BACKGROUND
arsenic is present in various environments, released from either anthropogenic or natural sources.
this element is toxic for living organisms and known to be a human carcinogen  <cit> .
its toxicological effects depend, at least in part, on its oxidation state and its chemical forms, inorganic species being considered as more toxic  <cit> .
the contamination of drinking water by the two inorganic forms, arsenite as and arsenate as, has been reported in different parts of the world  <cit>  and constitutes a major threat of public health.
microorganisms are known to take part in the transformation, i.e oxidation, reduction or methylation of the metalloid, having a deep impact on arsenic contamination in environment.
several bacteria and prokaryotes have developed adaptation, resistance and colonization mechanisms, which allow them to live in hostile arsenic contaminated environments.
h. arsenicoxydans is a gram-negative β-proteobacterium isolated from an industrial activated sludge plant and exhibiting a remarkable set of arsenic resistance determinants  <cit> .
the h. arsenicoxydans adaptive response to arsenic is organized in a complex and sophisticated network.
in particular, differential proteome studies have recently demonstrated the synthesis of several proteins encoded by the three ars resistance operons, e.g.
arsenate reductase arsc, flavoprotein arsh and regulator arsr  <cit>  and the induction of oxidative stress protein encoding genes, e.g.
catalase , superoxide dismutase  and alkyl hydroperoxide reductase   <cit> .
one of the most noticeable response to arsenic in h. arsenicoxydans is the ability of this bacterium to oxidize as to as, a less toxic and less mobile form, via an arsenite oxidase activity.
the two genes coding for this heterodimeric enzyme are organized in an operonic structure, and have been named aoxa and aoxb for the small and the large subunit, respectively  <cit> .
homologous genes have been since identified in various microorganisms  <cit> .
in agrobacterium tumefaciens, a complex transcriptional regulation has been recently suggested, involving as sensing, two-component signal transduction by an aoxs sensor kinase and an aoxr regulator, and quorum sensing  <cit> .
nevertheless, the molecular mechanisms involved in the control of arsenite oxidase expression remain largely unknown.
to extend our view of arsenic response  <cit> , in particular with respect to the arsenic oxidation process, we conducted a comprehensive transcriptomic, genetic and molecular analysis of h. arsenicoxydans following exposure to as.
these approaches allowed us to identify major determinants involved in the control of arsenite oxidation.
RESULTS
gene expression profiling in response to arsenic
the response to as was analyzed in exponentially growing cells using microarrays.
the data from three biological replicates were combined after normalization and statistical analysis carried out using the r software and packages http://www.r-project.org.
the set of genes was further refined to include only those genes that showed a valid p-value and whose expression was altered by a factor of  <dig> or more when compared to the level measured in the absence of arsenic.
this experiment led to the identification of  <dig> genes showing an arsenic-induced expression change ).
among these genes,  <dig>  were up-regulated and the remaining part, i.e.
<dig> genes, was down-regulated.
the relative changes in gene expression ranged from a 11-fold down-regulation  to a 126-fold up-regulation .
a list of those genes is shown in additional file  <dig>  table s <dig>  the corresponding hear gene numbers are available in the arsenoscope relational database http://www.genoscope.cns.fr/agc/mage/arsenoscopevia the mage web interface  <cit> .
the  <dig> genes differentially expressed were clustered according to the function of the corresponding encoded proteins.
among the  <dig> genes that were induced by at least a 2-fold factor, about 11% are involved in metabolism, 26% in transport and binding protein, 26% in cellular processes and 31% have no assigned function.
the high percentage of genes with unknown function is in accordance with the proportion of unknown function proteins identified in the genome of h. arsenicoxydans  <cit> .
in agreement with our previous results, genes involved in arsenic resistance, phosphate transport and flagellar biosynthesis were induced in the presence of as , further supporting the relationship between these physiological processes  <cit> .
interestingly, only one methyl-accepting chemotaxis protein  gene was induced in our microarray data, suggesting a role for this protein in the sensing of arsenic.
this mechanism is currently under investigation.
genes encoding the putative nitroreductase aoxc and the cytochrome c <dig> precursor aoxd as well as the response regulator aoxrs were found to be induced by as .
aoxr has been proposed to act as a positive regulator of the aox operon upon phosphorylation by aoxs in a. tumefaciens  <cit> .
our transcriptomic data suggest that the regulation machinery is, at least in part, similar in h. arsenicoxydans.
