
the highest induced gene  in planktonic cells was that encoding a nad+-dependent isocitrate dehydrogenase that we reported previously  <cit> .
the upregulation of this gene and other tca genes could be explained by increased expression of genes involved in carbon fixation in the planktonic phase using the pyruvate oxido-reductase pathway  and the reductive tca cycle, as we suggested previously  <cit> , and also proposed for another leptospirillum species  <cit>  .
the next most up-regulated genes in planktonic cells corresponded to abc transport systems for potassium  and phosphate .
the induction of kdpbac may be a consequence of the high salt concentration  in the water, which could be compensated by the accumulation of intracellular k+ that would play a role as osmoregulator, as in the halophilic bacteria halomonas elongata  <cit> .
this would imply that biofilm structures are playing a protective role against salinity that renders unnecessary the intracellular accumulation of k+ as osmoprotectant.
alternatively, a lower concentration of intracellular k+ in biofilm streamers may favor cellular aggregation, as it has been reported very recently in b. subtilis, where some natural products that caused potassium leakage triggered multicellularity  <cit> .
in addition, ueda and wood  <cit>  reported that potassium and sodium transporters regulate cellular adhesion and virulence to barley in p. aeruginosa.
in this scenario, the role of k+ as osmoprotectant may be replaced by the compatible solute trehalose in the sessile cells, in which the trehalose synthase gene  is up-regulated more than  <dig> fold .
the pstscab operon, encoding the specific phosphate abc transporter, is also preferentially expressed in planktonic cells.
in e. coli, transcription of the pst operon is induced under phosphate starvation and the full-length mrna is rapidly processed post-transcriptionally  <cit> .
the high amounts of fe3+ and al3+ ions in the río tinto samples may favor phosphate precipitation  <cit> , so that this nutrient is limited for planktonic cells.
in vitro experiments with p. aeruginosa demonstrated that acute phosphate depletion resulted in increased expression of psts and biofilm production  <cit> .
moreover, depletion of intestinal phosphate after operative injury activates the virulence of p. aeruginosa, causing lethal gut-derived sepsis.
the río tinto planktonic cells may be in similar situation: the continuous phosphate deficiency and high levels of psts shifts towards a biofilm producing phenotype and, once the biofilm is well established, the pstscab operon is repressed.
CONCLUSIONS
our results indicate that the main mechanisms governing biofilm formation and dispersion are operating in natural extremely acidophilic filamentous biofilms.
the identified genes corresponded to others known to be involved in different stages of biofilm formation in other bacteria  <cit> .
therefore, we conclude that the acidophilic biofilm streamers are dynamic structures in which mechanisms like quorum sensing, motility and chemotaxis, swarming and biofilm dispersion/formation are all operating .
specific transcriptomic fingerprints inferred for both planktonic and sessile cells showed metabolic differences, having the former a more active tca cycle, while the mixed acid fermentation process dominated in the latter.
the up-regulation of the mixed acid fermentation genes suggests that the acetate detected in the extremely acidic water of the sampling site  may be a consequence of acetic acid production by the acidophilic biofilms .
the excreted acetate could play an ecological role by controlling the growth of chemolithoautotrophs and as a source of electrons for heterotrophic fe3+ reducers like some alphaproteobacteria, acidobacterium spp.
and sulfobacillus spp., all detected with the pam microarray .
the newly produced fe2+ could be used as energy source for iron oxidizers like l. ferrooxidans and a. ferrooxidans, closing the iron cycle .

