BACKGROUND
bacteria live in changing environments and are subjected to a variety of environnmental stresses such as ph, temperature, osmolarity or heavy metals.
arsenic is found in numerous disturbed or natural ecosystems where it can exist in mutiple oxidation states, the most common being arsenite as and arsenate as  <cit> .
this metalloid is known to generate oxidative stress in cells by its capability to induce the formation of reactive oxygen species .
the damages caused by ros to lipids, proteins and dna are likely to contribute to as toxicity  <cit> .
in addition, one property of arsenite, which indicates that it behaves like a soft metal, consists in a high reactivity with sulphydryls groups and that affects the activity of many proteins.
microorganisms have developed remarkable capabilities to cope with arsenic.
the most common arsenic resistance mechanism depends on the presence on plasmid or chromosome of ars genes encoding proteins involved in the reduction and/or the efflux of the toxic element  <cit> .
nevertheless, other arsenic resistance mechanisms have been described, i.e.
arsenite oxidation and arsenic methylation  <cit> .
in addition, in various microbial species, arsenic stress is associated with arsenite oxidase activity, biofilm formation, motility, oxidative stress or sulfur assimilation  <cit> .
for example, the biofilm development by thiomonas arsenivorans has been described as a physical barrier decreasing as access to sessile cells  <cit> .
remarkably, some organisms such as rhizobium sp.
nt- <dig>  have evolved specific metabolic pathways allowing them to oxidize as as an energy source  <cit>  or others are known to use as in anaerobic respiration  <cit> .
in the heterotrophic prokaryote herminiimonas arsenicoxydans, genome sequencing revealed the presence of four ars operons involved in the reduction of as to as and of one aoxab operon involved in the oxidation of the most toxic form, as to the less toxic form as  <cit> .
in addition to this detoxification process, this bacterium exhibits positive chemotaxis and motility towards arsenic, and metalloid scavenging by exopolyssacharides.
the availability of the h. arsenicoxydans complete genome sequence offers an opportunity to study its physiology by functional genomic approaches  <cit> .
our previous transcriptomic studies have demonstrated that a large number of genes encoding proteins involved in oxidative stress, low affinity import of phosphate or dna repair are induced after  <dig> min as exposure.
however, no variation was found in the genes coding for arsenite oxidase, a key enzyme in arsenic response  <cit>  recently shown to be subjected to a complex regulation  <cit> .
in addition, little is known regarding the kinetics of arsenic stress response in microorganisms.
to address these processes, physiological analyses coupled with western immunoblotting experiments and dna microarrays were used to examine the temporal changes in transcriptome profiles during the transition from as to as species due to as oxidation.
our work represents, to our knowledge, the first kinetic analysis of transcription pattern in bacteria exposed to arsenic, leading to propose a global model of arsenic response in h. arsenicoxydans.
RESULTS
characterization of arsenic oxidoreduction kinetics
to study the oxidoreduction kinetic in h. arsenicoxydans, the two arsenic species  and as) were quantified by hplc-icp-aes from filtered culture supernatants at several times after as or as induction  .
in the presence of as , three distinct phases were observed: in phase a <dig> , no oxidation was observed since arsenic was present as 100% as.
in phase a <dig>  which corresponds to a partial oxidation of as into as, both species were present, a proportion of 50% as and 50% as being observed after  <dig> hours.
the transformation of as into as was complete after  <dig> hours .
overview of transcriptome profiles
to get insight into the mechanisms involved in arsenic response, including oxidoreduction processes, transcriptomic analyses were performed at different steps of the experimental time course.
cells were collected from cultures of h. arsenicoxydans induced  <dig> min ,  <dig> hours ,  <dig> hours  or not with  <dig>  mm as  or  <dig>  mm as .
these concentrations were choosen as they are far from h. arsenicoxydans mic for as or as, which is able to grow up to  <dig> mm and  <dig> mm of as and as, respectively .
moreover, this concentration of as had only a minor impact on growth rate and cells continued to divide immediately after its addition.
under as stress,  <dig> genes were up-regulated and  <dig> genes were down-regulated in the early phase  .
in the late phase  the number of genes up-regulated  and down-regulated  increased .
under as stress, the proportion was inverted.
the major shift in gene expression occured during the onset of the early phase  with  <dig> genes up-regulated and  <dig> genes down-regulated under as stress.
this represents approximatly 14% of the genes whose expression was significantly affected by as.
as cell growth progressed further into the late phase , the number of genes up-regulated  and down-regulated  decreased, representing 4% of the genome.
the large number of genes responding to as, especially in the early phase, suggests a major remodelling of cellular physiology.
in addition, oxidoreduction kinetic was only observed under as stress , suggesting that the aox-dependent oxidation largely predominate the ars-dependent reduction process.
this study therefore focused on the as response in h. arsenicoxydans.
hierachical clustering kinetics
to classify the transcripts on the basis of their temporal dynamics after exposure to arsenite, we performed a hierarchical clustering with the arrayminer® software http://www.optimaldesign.com/arrayminer/arrayminer.htm.
genes were sorted into groups that exhibited similar transcription kinetics as cells transitioned from a <dig> to a <dig> phases.
the set of genes regulated by as shown in figure  <dig> was classified into  <dig> clusters.