futhermore, genes coding for the arsenite oxidase aoxab subunits were found to be among the most induced genes in the presence of as.
however, even though these results extended our knowledge of the arsenic response in h. arsenicoxydans, they did not led to a better understanding of the molecular mechanisms involved in the control of arsenite oxidation.
this prompted us to perform a transposon mutagenesis experiment.
identification of arsenite oxidase accessory genes by screening an aox activity deficient mutant library
to identify genes possibly involved in the control of arsenite oxidation in h. arsenicoxydans, a library of  <dig>  kanamycin resistant mutants was constructed by transposon mutagenesis, as previously described  <cit> .
these clones were tested by silver nitrate staining  <cit>  for arsenate production on as-supplemented cdm agar plates.
as compared to the wild-type strain, whose arsenite oxidase activity was revealed by a brownish precipitate,  <dig> mutants with a lack of as oxidase activity were obtained.
these strains showed no precipitate , as observed for the m <dig> and m <dig> strains used as negative controls.
indeed, these strains carry a mutation in aoxa or aoxb genes coding for the small and the large subunit of arsenite oxidase, respectively  <cit> .
genes disrupted by transposon insertions were identified in these  <dig> new mutants.
as expected, four of the  <dig> mutants showed insertions in the aoxab operon .
more interestingly, six mutants carried a transposon insertion outside the aoxab operon.
two mutants were found to be affected in the aoxrs two-component signal transduction system  located upstream of the aoxab operon in h. arsenicoxydans  <cit>  .
these results further support our transcriptomic data suggesting that these two genes play a role in arsenic response.
two transposon insertions were shown to disrupt genes of the modeabc operon coding for a molybdenum high-affinity transport system  <cit> , i.e.
modc encoding an atp-binding cassette transport protein  and modb encoding a molybdenum transport system permease  .
remarkably, transposon insertions were also located in dnaj encoding a heat shock protein ,   and in rpon encoding the alternative nitrogen sigma factor  of rna polymerase  .
two additional experiments were performed to complete the physiological characterization of these mutants with respect to arsenite oxidation.
first, arsenic species were quantified by hplc-icp-aes on filtered culture supernatants.
h. arsenicoxydans was grown in liquid medium supplemented with  <dig>  mm arsenite and showed 100% transformation of as into as after  <dig> h, whereas m <dig>  and m <dig>  mutants used as controls were not able to transform as into as.
the same loss of arsenite oxidase activity was measured in ha <dig> , ha <dig> , ha <dig>  and ha <dig>  mutants.
in contrast to the results obtained on agar plates, ha <dig>  and ha <dig>  strains showed 100% transformation of arsenite .
previous studies have demonstrated that the bioavailability of metals or trace elements considerably varies according to the type of matrix used for microbial growth  <cit> .
we therefore assumed that mo might be partly sequestred on cdm agar medium, resulting in a lack of arsenite oxidase activity on plate.
to test this hypothesis, as oxidase tests were performed on cdm agar plates supplemented with  <dig> μm mo.
the addition of mo to the solid medium restored as oxidase activity in both ha <dig>  and ha <dig>  mutants while it had no effect on other mutant strains .
determined by hplc-icp-aes after  <dig> h growth in cdm medium containing  <dig> mg.liter of arsenite.
b  <cit>
second, we have previously demonstrated that the polar flagellum-dependent motility of h. arsenicoxydans is increased in the presence of as, suggesting that arsenite oxidation may result in a gain of energy  <cit> .
the motility of mutant strains was therefore tested on plates containing different concentrations of as, i. e.  <dig>  mm,  <dig>  mm and  <dig> mm.
the diameter of the swarming rings was measured after  <dig> h. as shown in figure  <dig>  the disruption of aoxa, aoxb, aoxr, aoxs or rpon genes abolished the improvement of swarming performances in the presence of as.
unlike those mutants, a disruption in dnaj completely abolished the motility of h. arsenicoxydans in the presence or the absence of as.
dnaj is known to be essential for the expression of the flhdc flagellar master operon in escherichia coli  <cit> .
the lack of motility observed in the dnaj mutant suggests the existence of a similar flhdc-dependent regulation of flagellar genes in h. arsenicoxydans.
more importantly, the dnaj mutant was affected in both the motility and arsenite oxidation, suggesting that these two mechanisms are co-regulated.