 the prokaryotic diversity of the extremely acidic waters of the río tinto has been studied for many years and is well characterized. among the most abundant microorganisms are the gram-negative iron oxidizing bacteria leptospirillum ferrooxidans and acidithiobacillus ferrooxidans, both accounting for more than 70% of the prokaryotic population in the water column. these bacteria have special relevance for the biomining industry because they are used to extract metals through the bioleaching of sulfide ores, and may produce acidification of mine drainages. in addition, due to their very limited nutrient requirements and their association with iron and sulfur minerals, they are good models for the study of the origin, evolution and adaptation of life on earth and elsewhere, particularly mars. the acidophile prokaryotic communities play a critical ecological role because they are responsible for maintaining the low ph and, as a consequence, other physicochemical conditions of the ecosystem, like the elevated heavy metal concentration. they are also critical for the existence of fully operative iron and sulfur cycles in the río tinto ecosystem. we previously reported the environmental transcriptomic fingerprint of l. ferrooxidans in its natural planktonic microbial community under high iron and sulfur content, as well as high oxidative stress. the cell density in the water column is relatively low, however, there are several types of macroscopic filaments in the river or attached to solid substrates forming true biofilms with similar prokaryotic diversity as that found in the water column. many bacterial species live predominantly in biofilms in both natural and artificial environments. biofilms can be defined as matrix-enclosed bacterial populations, dynamic structures in which transitions between the planktonic and biofilm modes of growth occur as a response to different environmental signals. biofilms constitute habitats where microorganisms exhibit physiological heterogeneity and behavioral characteristics that makes them different of those with a free living lifestyle. it has been reported that ore bioleaching requires the formation of biofilms as the mode for bacterium-mineral interaction. extracellular polymeric substances from biofilms seem to mediate in the attachment of cells to the solid substrates and the electrochemical reactions operating in biocorrosion and bioleaching processes. among the factors affecting the biofilm formation and maintenance is the so called quorum sensing, by which microorganisms communicate via the secretion of chemical signaling molecules like acyl homoserine lactone, principally used by gram-negative bacteria. up to two different acyl homoserine lactone production systems have been identified in a. ferrooxidans, however very little is known about quorum sensing in leptospirillum spp. metaproteomic and metagenomic studies in acidic biofilms dominated by leptospirillum ferriphilum reported the presence of the luxri-like genes, involved in quorum sensing through acyl homoserine lactone. due to the important role of extremely acidophilic biofilms, both in microbial ecology and in industrial bioleaching, the aim of this work was to gain insights into the physiological differences between the planktonic and sessile modes of life through the analysis of the leptospirillum spp. environmental transcriptomes.  physicochemical and microbiological characterization of sampling sites biomass was collected from water and filamentous biofilms. the sampling site was mainly characterized by a high metal and salt content. however, differences in some parameters could be observed between the two sampling times. in general, the samples contained higher concentrations of iron, sulfate, al, cu, mg, mn or zn, while they had a lower amount of k+ and acetate than the ones. significant concentrations of acetate in water were also determined in other campaigns: mm in june or mm in july other acetate measurements: mm on june or mm on july uld, under the limit of detection. we estimated the microbial community composition by two color simultaneous hybridization of total rna to a prokaryotic acidophile microarray already tested and validated with this type of samples. the pam analysis indicated the predominance of a. ferrooxidans and leptospirillum spp., both in water and in filaments along the two sampling times. other groups of the microbial community with significant representation were the firmicutes, some alpha- and beta-proteobacteria, acidimicrobium spp., and some eukaryotes. in addition, the pam analysis allowed us to estimate the relative proportion of the rna of each strain between the plankton and biofilm, which was considered for normalization purposes. the composition of the free-living and the sessile prokaryotic communities was very similar in while it showed clear differences in when some groups like firmicutes, alpha and betaproteobacteria, and acidomicrobium predominated in plankton, while leptospirillum ferrodiazotrophum was only detected in the biofilm. we assume that other leptospirillum spp. present in the biofilm may be contributing to the microarray