the profiles similarity of those clusters enabled us to identify two major classes: 1) class i:  containing genes whose expression increased immediately after  <dig> min induction and decreased after  <dig> and  <dig> hours and class ii:  containing genes whose expression levels increased only after  <dig> and  <dig> hours induction.
the product of each upregulated or downregulated gene by a fold change of two or more was identified to gain insight into the changes in physiology .
next, to examine how the two classes are distributed with regard to their functions, we further classified these genes according to the gene classification based on cog functional categories .
the overall gene expression pattern suggests the existence of two major physiologic phases  during the adaptation to as stress.
i) early phase
our microarray analysis highlighted a set of stress responsive genes whose transcription was early affected by as.
this phase contained the early up-regulated genes whose expression levels gradually decreased after the induction  or down-regulated genes whose expression levels gradually increased after the induction .
first, σe and several genes encoding proteins involved in the synthesis of cell envelope components were down-regulated ) whereas genes encoding proteins involved in transport were up-regulated ).
the bacterial envelope is a major defense against threats from the environment.
due to its crucial importance, the integrity of the cell envelope, including the presence of outer membrane proteins , is closely monitored to ensure its functionality.
the σe system is the major pathway  <cit>  required for proper folding of omps and their turnover  <cit> , phospholipids and lipopolysaccharide  biosynthesis, signal transduction and the expression of inner and outer membrane proteins  <cit> .
in escherichia coli, the σe regulon is induced specifically in response to the unbalanced synthesis of outer membrane proteins  <cit>  and to the misfolding of polypeptides that have been translocated across the cytoplasmic membrane  <cit> .
our results suggest that both the organization  and the functioning  of the bacterial envelope may be changed in the early phase of the response to as.
second, early as stress strongly stimulated expression of genes encoding components of sulfur assimilation, sulfur oxidation and glutathione  biosynthesis pathways.
in fact, mrna levels of genes encoding all the enzymatic steps required for sulfate uptake, its conversion into cysteine and gsh were increased.
the expression of these genes was induced about  <dig> to  <dig> fold after  <dig> min induction with as.
the expression of some of these genes was down-regulated at  <dig> and  <dig> hours .
in eukaryotic cells, as exposure results in increased levels of reduced glutathione  <cit>  and an as-triglutathione complex has been found in human liver excreta  <cit> , leading to the conclusion that gsh plays an important role in reducing as toxicity.
most effects of arsenite result from its interaction with thiol groups of proteins, its iniophoretic properties and its ability to directly or indirectly generate free radicals and hence induce oxidative stress  <cit> .
microorganisms have therefore developed defense mechanisms that either keep the concentration of the o2-derived radicals at acceptable levels or repair oxidative damages.
in our previous proteomic experiments, oxidative stress proteins, such as kata and sodb, were shown to be induced in response to as stress in h. arsenicoxydans  <cit> .
similarly, we observed in the present study a positive regulation of three genes  involved in the protection against oxidative stress.
the sodc gene encodes the superoxide dismutase catalyzing the dismutation of the superoxide radical to h2o <dig>  which is less toxic, and o <dig>  in addition, kata and ahpc genes encoding a catalase and a peroxidase, respectively, are responsible for scarvenging endogenously produced h2o <dig>  finally, as early exposure induced a glutaredoxin and various thioredoxin encoding transcripts .
in h. arsenicoxydans the induction of genes encoding proteins involved in sulfate and gsh pathways suggests that gsh may play a key role in the cell defense against oxidative stress and metalloid toxicity by maintaining an intracellular reducing environment.
gsh may detoxify arsenite by i) chelation and then removing of arsenite from the cell, ii) protection against oxidation caused by metals since gsh serves as the main redox buffer in the cell, and iii) binding to reactive sulfhydryl groups of proteins thereby protecting them from irreversible metalloid binding and/or oxidative damages  <cit> .
thioredoxin is believed to serve as a cellular antioxidant by maintaining the intracellular redox state, which is greatly influenced by the presence or absence of intracellular ros, by reducing disulfide bonds produced by various oxidants and by interacting with proteins by direct association or thiol reactivity.
glutaredoxin is also known as a thioltransferase and like thioredoxin possesses an active center disulphide bond.
finally, arsenic could indirectly produce ros by inactivating catalytic iron-sulfur clusters of enzymes as it has been shown for enzymes of the deshydratase family in e. coli in the presence of copper.
in this organism, iron-sulfur clusters are the primary targets of copper and the resulting damages lead to the release of iron atoms in the cell and the generation of hydroxyl radicals  <cit> .
all these cellular activities  are thought to be part of a general set of reactions involved in the direct or indirect response to as-mediated production of reactive oxygen species in h. arsenicoxydans.
third, the adaptive responses of bacteria to sudden environmental changes, usually involve the induction of many major heat shock proteins mhsps, including chaperones, proteases, transcriptional regulators and other structural proteins.
as induced the expression of several genes encoding mhsps .
the mhsps play a role in repairing and preventing damages caused by an accumulation of unfolded proteins resulting from diverse environmental stresses.
in agreement with this, our microarray data showed that as early exposure induced the transcription of two uspa genes that might be involved in protecting bacterial cells against as stress.
the protein uspa has previously shown to be induced by as  <cit> .
the expression of the universal stress protein a  is known to be induced by a large variety of stress conditions  <cit> , our results show that these conditions include as.
finally, arsenic resistance in h. arsenicoxydans is partially mediated by proteins encoded by three different ars operons and one aox operon  <cit> .