effect of aoxr, aoxs, rpon and dnaj on arsenite oxidase synthesis
to get further insight into the involvement of aoxr, aoxs, rpon and dnaj in arsenite oxidase activity, western immunoblotting experiments were performed using antibodies raised against aoxb.
the abundance of this protein was evaluated from total protein extracts of h. arsenicoxydans wild-type and mutant strains grown in the presence or not of as.
aoxb was detected as a single band corresponding to a molecular mass of  <dig> kda in as-challenged h. arsenicoxydans strain .
this single band was not observed in the various mutant strains.
furthermore, arsenite oxidase activity on native gel was only detected in as-challenged wild type total extract .
taken together these results suggest that the lack of activity in the mutant strains is due to the absence of aoxb protein, which may result from an effect of aoxr, aoxs, rpon and dnaj on aoxab expression.
effect of aoxr, aoxs, rpon and dnaj on the control of arsenite oxidase operon expression
to determine the involvement of aoxr, aoxs, dnaj and rpon on aoxab transcription, we performed quantitative rt-pcr experiments.
for each strain, changes in aoxb transcript abundance were compared to two internal controls, i.e.
the putative rna methyltransferase gene and the peptide deformylase gene, in cultures challenged or not by as.
the expression of aoxb mrna was increased by a  <dig>  fold factor after as exposure in the h. arsenicoxydans wild-type strain.
in contrast, aoxb expression was not increased in ha <dig> , ha <dig> , ha <dig>  and ha <dig>  mutant strains, suggesting that the corresponding proteins play a crucial role in aoxab operon expression .
strain
ratio
error
expression ratios of aoxb in h. arsenicoxydans wild-type and mutant strains without as versus an as  <dig> hours induction , as measured by quantitative rt-pcr.
expression of each gene was normalized to the expression of the two housekeeping genes hear <dig> and hear <dig> coding for a peptide deformylase and a putative rna methyltransferase, respectively.
standard errors were calculated from the data of two quantitative pcr replicates obtained from two independent biological replicates.
bold text indicates statistically significant induction.
molecular mechanisms of arsenite oxidase transcription
the aoxr and aoxs genes encode a two-component system while rpon encodes a sigma factor which recognizes a particular promoter with a specific -12/- <dig> binding site.
these three proteins may therefore play a role in the initiation of aoxab transcription.
to get further insight into the molecular interactions between those regulators and the aoxab promoter, we mapped the transcriptional start site of this operon by the amplification of aoxab cdna ends and 5'race.
messenger rnas were extracted from induced ) and non induced h. arsenicoxydans wild-type strain cultures.
a single transcriptional start site was identified from induced cells at - <dig> bp relative to the translation start codon, while no transcriptional start site was identified from non induced cells.
in agreement with this, a tggcacgcagtttgc putative -12/- <dig> σ54-dependent promoter motif was identified upstream of the aoxab transcriptional start site .
in addition, multiple alignment of aoxab promoter sequences present in databases revealed a similarity to promoters recognized by σ <dig> in a. tumefaciens, thiomonas sp., rhizobium sp.
nt- <dig>  achromobacter sp., rhodoferax ferrireducens, ochrobactrum tritici .
in contrast, no such σ54-dependent promoter motif was found in several strains containing the aoxab operon but lacking the two-component transduction system aoxrs operon, such as chloroflexus aurantiacus, chlorobium limicola, thermus thermophilus, burkholderia multivorans, roseobacter litoralis, pseudomonas sp.ts <dig>  chlorobium phaeobacteroides and chloroflexus aggregans .
remarkably, a multiple alignment of amino acid sequences revealed that aoxr shares significant homology with a number of σ <dig> rna polymerase transcriptional activators, i.e.
<dig> % identity with zrar and  <dig> % identity with atoc from e. coli k <dig>  aoxr contains three conserved domains shared by most enhancer binding proteins , namely a n-terminal response regulator receiver domain , a central σ <dig> interaction domain  common to all σ <dig> dependent ebps  and a c-terminal dna binding helix-turn-helix  domain  enable to bind to specific upstream activation sequences  <cit> .
aoxr shares similarities with several ebps of σ <dig> essential for the formation of an open complex formation during σ54-dependent transcriptional initiation, in particular the σ <dig> activator sequence gaftga loop  <dig> which directly binds to σ <dig> conserved region iii   <cit> .