hybridization, as deduced from comparative genome hybridization of l. ferriphilum using the l. ferrooxidans microarray. by comparing the genome sequences of l. ferrooxidans to that of l. rubarum and l. ferrodiazotrophum, we estimated a degree of sequence identity around 70-75%. this level of sequence identity has, in fact, allowed us to perform transcriptome studies of l. ferriphilum using the l. ferrooxidans microarray. we hybridized at a temperature 5°c lower than that used for l. ferrooxidans transcriptome studies and the environmental transcriptome reported herein. finally, the possible interference on the transcriptome studies by the other major component of the microbial community, the a. ferrooxidans group, was considered negligible following the cgh results. comparing the leptospirillum spp. environmental biofilm and planktonic transcriptomes total amplified rna from plankton and biofilm was fluorescently labeled, mixed and set to hybridize with the genomic l. ferrooxidans dna microarray. the signal intensities and the quality of the microarrays assayed with the samples collected in were higher than those of probably due to a better quality of the extracted rna or to a higher metabolic activity in the samples. as expected, most of the probes on the microarray showed a log ratio between - and + in the experiments with samples from the two campaigns. more than probes bearing the l. ferrooxidans 16s rrna gene showed strong reproducible signals and log ratios between - and + from the and microarrays after normalization. these consistent results suggested the absence of rna amplification bias in any of the samples, although we cannot exclude the possibility of some preference in the amplification of particular genes or regions. we selected different clone probes having a significant intensity in at least one of the microarrays for further analysis. then, up to probes were selected having a biofilm vs plankton ratio higher than in both campaign experiments, suggesting that the up-regulation of these genes was tightly related to the biofilm architecture and organization processes, rather than to differences in the environmental conditions. in previous works, those spots showing special up or down-regulation were selected and the corresponding clones were sequenced from both ends. we identified different open reading frames preferentially expressed in biofilm, others preferentially expressed in free living cells, and others with high expression levels in both sites. many of them were assigned to already known genes and some corresponded to hypothetical conserved proteins of unknown function. as expected, the genes whose transcription was induced in biofilm corresponded preferentially to several functional categories related to biofilm formation and maintenance or key steps of central metabolism: cell envelope biogenesis and exopolysaccharide synthesis; quorum-sensing and chemotaxis; specific endopeptidase systems; two-component sensor kinase systems; cofactor biosynthesis; carbohydrate transport and metabolism; pentose phosphate pathway; deoxynucleotide biosynthesis; and mixed acid fermentation. on the other hand, the functional categories up-regulated in free living cells were related to: ion transport, like po4= and k+; amino acids and fatty acids biosynthesis; and tricarboxylic acid cycle enzymes. some of the more relevant up-regulated genes were also checked by rt-pcr and qrt-pcr using total amplified rna from biofilm and planktonic cells from the campaign. the preferential transcription detected by genome microarray experiments of mqsr, sira-like, poxb, acsa, pta, clpx, rpfg-like, acka and the unknown orf ygit-like, was confirmed by rt-pcr. the qrt-pcr results further confirmed the induction of mqsr, sira-like and poxb in the biofilm, and pora and pta in planktonic cells. effect of monosaccharides and small organic acids on the growth of l. ferrooxidans the toxic effect of small organic acids and other organic compounds on the growth of acidophile chemolithoautotrophs is well known. the presence of significant amounts of acetate in the acidic water of the río tinto spring suggested that leptospirillum spp. were able either to grow or at least to tolerate this relatively high concentrations. additionally, the induction of genes like poxb, acka, or acsa also suggested that l. ferrooxidans and/or other leptospirillum spp. are capable to produce acetate by mixed acid fermentation. acetate may be produced from the hexoses and pentoses in the biofilm matrix to render pyruvate via the embden-meyerhof-parnas pathway and then to produce acetyl phosphate and acetate through reactions catalyzed by poxb and acka, as it was described in lactobacillus plantarum. to check whether the presence of hexoses and pentoses as well as organic acids were toxic, we cultivated our l. ferrooxidans strain under different concentrations of glucose, arabinose, pyruvate and acetate. the results showed a concentration-dependent growth on glucose and arabinose, as monitored by following the iron oxidation and cell counting. no growth was detected in media containing pyruvate or acetate. discussion