the clusters of ars genes encode an arsr regulator, an as extrusion pump arsb/acr <dig>  an arsh putative flavoprotein and one or two arsenate reductases arsc.
one of the loci contains an acr3-type transporter, the others associate an arsb-type transporter with arsc reductases.
arsc is an enzyme that reduces as to as which can be pumped out by the arsb or acr <dig> membrane proteins.
both acr <dig> and arsb genes were induced in the presence of as.
however, acr <dig> mrnas were only detected in  <dig> min as exposed cells, which demonstrates that acr <dig> is specific to the early response.
remarkably, the arsenite oxidase genes were not induced in early phase.
the lack of induction of the aox genes is in agreement with the detection by hplc-ic-aes of 100% as .
ii) late phase
this phase contains genes whose expression levels gradually increased  or decreased , after arsenic exposure.
multiple genes of cellular processes, cell envelope and metabolism  were highly downregulated in class i .
class ii was characterized by the induction of several functional categories, i.e.
genes involved in metabolism, regulatory functions and cellular processes .
these genes are suggested to be specific for the late response to as.
first, h. arsenicoxydans reacted to a late as exposure by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds .
indeed, after late exposure, phosphate specific transporter  and phosphate inorganic transporter  were induced.
although we cannot exclude that the various genes involved in phosphate uptake were strongly induced because of a partial depletion of phophate in the medium, this induction could also result from the accumulation of as.
indeed, as produced by the oxidation of as, which only occurred in late phase , may compete with phosphate because of their structural similarity.
so, h. arsenicoxydans may preferentially transport phosphate via the specific pst phosphate transport system rather than the pit general transport mechanism, in order to reduce the entry of as.
these observations suggest that a complex regulation of the pst and pit operons allows h. arsenicoxydans to maintain its intracellular phosphate level despite the accumulation of as.
this further supports our previous hypothesis on the physiological link between arsenic and phosphate metabolism  <cit> .
second, genes involved in chemotactic machinery and flagellum assembly were up-regulated after late exposure to as.
our results from the microarray experiments during exposure to as combined to previous observations  <cit>  clearly demonstrate that the chemotactic response is induced by as.
in h. arsenicoxydans the flagellar machinery is organized in a mixed peritrichous/polar cascade which is most probably synthesized through sequential hierarchy of gene activation events initiated by the expression of the master transcriptional regulator flhdc  <cit> .
the microarray results strongly suggest that motility is gradually induced in response to as in h. arsenicoxydans.
finally, the aox operon was induced after late exposure.
the aox operon contains aoxa encoding the small subunit of arsenite oxidase, aoxb encoding the large subunit, aoxc encoding a nitroreductase and aoxd encoding a cytochrome c <dig>  the aoxrs operon is involved in the regulation of aoxabcd operon  <cit> .
all of them were induced by late as stress , which strongly suggests that arsenite oxidase activity is specific to late exposure.
transcriptional and physiological analysis of the major metabolic changes
our microarray data suggest that several genes play a major role in the transition from the absence of as to early or late arsenic exposure.
to confirm this hypothesis, chemical, transcriptional, physiological and biochemical experiments were performed.
first, to further support the involvement of glutathione synthesis and sulfate metabolism in early as response  in h. arsenicoxydans, we performed quantitative rt-pcr experiments.
transcripts abundance of gloa and soxc were compared to two internal controls, i.e the putative rna methyltransferase gene and the peptide deformylase gene, in cultures challenged  <dig> min,  <dig> hours or not by as.
the expression of soxc and gloa mrna was increased by a  <dig> and  <dig> fold factor, respectively, after  <dig> min as exposure.
no such induction was observed in late exposure.
to address the possible role of as in the gsh pathway, the total content of gsh was measured.
the pool of gsh increased after  <dig> min  and further rose over time .
after  <dig> hours the level remained unchanged, as compared with  <dig> hours.
these observations suggest that the accumulation of gsh in response to as was specific to the early phase.
in addition, although we did not observed any change in the extracellular concentration of sulfate, genes encoding sulfate uptake were up-regulated after  <dig> min and down regulated after  <dig> and  <dig> hours .
moreover, genes involved in the sulfur oxidation pathway, i.e.
soxc, soxd, were also induced .
a similar induction of enzymes involved in sulfur metabolism has already been shown under oxidative stress in pseudomonas aeruginosa  <cit> .
oxidative stress enzymes, i.e.
thioredoxin and superoxide dismutase, are known to be implicated in sulfur assimilation and metabolism  <cit> .
our results suggest that the intracellular concentration of different species of sulfur compounds is altered in h. arsenicoxydans under arsenic stress, demonstrating for the first time that sulfur metabolism is also impaired in response to arsenic stress.
on the other hand, to confirm the oxidative stress response resulting from arsenic exposure, peroxidase activity was measured from cultures induced or not,  <dig> min,  <dig>   <dig> hours with as.
the activity was induced  <dig> fold after  <dig> min and remained unchanged after late exposure.
this observation demonstrates that peroxidase activity is also specific to the early exposure to as.
second, the induction of arsenite oxidase activity in the late phase was demonstrated in this study .
the oxidation was complete after  <dig> hours exposed cells.
on agar plate, the activity was detectable after  <dig> hours growth in presence of as, by a silver nitrate staining test .