taken together, these observations strongly suggest that aoxr interacts directly with rpon to initiate the transcription of aoxab operon in h. arsenicoxydans.
discussion
despite many works devoted to arsenic metabolism in microorganisms, little is known about the regulation of arsenite oxidase activity.
in the present study, the combination of transcriptomic, genetic and molecular data provided a comprehensive view of the role of various proteins in the control of arsenite oxidation in h. arsenicoxydans .
we showed that some proteins play an indirect role in this process, as their presence is not essential for aoxab synthesis.
in this respect, the aoxb large subunit contains a mo site required in arsenite oxidase enzymatic activity  <cit> .
ha <dig>  and ha <dig>  mutations were located in the molybdenum high-affinity transport system operon, which further support the key role of this element in enzyme activity.
in addition, the recovery of as oxidase activity in these two mutants in the presence of an excess molybdenum suggests that mo may also be transported through an alternative uptake system in mod mutants, e.g.
a low-affinity uptake system involving non specific permeases such as hear <dig>  hear <dig>  hear <dig> or hear <dig> or a sulfate transport system, as described in e. coli mod mutants  <cit> .
more importantly, our results suggest that aoxr and rpon constitute a transcriptional complex that play a major role in the initiation of aoxab operon transcription.
three mutants, i.e.
ha <dig> , ha <dig>  and ha <dig> , were affected in this process.
the amino acids sequence analysis of h. arsenicoxydans aoxr and aoxs revealed the existence in these proteins of structural features common to partners of two-component signal transduction systems, which are composed of a sensor kinase and a response regulator  <cit> .
moreover, the comparison of aoxs and aoxr protein sequences with those of a. tumefaciens revealed similarities.
indeed, the aoxs protein sequence contains short blocks of conserved motifs that are consistent with a role of sensor histidine kinase, e.g.
the "h" , the "g2"  and the "n"  domains.
in addition, four highly invariant residues playing a central role in phosphorylation correspond to asp <dig>  asp <dig>  asp <dig> and lys <dig> in the h. arsenicoxydans aoxr protein.
the three asparagine residues are known to interact together to form an "acid pocket", characteristic of orthodox receivers, into which the side chain of lys <dig> protrudes  <cit> .
finally, a transmembrane region and a  <dig> amino acids residue cluster possibly exposed to the periplasm are present in aoxs and could serve as a signal receptor in the presence of as in the medium.
the detection of as would then lead to aoxs autophosphorylation at a histidine residue via atp hydrolysis and phosphotransfer to an aspartate residue in the response regulator aoxr, as recently proposed in a. tumefaciens  <cit> .
remarkably, our results demonstrated for the first time that the alternative n sigma factor  is essential for the initiation of arsenite oxidase transcription.
indeed, a mutation in the corresponding gene led to a complete loss of as oxidation and aoxb transcription in ha <dig> .
σ <dig> is one of the alternative sigma subunits of rna polymerase responsible for specific binding to dna.
the core rna polymerase complexed with σ <dig> is usually associated with nitrogen assimilation and fixation, but is also known to play a role in various physiological processes, e.g.
flagellar synthesis, carbon source utilization or bacterial virulence  <cit> .
to date, only one report has shown that σ <dig> participates in the transcription of genes possibly involved in metal tolerance, i.e.
the zrar/s genes that code for a zinc and lead responsive two-component regulatory system in e. coli  <cit> .
rna polymerase together with σ <dig> binds to a specific promoter site, with the consensus dna sequence ytggcacgnnnnttgcwnnw  <cit> , forming a transcriptionally inactive closed complex.
such a characteristic -12/- <dig> σ54-dependent promoter motif, i.e.
tggcacgcagtttgc, was identified  <dig> pb upstream of the transcriptional initiation codon of aoxab with respect to the + <dig> transcriptional start site , which confirmed the need for rpon in the initiation of aoxab transcription.
changes in the conformation of σ54-rna polymerase are nucleotide dependent.
indeed, the dna melting step absolutely requires the interaction with a transcriptional activator protein.
most of these σ54-dependent activators share three domains found in aoxr, i.e.
a c-terminal dna binding domain that binds to upstream activation sequences, a conserved central domain belonging to the aaa+  protein family to proceed with initiation of transcription and a n-terminal receiver domain that regulates its own aaa+ domain  <cit> .
a multiple alignment of the central domain revealed a conservation of a common architecture between aoxr and σ <dig> ebps.