transcriptomic fingerprints in extremely acidophilic biofilms the analysis of the environmental transcriptomes of the leptospirillum spp. in planktonic and biofilm natural communities resulted in the detection of several genes preferentially up-regulated in the biofilm. most of them are related to functional categories and mechanisms known to be involved in biofilm formation and maintenance in other bacteria. in addition, genes involved in mixed acid fermentation were also induced in the biofilm. some of these genes and their functional categories are discussed below and in additional file. genes involved in biofilm production and maintenance several small size genes encoding hypothetical proteins were highly up-regulated in the streamers. a sira-like gene that may be part of a two-component regulatory system was induced in the biofilm. in several species, sira/bara orthologues are required for virulence gene expression, exoenzyme and antibiotic production, motility, and biofilm formation. a bdla-like gene is highly expressed both in biofilm and plankton. this gene is involved in regulating chemotaxis and biofilm dispersion in pseudomonas aeruginosa. the authors proposed a model by which the bdla protein acts as a sensor of environmental cues and controls both biofilm formation and biofilm dispersion/detachment by modulating the enzymatic activity of c-di-gmp-forming and hydrolyzing ggdef/eal diguanylate cyclase containing domains. similarly, an rpfg-rpfc two-component system is being expressed in both lifestyles, with a slight preference for biofilm. the rpfgc operon is involved in the positive regulation of the synthesis of extracellular enzymes and polysaccharides, in the regulation of the formation and dispersal of biofilms, and is required for full virulence in xanthomonas campestris. several proteins containing ggdef or ggdef/eal domains are being expressed, some of them preferentially in the biofilm, and others in both lifestyles. genes for quorum sensing and motility we identified a putative mqsr /mqsa -like operon and both genes were highly up-regulated in the biofilm. the mqsr-like protein shares 60% identity to the e. coli homologue, while mqsa has 45% identity to that of e. coli. mqsr is directly associated with biofilm development and is linked to the development of persister cells. in addition, and relevant for the streamer biofilms, deletion of mqsr leads to a reduction in biomass, substratum coverage, and changes the biofilm architecture when cultured in flow cell devices. mqsr transcriptionally regulates expression of genes necessary for motility, like flia or mota. very recently, kim et al. have reported that mqsr and mqsa are in fact a toxin/antitoxin pair which regulates additional loci besides its own. the system may be additionally regulated through the degradation of mqsa by clpx/p and lon proteases. the genes encoding these proteases are also up-regulated in the rio tinto biofilm. in our study, the l. ferrooxidans mqsr-like protein might be the responsible for more than two-fold upregulation of a chemotaxis and motility operon consisting of flia-chey-chea-orf-mcp-chew-mota-motb, where flia is a sigma 28-like encoding gene. a flagellar motor switch flim-like gene, located downstream of a methyl-accepting chemotaxis gene bdla is also induced in biofilm streamers. on the contrary, a putative operon encoding a sigma- like factor followed by flgb-flgc-flie-flifg-orf is preferentially expressed in plankton, which may indicate different peculiarities in motility between both lifestyles: "swimming motility" in plankton and "swarming motility" in biofilm. a bioinformatic search of mqsr-like genes in public leptospirillum spp. genome sequences was unsuccessful, so it seems to be only present in our l. ferrooxidans strain. genes for the synthesis of cell wall structures among the highest up-regulated genes was lnt, encoding an apolipoprotein n-acyltransferase. this enzyme is involved in lipoprotein biosynthesis by transferring the acyl group to the apolipoproteins. lipoproteins are integral membrane proteins that bind to specific enzymes or transport proteins across the cell membrane. the lnt protein is essential in e. coli, and its depletion causes mislocalization of outer membrane lipoproteins. lipoproteins are involved in functions like cell wall synthesis, secretion, or flagellar assembly and motility in salmonella sp.. thermosensitive lnt mutants in salmonella sp. are non-flagellate at 42°c. however, it has not been so far associated to biofilm formation or maintenance. genes encoding key enzymes for the peptidoglycan synthesis are also up-regulated in biofilm, like murb, involved in the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. additionally, a slt-like gene, encoding a soluble lytic murein transglycosylase for recycling of muropeptides during cell elongation and/or cell division is also up-regulated. several putative glycosyl transferases involved in eps and lipoplysaccharide biosynthesis as well as cell wall biogenesis are being expressed both in biofilm and planktonic cells, some of them with slight preference for biofilm, like an o antigen