the accumulation of arsenite oxidase was tested by western immunoblotting experiments with anti-aoxb antibodies.
aoxb was detected in protein extract from cells induced for  <dig> and  <dig> hours.
in contrast, no protein was detected after  <dig> min induction  which explains the lack of aox gene expression after  <dig> min in the presence of as observed in a previous study  <cit> .
arsenite oxidase activity is therefore specific to late exposure to as in h. arsenicoxydans.
in addition, in as non exposed cultures of some bacteria, as oxidation did not occur until cultures reached stationary phase, as suggested in agrobacterium tumefaciens  <cit> , indicating that quorum sensing is involved in the regulation of as oxidation.
however, in h. arsenicoxydans, no significant induction of genes involved in quorum sensing was observed in the time course experiment.
similarly, to support the effect of arsenic on flagellar motility and phosphate transport in the late phase, the transcript abundance of flic and pstsb was measured.
pstsb mrnas increased by a  <dig> fold factor after  <dig> hours as exposure but no such induction was observed in early exposure.
remarkably, flic was induced by a  <dig> and  <dig> fold change after  <dig> min and  <dig> hours, respectively.
the motility of h. arsenicoxydans was analyzed in the presence and the absence of as.
as shown in figure  <dig> a swarming diameter of  <dig> mm was measured for cells incubated  <dig> hours whithout arsenic on the semi-solid medium.
in the presence of as, cells were mobile on semi-solid medium only after  <dig> hours incubation , which corresponds to the late phase in liquid medium.
these results suggest that the presence of as delays cell motility.
however, no significant difference in the expression of flagellar genes was observed between early and late exposure .
moreover, the expression of mota and motb genes, encoding proteins responsible for the rotary activity of flagellum motor  <cit> , was not affected by early or late exposure to as.
these observations clearly demonstrate that the flagellum biosynthesis was not altered in response to as, unlike its functioning.
in h. arsenicoxydans, the rotation of this flagellar appendange is driven by a sodium motive force  <cit> .
because the arsenite oxidase is known to be a periplamic enzyme, in agreement with western immunoblotting experiments on cell fractions , and the oxidation of arsenite releases four protons  <cit> , we proposed that arsenite oxidase activity may indirectly increase motility.
indeed, a na+/h+ antiporter has been shown to be involved in a. tumefaciens as oxidation  <cit> .
in h. arsenicoxydans, the genome exploration revealed the possible synthesis of such a na+/h+ transporter.
the protons released from arsenite oxidation may thus extrude na+ through this antiporter and generate the electron potential for the na+-driven flagellar motor, as described in the differentiation of flagellar system of vibrio alginolyticus  <cit> .
this hypothesis could explain how arsenite oxidase contributes to the motility of h. arsenicoxydans after as late exposure.
CONCLUSIONS
many microorganisms have acquired the capacity to survive in the presence of toxic metals or metalloids by inducing the expression of resistance genes.
the specific nature of these resistance mechanisms is the result of a cleverly designed genetic circuit that is tightly controlled by regulatory proteins.
however, the multiple physiological responses induced by arsenite stress in h. arsenicoxydans are not exclusively associated with the expression of classical arsenic resistance operons.
indeed, in this study we demonstrated that the adaptation to as is characterized by an early and a late response.
a highly sophisticated regulatory network may thus modulate the expression of genes in response to arsenic.
these effects are mediated in part through the activation or repression of mrna transcripts by dna-binding proteins and signal transduction systems, such as arsr, aoxrs and others.
all these transcriptomic observations are summarized in a kinetic and metabolic model .
the early response is characterized by the induction of genes involved in general stress.
first, gsh is a protagonist of the primary cellular responses to as.
the genes encoding enzymes involved in sulfate uptake , sulfur oxidation , cysteine synthesis  leading to cysteine containing proteins and their conversion in gsh  were induced.
as-exposed cells may thus channel a large part of the assimilated sulfur into gsh for metal chelation, cellular redox buffering and possibly also protein glutathionylation.
as may react with gsh to form an as <dig> complex .
interactions of these as-thiol group non specific complexes with molecular oxygen lead to the formation of reactive oxygen species such as h2o <dig>  resulting in oxidative stress within the cell.
the as is extruded by arsb or acr <dig>  and acr <dig> induction was demonstrated to be specific to early exposure.
the mechanism proposed here is in agreement with the observations made with hplc-icp-aes, showing that no oxidation occurred in early as stress and only as species was present.
the late response was characterized by the induction of genes specific for arsenic.
indeed, the gsh response is downregulated  and the transcripts encoding arsenic resistance , especially arsenite oxidase and its regulation  , and associated mechanisms such as phosphate uptake  and flagellum biosynthesis  were upregulated.
the arsenite oxidase catalyses the oxidation of as in as .
as may directly enter the cell via the pst or pit transporter , where it is reduced to as by the arsenate reductase and extruded into the periplasm via arsb .
then as produced by the oxidation of as is extruded out of the cell .
the mechanism proposed here for the late response to as is in agreement with the oxidoreduction kinetics and underlies the links between arsenic, phosphate and motility.
in this study, we demonstrated that the detoxification and metabolism processes are gradually expressed in h. arsenicoxydans to adapt to arsenic-rich environments.
in bacteria, the response to as seems therefore to be characterized by a stereotypic just-in-time transcription of specific metabolic pathways, resulting in a two-step response.