indeed, seven highly conserved sequence motifs corresponding to a σ <dig> interaction domain of aoxr further support the direct interaction of aoxr with rpon to stimulate the transcription of aoxab operon in h. arsenicoxydans .
this central σ <dig> interaction domain has been already used to identify new σ <dig> ebps  <cit> .
it contains the highly conserved signature sequence gaftga loop  proved to be required for direct interaction with σ <dig>  <cit> , the walker a  and b  atpase motifs necessary for the open complex formation.
both multiple sequences alignment and genetic environment analysis of aox promoters suggest the existence of a wide diversity in the transcriptional control of the aox operon.
indeed, as in h. arsenicoxydans, a σ54-dependent promoter signature was identified in bacteria possessing a two-component transduction system aoxrs operon downstream of the aoxab operon, e.g.
a. tumefaciens and o. tritici .
in contrast, no σ54-dependent promoter motif and no aoxr homologous gene were found in other bacteria, e.g.
c. aurantiacus or c. aggregans .
these observations suggest that the transcription of the aox operon in these bacteria may involve other regulatory proteins and that aoxr may represent a specific co-activator of rpon in the initiation of the aox operon transcription.
finally, our results provide evidence that the dnaj co-chaperone is required for as oxidation.
dnaj is part of the dnak-dnaj-grpe hsp <dig> machinery.
hsp <dig> chaperones represent one of the most potent defence cellular mechanism against environmental insults as dnak-dnaj-grpe are known to assist protein folding  <cit>  or to be involved in mrna stability  <cit> .
in the present study we showed that there is no induction of aoxab transcription in the dnaj mutant, resulting in a loss of aoxab synthesis.
several possible mechanisms involving dnaj in the regulation of arsenite oxidase can be hypothesized.
dnaj may be required for the proper folding or activity of the aoxr regulator.
such a function has been demonstrated for the positive regulator crp in a dnaj deletion mutant in e. coli  <cit> .
similarly, a post-transcriptional regulation of the arsenite oxidase itself can not be excluded.
moreover, a tat  signal has been detected in the aoxa sequence of h. arsenicoxydans  <cit> .
proteins secreted to the periplasm via a tat protein export pathway are known to require a folding by hsp <dig> chaperones before their secretion.
dnaj could be one of these chaperones  <cit> .
another possible target of dnaj may be the rpon sigma factor, as this chaperone has been demonstrated to play a role in the regulation of σs in various species  <cit> .
alternatively, several mechanisms are known to be involved in the stability of messenger rna.
for example, in e. coli, a long 5' untranslated region  has been observed upstream of the transcriptional start site of the flhdc flagellum master operon.
this region plays a crucial role in the stability of the mrna controlled by csra  <cit> .
in the present report, the aoxab transcriptional start site was located  <dig> bp upstream of the translational start codon, providing evidence that such a long 5'utr does not exist upstream of the aox operon.
however, this does not rule out a role of dnaj on the stability of aoxab mrna, as hsp proteins are known to function as mrna stabilizers and to protect them against nuclease degradation  <cit> .
further experiments will therefore be required to fully elucidate the molecular mechanisms of arsenite oxidase regulation in h. arsenicoxydans.
CONCLUSIONS
taken together, our observations provide evidence that multiple proteins play a role in the control of arsenite oxidation in h. arsenicoxydans.
the following regulatory model is proposed: aoxs responds to the presence of as in the environment and autophosphorylates.
the phosphate is then transferred to aoxr, which acts as a positive regulator of the aox operon and activates the initiation of the transcription in association with rpon.
in addition, dnaj acts on the expression or the stability of both arsenite oxidation and motility genes, demonstrating that these two functions are strongly linked.
our results include the role of rpon and dnaj in arsenite oxidase synthesis, which provide further insight into the molecular mechanisms used by h. arsenicoxydans to cope with the most toxic form of arsenic in its environment.