polymerase and a putative lipoplysaccharide biosynthesis protein, and others with more than two fold induction in streamer biofilm. genes involved in mixed acid fermentation some genes involved in mixed acid fermentation showed up-regulation patterns as determined by microarray hybridization, rt-pcr and q-rt-pcr analysis. this is the case for the genes poxb, pta and acka encoding a pyruvate oxidase, phosphotransacetylase and acetate kinase enzymes, respectively. the pta gene is up-regulated in planktonic cells, while poxb and acka are preferentially expressed in biofilm. these enzymes are critical for the control of the acetate, acetyl-phosphate and acetyl-coa levels. wolfe et al. showed evidence for an important role of acp as a global signal during the first steps of biofilm formation. the pta and acka mutants showed aberrant biofilms and affected the expression of other genes involved in flagella, pili and fimbriae synthesis, stress proteins or colanic acid biosynthesis. very recently, gueriri et al. reported that acp is an efficient phosphodonor for the response regulator degu in listeria monocytogenes, and that a double pta-acka mutant unable to synthesize acp was strongly affected in chemotaxis, motility and biofilm formation. the high induction of poxb in the biofilm, may be the responsible for the significant amounts of acetate measured in the water stream. no poxb genes have been identified in other leptospirillum genomes sequenced so far. the l. ferrooxidans poxb protein is also very similar to other putative poxb proteins from the gammaproteobacteria and firmicutes groups, like lactobacillus plantarum. the location of poxb and mqsr close to each other, next to transposase coding genes and at the end of a contig, suggests that both genes may have been acquired by a horizontal gene transfer event from another microorganism. the oxygen concentration drops drastically in the water column, so most of the submerged streamers are microaerophilic. under these conditions, the primary fermenters in the biofilm community can use the structural components of the biofilm to produce pyruvate via the embden-meyerhof-parnas pathway. then, the same or other bacteria may oxidize and phosphorilate pyruvate by poxb to acetylphosphate which, in turn, can be transformed to acetate by acka. a recent work reported the high hexose content in the extremely acidophilic biofilms from río tinto, which indicates that sugars from the same biofilm can be a primary source of fermentable material. it is known that acetic acid is toxic to chemolithoautotrophs like a. ferrooxidans and l. ferrooxidans, however it can also be assimilated, like in a. caldus, or consumed by heterotrophic iron reducer microorganisms present in the community. a recent work by nancucheo and johnson reported the presence of glycolic acid as an exudate in actively growing cultures of three chemolithotrophic acidophiles. the glycolic acid showed similar toxicity as acetic acid with strains of acidophiles screened, while only members of the firmicutes group were capable to metabolize it. similarly, the acetic acid we detected in the river's water can be consumed by sulfobacillus spp. or other firmicutes. we did not detect glycolic acid nor acetic acid production in the culture media of l. ferrooxidans even when it was cultivated in the presence of % glucose or arabinose. this result, together with the environmental transcriptomic studies shown herein suggest that only sessile l. ferrooxidans cells in the biofilm are capable to produce acetate. the transcriptomic fingerprint of l. ferrooxidans planktonic cells the highest induced gene in planktonic cells was that encoding a nad+-dependent isocitrate dehydrogenase that we reported previously. the upregulation of this gene and other tca genes could be explained by increased expression of genes involved in carbon fixation in the planktonic phase using the pyruvate oxido-reductase pathway and the reductive tca cycle, as we suggested previously, and also proposed for another leptospirillum species. the next most up-regulated genes in planktonic cells corresponded to abc transport systems for potassium and phosphate. the induction of kdpbac may be a consequence of the high salt concentration in the water, which could be compensated by the accumulation of intracellular k+ that would play a role as osmoregulator, as in the halophilic bacteria halomonas elongata. this would imply that biofilm structures are playing a protective role against salinity that renders unnecessary the intracellular accumulation of k+ as osmoprotectant. alternatively, a lower concentration of intracellular k+ in biofilm streamers may favor cellular aggregation, as it has been reported very recently in b. subtilis, where some natural products that caused potassium leakage triggered multicellularity. in addition, ueda and wood reported that potassium and sodium transporters regulate cellular adhesion and virulence to barley in p. aeruginosa. in this scenario, the role of k+ as osmoprotectant may be replaced by the compatible solute trehalose in the sessile cells, in which the trehalose synthase gene is up-regulated