 bacteria live in changing environments and are subjected to a variety of environnmental stresses such as ph, temperature, osmolarity or heavy metals. arsenic is found in numerous disturbed or natural ecosystems where it can exist in mutiple oxidation states, the most common being arsenite as and arsenate as. this metalloid is known to generate oxidative stress in cells by its capability to induce the formation of reactive oxygen species. the damages caused by ros to lipids, proteins and dna are likely to contribute to as toxicity. in addition, one property of arsenite, which indicates that it behaves like a soft metal, consists in a high reactivity with sulphydryls groups and that affects the activity of many proteins. microorganisms have developed remarkable capabilities to cope with arsenic. the most common arsenic resistance mechanism depends on the presence on plasmid or chromosome of ars genes encoding proteins involved in the reduction and/or the efflux of the toxic element. nevertheless, other arsenic resistance mechanisms have been described, i.e. arsenite oxidation and arsenic methylation. in addition, in various microbial species, arsenic stress is associated with arsenite oxidase activity, biofilm formation, motility, oxidative stress or sulfur assimilation. for example, the biofilm development by thiomonas arsenivorans has been described as a physical barrier decreasing as access to sessile cells. remarkably, some organisms such as rhizobium sp. nt- have evolved specific metabolic pathways allowing them to oxidize as as an energy source or others are known to use as in anaerobic respiration. in the heterotrophic prokaryote herminiimonas arsenicoxydans, genome sequencing revealed the presence of four ars operons involved in the reduction of as to as and of one aoxab operon involved in the oxidation of the most toxic form, as to the less toxic form as. in addition to this detoxification process, this bacterium exhibits positive chemotaxis and motility towards arsenic, and metalloid scavenging by exopolyssacharides. the availability of the h. arsenicoxydans complete genome sequence offers an opportunity to study its physiology by functional genomic approaches. our previous transcriptomic studies have demonstrated that a large number of genes encoding proteins involved in oxidative stress, low affinity import of phosphate or dna repair are induced after min as exposure. however, no variation was found in the genes coding for arsenite oxidase, a key enzyme in arsenic response recently shown to be subjected to a complex regulation. in addition, little is known regarding the kinetics of arsenic stress response in microorganisms. to address these processes, physiological analyses coupled with western immunoblotting experiments and dna microarrays were used to examine the temporal changes in transcriptome profiles during the transition from as to as species due to as oxidation. our work represents, to our knowledge, the first kinetic analysis of transcription pattern in bacteria exposed to arsenic, leading to propose a global model of arsenic response in h. arsenicoxydans.  characterization of arsenic oxidoreduction kinetics to study the oxidoreduction kinetic in h. arsenicoxydans, the two arsenic species and as) were quantified by hplc-icp-aes from filtered culture supernatants at several times after as or as induction. in the presence of as, three distinct phases were observed: in phase a, no oxidation was observed since arsenic was present as 100% as. in phase a which corresponds to a partial oxidation of as into as, both species were present, a proportion of 50% as and 50% as being observed after hours. the transformation of as into as was complete after hours. overview of transcriptome profiles to get insight into the mechanisms involved in arsenic response, including oxidoreduction processes, transcriptomic analyses were performed at different steps of the experimental time course. cells were collected from cultures of h. arsenicoxydans induced min, hours, hours or not with mm as or mm as. these concentrations were choosen as they are far from h. arsenicoxydans mic for as or as, which is able to grow up to mm and mm of as and as, respectively. moreover, this concentration of as had only a minor impact on growth rate and cells continued to divide immediately after its addition. under as stress, genes were up-regulated and genes were down-regulated in the early phase. in the late phase the number of genes up-regulated and down-regulated increased. under as stress, the proportion was inverted. the major shift in gene expression occured during the onset of the early phase with genes up-regulated and genes down-regulated under as stress. this represents approximatly 14% of the genes whose expression was significantly affected by as. as cell growth progressed further into the late phase, the number of genes up-regulated and down-regulated decreased, representing 4% of the genome. the large number of genes responding to as, especially in the early phase, suggests a major remodelling of cellular physiology. in addition, oxidoreduction kinetic was only observed under as stress, suggesting that the aox-dependent oxidation largely predominate the ars-dependent reduction process. this study therefore focused on the as response in h. arsenicoxydans. hierachical clustering kinetics to classify the transcripts on the basis of their temporal dynamics after exposure to arsenite, we performed a hierarchical clustering with the arrayminer® software http://www.optimaldesign.com/arrayminer/arrayminer.htm. genes were sorted into groups that exhibited similar transcription kinetics as cells transitioned from a to a phases. the set of genes regulated by as shown in figure was classified into clusters. the profiles similarity of those clusters enabled us to identify two major classes: 1) class i: containing genes whose expression increased immediately after min induction and decreased after and hours and class ii: containing genes whose expression levels increased only after and hours induction. the product of each upregulated or downregulated gene by a fold change of two or more was identified to gain insight into the changes in physiology. next, to examine how the two classes are distributed with regard to their functions, we further classified these genes according to the gene classification based on cog functional categories. the overall gene expression pattern suggests the existence of two major physiologic phases during the adaptation to as stress. i) early phase our microarray analysis highlighted a set of stress responsive genes whose transcription was early affected by as. this phase contained the early up-regulated genes whose expression levels gradually decreased after the induction or down-regulated genes whose expression levels gradually increased after the induction. first, σe and several genes encoding proteins involved in the synthesis of cell envelope components were down-regulated ) whereas genes encoding proteins involved in transport were up-regulated ). the bacterial envelope is a major defense against threats from the environment. due to its crucial importance, the integrity of the cell envelope, including the presence of outer membrane proteins, is closely monitored to ensure its functionality. the σe system is the major pathway required for proper folding of omps and their turnover, phospholipids and lipopolysaccharide biosynthesis, signal transduction and the expression of inner and outer membrane proteins. in escherichia coli, the σe regulon is induced specifically in response to the unbalanced synthesis of outer membrane proteins and to the misfolding of polypeptides that have been translocated across the cytoplasmic membrane. our results suggest that both the organization and the functioning of the bacterial envelope may be changed in the early phase of the response to as. second, early as stress strongly stimulated expression of genes encoding components of sulfur assimilation, sulfur oxidation and glutathione biosynthesis pathways. in fact, mrna levels of genes encoding all the enzymatic steps required for sulfate uptake, its conversion into cysteine and gsh were increased. the expression of these genes was induced about to fold after min induction with as. the expression of some of these genes was down-regulated at and hours. in eukaryotic cells, as exposure results in increased levels of reduced glutathione and an as-triglutathione complex has been found in human liver excreta, leading to the conclusion that gsh plays an important role in reducing as toxicity. most effects of arsenite result from its interaction with thiol groups of proteins, its iniophoretic properties and its ability to directly or indirectly generate free radicals and hence induce oxidative stress. microorganisms have therefore developed defense mechanisms that either keep the concentration of the o2-derived radicals at acceptable levels or repair oxidative damages. in our previous proteomic experiments, oxidative stress proteins, such as kata and sodb, were shown to be induced in response to as stress in h. arsenicoxydans. similarly, we observed in the present study a positive regulation of three genes involved in the protection against oxidative stress. the sodc gene encodes the superoxide dismutase catalyzing the dismutation of the superoxide radical to h2o which is less toxic, and o in addition, kata and ahpc genes encoding a catalase and a peroxidase, respectively, are responsible for scarvenging endogenously produced h2o finally, as early exposure induced a glutaredoxin and various thioredoxin encoding transcripts. in h. arsenicoxydans the induction of genes encoding proteins involved in sulfate and gsh pathways suggests that gsh may play a key role in the cell defense against oxidative stress and metalloid toxicity by maintaining an intracellular reducing environment. gsh may detoxify arsenite by i) chelation and then removing of arsenite from the cell, ii) protection against oxidation caused by metals since gsh serves as the main redox buffer in the cell, and iii) binding to reactive sulfhydryl groups of proteins thereby protecting them from irreversible metalloid binding and/or oxidative damages. thioredoxin is believed to serve as a cellular antioxidant by maintaining the intracellular redox state, which is greatly influenced by the presence or absence of intracellular ros, by reducing disulfide bonds produced by various oxidants and by interacting with proteins by direct association or thiol reactivity. glutaredoxin is also known as a thioltransferase and like thioredoxin possesses an active center disulphide bond. finally, arsenic could indirectly produce ros by inactivating catalytic iron-sulfur clusters of enzymes as it has been shown for enzymes of the deshydratase family in e. coli in the presence of copper. in this organism, iron-sulfur clusters are the primary targets of copper and the resulting damages lead to the release of iron atoms in the cell and the generation of hydroxyl radicals. all these cellular activities are thought to be part of a general set of reactions involved in the direct or indirect response to as-mediated production of reactive oxygen species in h. arsenicoxydans. third, the adaptive responses of bacteria to sudden environmental changes, usually involve the induction of many major heat shock proteins mhsps, including chaperones, proteases, transcriptional regulators and other structural proteins. as induced the expression of several genes encoding mhsps. the mhsps play a role in repairing and preventing damages caused by an accumulation of unfolded proteins resulting from diverse environmental stresses. in agreement with this, our microarray data showed that as early exposure induced the transcription of two uspa genes that might be involved in protecting bacterial cells against as stress. the protein uspa has previously shown to be induced by as. the expression of the universal stress protein a is known to be induced by a large variety of stress conditions, our results show that these conditions include as. finally, arsenic resistance in h. arsenicoxydans is partially mediated by proteins encoded by three different ars operons and one aox operon. the clusters of ars genes encode an arsr regulator, an as extrusion pump arsb/acr an arsh putative flavoprotein and one or two arsenate reductases arsc. one of the loci contains an acr3-type transporter, the others associate an arsb-type transporter with arsc reductases. arsc is an enzyme that reduces as to as which can be pumped out by the arsb or acr membrane proteins. both acr and arsb genes were induced in the presence of as. however, acr mrnas were only detected in min as exposed cells, which demonstrates that acr is specific to the early response. remarkably, the arsenite oxidase genes were not induced in early phase. the lack of induction of the aox genes is in agreement with the detection by hplc-ic-aes of 100% as. ii) late phase this phase contains genes whose expression levels gradually increased or decreased, after arsenic exposure. multiple genes of cellular processes, cell envelope and metabolism were highly downregulated in class i. class ii was characterized by the induction of several functional categories, i.e. genes involved in metabolism, regulatory functions and cellular processes. these genes are suggested to be specific for the late response to as. first, h. arsenicoxydans