more than fold. the pstscab operon, encoding the specific phosphate abc transporter, is also preferentially expressed in planktonic cells. in e. coli, transcription of the pst operon is induced under phosphate starvation and the full-length mrna is rapidly processed post-transcriptionally. the high amounts of fe3+ and al3+ ions in the río tinto samples may favor phosphate precipitation, so that this nutrient is limited for planktonic cells. in vitro experiments with p. aeruginosa demonstrated that acute phosphate depletion resulted in increased expression of psts and biofilm production. moreover, depletion of intestinal phosphate after operative injury activates the virulence of p. aeruginosa, causing lethal gut-derived sepsis. the río tinto planktonic cells may be in similar situation: the continuous phosphate deficiency and high levels of psts shifts towards a biofilm producing phenotype and, once the biofilm is well established, the pstscab operon is repressed.  our results indicate that the main mechanisms governing biofilm formation and dispersion are operating in natural extremely acidophilic filamentous biofilms. the identified genes corresponded to others known to be involved in different stages of biofilm formation in other bacteria. therefore, we conclude that the acidophilic biofilm streamers are dynamic structures in which mechanisms like quorum sensing, motility and chemotaxis, swarming and biofilm dispersion/formation are all operating. specific transcriptomic fingerprints inferred for both planktonic and sessile cells showed metabolic differences, having the former a more active tca cycle, while the mixed acid fermentation process dominated in the latter. the up-regulation of the mixed acid fermentation genes suggests that the acetate detected in the extremely acidic water of the sampling site may be a consequence of acetic acid production by the acidophilic biofilms. the excreted acetate could play an ecological role by controlling the growth of chemolithoautotrophs and as a source of electrons for heterotrophic fe3+ reducers like some alphaproteobacteria, acidobacterium spp. and sulfobacillus spp., all detected with the pam microarray. the newly produced fe2+ could be used as energy source for iron oxidizers like l. ferrooxidans and a. ferrooxidans, closing the iron cycle. 
the aim of this study was to investigate the physiological differences between the free living and the sessile lifestyles of leptospirillum spp. genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing, synthesis of cell wall structures, specific proteases, stress response chaperons, etc. our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active tca cycle, while the mixed acid fermentation process dominate in the latter. macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates. additionally, genes involved in mixed acid fermentation were up-regulated in the biofilm. in the río tinto, brown filaments develop under the water stream where the gram-negative iron-oxidizing bacteria leptospirillum spp. this result, together with the presence of small organic acids like acetate and formate in the acidic water stream, suggests that either l. ferrooxidans or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions. the excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic fe3+ reducers like some alphaproteobacteria, acidobacterium spp. total rna extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of l. ferrooxidans. and sulfobacillus spp., also present in the biofilm.
 extreme acidic environments are characterized by their high metal content and lack of nutrients. macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates. in the río tinto, brown filaments develop under the water stream where the gram-negative iron-oxidizing bacteria leptospirillum spp. and acidithiobacillus ferrooxidans are abundant. these microorganisms play a critical role in bioleaching processes for industrial and environmental applications. the aim of this study was to investigate the physiological differences between the free living and the sessile lifestyles of leptospirillum spp. as part of its natural extremely acidophilic community.  total rna extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of l. ferrooxidans. genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing, synthesis of cell wall structures, specific proteases, stress response chaperons, etc. additionally, genes involved in mixed acid fermentation were up-regulated in the biofilm. this result, together with the presence of small organic acids like acetate and formate in the acidic water stream, suggests that either l. ferrooxidans or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions.  our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active tca cycle, while the mixed acid fermentation process dominate in the latter. the excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic fe3+ reducers like some alphaproteobacteria, acidobacterium spp. and sulfobacillus spp., also present in the biofilm. additionally, acetate may have a negative effect on bioleaching by inhibiting the growth of chemolithotrophic bacteria. 