reacted to a late as exposure by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. indeed, after late exposure, phosphate specific transporter and phosphate inorganic transporter were induced. although we cannot exclude that the various genes involved in phosphate uptake were strongly induced because of a partial depletion of phophate in the medium, this induction could also result from the accumulation of as. indeed, as produced by the oxidation of as, which only occurred in late phase, may compete with phosphate because of their structural similarity. so, h. arsenicoxydans may preferentially transport phosphate via the specific pst phosphate transport system rather than the pit general transport mechanism, in order to reduce the entry of as. these observations suggest that a complex regulation of the pst and pit operons allows h. arsenicoxydans to maintain its intracellular phosphate level despite the accumulation of as. this further supports our previous hypothesis on the physiological link between arsenic and phosphate metabolism. second, genes involved in chemotactic machinery and flagellum assembly were up-regulated after late exposure to as. our results from the microarray experiments during exposure to as combined to previous observations clearly demonstrate that the chemotactic response is induced by as. in h. arsenicoxydans the flagellar machinery is organized in a mixed peritrichous/polar cascade which is most probably synthesized through sequential hierarchy of gene activation events initiated by the expression of the master transcriptional regulator flhdc. the microarray results strongly suggest that motility is gradually induced in response to as in h. arsenicoxydans. finally, the aox operon was induced after late exposure. the aox operon contains aoxa encoding the small subunit of arsenite oxidase, aoxb encoding the large subunit, aoxc encoding a nitroreductase and aoxd encoding a cytochrome c the aoxrs operon is involved in the regulation of aoxabcd operon. all of them were induced by late as stress, which strongly suggests that arsenite oxidase activity is specific to late exposure. transcriptional and physiological analysis of the major metabolic changes our microarray data suggest that several genes play a major role in the transition from the absence of as to early or late arsenic exposure. to confirm this hypothesis, chemical, transcriptional, physiological and biochemical experiments were performed. first, to further support the involvement of glutathione synthesis and sulfate metabolism in early as response in h. arsenicoxydans, we performed quantitative rt-pcr experiments. transcripts abundance of gloa and soxc were compared to two internal controls, i.e the putative rna methyltransferase gene and the peptide deformylase gene, in cultures challenged min, hours or not by as. the expression of soxc and gloa mrna was increased by a and fold factor, respectively, after min as exposure. no such induction was observed in late exposure. to address the possible role of as in the gsh pathway, the total content of gsh was measured. the pool of gsh increased after min and further rose over time. after hours the level remained unchanged, as compared with hours. these observations suggest that the accumulation of gsh in response to as was specific to the early phase. in addition, although we did not observed any change in the extracellular concentration of sulfate, genes encoding sulfate uptake were up-regulated after min and down regulated after and hours. moreover, genes involved in the sulfur oxidation pathway, i.e. soxc, soxd, were also induced. a similar induction of enzymes involved in sulfur metabolism has already been shown under oxidative stress in pseudomonas aeruginosa. oxidative stress enzymes, i.e. thioredoxin and superoxide dismutase, are known to be implicated in sulfur assimilation and metabolism. our results suggest that the intracellular concentration of different species of sulfur compounds is altered in h. arsenicoxydans under arsenic stress, demonstrating for the first time that sulfur metabolism is also impaired in response to arsenic stress. on the other hand, to confirm the oxidative stress response resulting from arsenic exposure, peroxidase activity was measured from cultures induced or not, min, hours with as. the activity was induced fold after min and remained unchanged after late exposure. this observation demonstrates that peroxidase activity is also specific to the early exposure to as. second, the induction of arsenite oxidase activity in the late phase was demonstrated in this study. the oxidation was complete after hours exposed cells. on agar plate, the activity was detectable after hours growth in presence of as, by a silver nitrate staining test. the accumulation of arsenite oxidase was tested by western immunoblotting experiments with anti-aoxb antibodies. aoxb was detected in protein extract from cells induced for and hours. in contrast, no protein was detected after min induction which explains the lack of aox gene expression after min in the presence of as observed in a previous study. arsenite oxidase activity is therefore specific to late exposure to as in h. arsenicoxydans. in addition, in as non exposed cultures of some bacteria, as oxidation did not occur until cultures reached stationary phase, as suggested in agrobacterium tumefaciens, indicating that quorum sensing is involved in the regulation of as oxidation. however, in h. arsenicoxydans, no significant induction of genes involved in quorum sensing was observed in the time course experiment. similarly, to support the effect of arsenic on flagellar motility and phosphate transport in the late phase, the transcript abundance of flic and pstsb was measured. pstsb mrnas increased by a fold factor after hours as exposure but no such induction was observed in early exposure. remarkably, flic was induced by a and fold change after min and hours, respectively. the motility of h. arsenicoxydans was analyzed in the presence and the absence of as. as shown in figure a swarming diameter of mm was measured for cells incubated hours whithout arsenic on the semi-solid medium. in the presence of as, cells were mobile on semi-solid medium only after hours incubation, which corresponds to the late phase in liquid medium. these results suggest that the presence of as delays cell motility. however, no significant difference in the expression of flagellar genes was observed between early and late exposure. moreover, the expression of mota and motb genes, encoding proteins responsible for the rotary activity of flagellum motor, was not affected by early or late exposure to as. these observations clearly demonstrate that the flagellum biosynthesis was not altered in response to as, unlike its functioning. in h. arsenicoxydans, the rotation of this flagellar appendange is driven by a sodium motive force. because the arsenite oxidase is known to be a periplamic enzyme, in agreement with western immunoblotting experiments on cell fractions, and the oxidation of arsenite releases four protons, we proposed that arsenite oxidase activity may indirectly increase motility. indeed, a na+/h+ antiporter has been shown to be involved in a. tumefaciens as oxidation. in h. arsenicoxydans, the genome exploration revealed the possible synthesis of such a na+/h+ transporter. the protons released from arsenite oxidation may thus extrude na+ through this antiporter and generate the electron potential for the na+-driven flagellar motor, as described in the differentiation of flagellar system of vibrio alginolyticus. this hypothesis could explain how arsenite oxidase contributes to the motility of h. arsenicoxydans after as late exposure.  many microorganisms have acquired the capacity to survive in the presence of toxic metals or metalloids by inducing the expression of resistance genes. the specific nature of these resistance mechanisms is the result of a cleverly designed genetic circuit that is tightly controlled by regulatory proteins. however, the multiple physiological responses induced by arsenite stress in h. arsenicoxydans are not exclusively associated with the expression of classical arsenic resistance operons. indeed, in this study we demonstrated that the adaptation to as is characterized by an early and a late response. a highly sophisticated regulatory network may thus modulate the expression of genes in response to arsenic. these effects are mediated in part through the activation or repression of mrna transcripts by dna-binding proteins and signal transduction systems, such as arsr, aoxrs and others. all these transcriptomic observations are summarized in a kinetic and metabolic model. the early response is characterized by the induction of genes involved in general stress. first, gsh is a protagonist of the primary cellular responses to as. the genes encoding enzymes involved in sulfate uptake, sulfur oxidation, cysteine synthesis leading to cysteine containing proteins and their conversion in gsh were induced. as-exposed cells may thus channel a large part of the assimilated sulfur into gsh for metal chelation, cellular redox buffering and possibly also protein glutathionylation. as may react with gsh to form an as complex. interactions of these as-thiol group non specific complexes with molecular oxygen lead to the formation of reactive oxygen species such as h2o resulting in oxidative stress within the cell. the as is extruded by arsb or acr and acr induction was demonstrated to be specific to early exposure. the mechanism proposed here is in agreement with the observations made with hplc-icp-aes, showing that no oxidation occurred in early as stress and only as species was present. the late response was characterized by the induction of genes specific for arsenic. indeed, the gsh response is downregulated and the transcripts encoding arsenic resistance, especially arsenite oxidase and its regulation, and associated mechanisms such as phosphate uptake and flagellum biosynthesis were upregulated. the arsenite oxidase catalyses the oxidation of as in as. as may directly enter the cell via the pst or pit transporter, where it is reduced to as by the arsenate reductase and extruded into the periplasm via arsb. then as produced by the oxidation of as is extruded out of the cell. the mechanism proposed here for the late response to as is in agreement with the oxidoreduction kinetics and underlies the links between arsenic, phosphate and motility. in this study, we demonstrated that the detoxification and metabolism processes are gradually expressed in h. arsenicoxydans to adapt to arsenic-rich environments. in bacteria, the response to as seems therefore to be characterized by a stereotypic just-in-time transcription of specific metabolic pathways, resulting in a two-step response. 
upon a longer arsenic exposure, a broad metabolic response was induced. the early response was characterized by arsenic resistance, oxidative stress, chaperone synthesis and sulfur metabolism. in particular our previous studies have suggested the existence of a temporal induction of arsenite oxidase, a key enzyme in arsenic metabolism, in the presence of as. the major metabolic changes were confirmed by chemical, transcriptional, physiological and biochemical experiments. the late response was characterized by arsenic metabolism and associated mechanisms such as phosphate transport and motility. herminiimonas arsenicoxydans, a bacterium isolated from arsenic contaminated sludge, has acquired remarkable capabilities to cope with arsenic. transcriptome kinetic analysis demonstrated the existence of two phases in arsenic response. these early and late responses were defined as general stress response and specific response to as, respectively. the expression of approximatively 14% of the whole genome was significantly affected by an as early stress and 4% by an as late exposure.
 arsenic is present in numerous ecosystems and microorganisms have developed various mechanisms to live in such hostile environments. herminiimonas arsenicoxydans, a bacterium isolated from arsenic contaminated sludge, has acquired remarkable capabilities to cope with arsenic. in particular our previous studies have suggested the existence of a temporal induction of arsenite oxidase, a key enzyme in arsenic metabolism, in the presence of as.  microarrays were designed to compare gene transcription profiles under a temporal as exposure. transcriptome kinetic analysis demonstrated the existence of two phases in arsenic response. the expression of approximatively 14% of the whole genome was significantly affected by an as early stress and 4% by an as late exposure. the early response was characterized by arsenic resistance, oxidative stress, chaperone synthesis and sulfur metabolism. the late response was characterized by arsenic metabolism and associated mechanisms such as phosphate transport and motility. the major metabolic changes were confirmed by chemical, transcriptional, physiological and biochemical experiments. these early and late responses were defined as general stress response and specific response to as, respectively.  gene expression patterns suggest that the exposure to as induces an acute response to rapidly minimize the immediate effects of as. upon a longer arsenic exposure, a broad metabolic response was induced. these data allowed to propose for the first time a kinetic model of the as response in bacteria. 