 arsenic is present in various environments, released from either anthropogenic or natural sources. this element is toxic for living organisms and known to be a human carcinogen. its toxicological effects depend, at least in part, on its oxidation state and its chemical forms, inorganic species being considered as more toxic. the contamination of drinking water by the two inorganic forms, arsenite as and arsenate as, has been reported in different parts of the world and constitutes a major threat of public health. microorganisms are known to take part in the transformation, i.e oxidation, reduction or methylation of the metalloid, having a deep impact on arsenic contamination in environment. several bacteria and prokaryotes have developed adaptation, resistance and colonization mechanisms, which allow them to live in hostile arsenic contaminated environments. h. arsenicoxydans is a gram-negative β-proteobacterium isolated from an industrial activated sludge plant and exhibiting a remarkable set of arsenic resistance determinants. the h. arsenicoxydans adaptive response to arsenic is organized in a complex and sophisticated network. in particular, differential proteome studies have recently demonstrated the synthesis of several proteins encoded by the three ars resistance operons, e.g. arsenate reductase arsc, flavoprotein arsh and regulator arsr and the induction of oxidative stress protein encoding genes, e.g. catalase, superoxide dismutase and alkyl hydroperoxide reductase. one of the most noticeable response to arsenic in h. arsenicoxydans is the ability of this bacterium to oxidize as to as, a less toxic and less mobile form, via an arsenite oxidase activity. the two genes coding for this heterodimeric enzyme are organized in an operonic structure, and have been named aoxa and aoxb for the small and the large subunit, respectively. homologous genes have been since identified in various microorganisms. in agrobacterium tumefaciens, a complex transcriptional regulation has been recently suggested, involving as sensing, two-component signal transduction by an aoxs sensor kinase and an aoxr regulator, and quorum sensing. nevertheless, the molecular mechanisms involved in the control of arsenite oxidase expression remain largely unknown. to extend our view of arsenic response, in particular with respect to the arsenic oxidation process, we conducted a comprehensive transcriptomic, genetic and molecular analysis of h. arsenicoxydans following exposure to as. these approaches allowed us to identify major determinants involved in the control of arsenite oxidation.  gene expression profiling in response to arsenic the response to as was analyzed in exponentially growing cells using microarrays. the data from three biological replicates were combined after normalization and statistical analysis carried out using the r software and packages http://www.r-project.org. the set of genes was further refined to include only those genes that showed a valid p-value and whose expression was altered by a factor of or more when compared to the level measured in the absence of arsenic. this experiment led to the identification of genes showing an arsenic-induced expression change ). among these genes, were up-regulated and the remaining part, i.e. genes, was down-regulated. the relative changes in gene expression ranged from a 11-fold down-regulation to a 126-fold up-regulation. a list of those genes is shown in additional file table s the corresponding hear gene numbers are available in the arsenoscope relational database http://www.genoscope.cns.fr/agc/mage/arsenoscopevia the mage web interface. the genes differentially expressed were clustered according to the function of the corresponding encoded proteins. among the genes that were induced by at least a 2-fold factor, about 11% are involved in metabolism, 26% in transport and binding protein, 26% in cellular processes and 31% have no assigned function. the high percentage of genes with unknown function is in accordance with the proportion of unknown function proteins identified in the genome of h. arsenicoxydans. in agreement with our previous results, genes involved in arsenic resistance, phosphate transport and flagellar biosynthesis were induced in the presence of as, further supporting the relationship between these physiological processes. interestingly, only one methyl-accepting chemotaxis protein gene was induced in our microarray data, suggesting a role for this protein in the sensing of arsenic. this mechanism is currently under investigation. genes encoding the putative nitroreductase aoxc and the cytochrome c precursor aoxd as well as the response regulator aoxrs were found to be induced by as. aoxr has been proposed to act as a positive regulator of the aox operon upon phosphorylation by aoxs in a. tumefaciens. our transcriptomic data suggest that the regulation machinery is, at least in part, similar in h. arsenicoxydans. futhermore, genes coding for the arsenite oxidase aoxab subunits were found to be among the most induced genes in the presence of as. however, even though these results extended our knowledge of the arsenic response in h. arsenicoxydans, they did not led to a better understanding of the molecular mechanisms involved in the control of arsenite oxidation. this prompted us to perform a transposon mutagenesis experiment. identification of arsenite oxidase accessory genes by screening an aox activity deficient mutant library to identify genes possibly involved in the control of arsenite oxidation in h. arsenicoxydans, a library of kanamycin resistant mutants was constructed by transposon mutagenesis, as previously described. these clones were tested by silver nitrate staining for arsenate production on as-supplemented cdm agar plates. as compared to the wild-type strain, whose arsenite oxidase activity was revealed by a brownish precipitate, mutants with a lack of as oxidase activity were obtained. these strains showed no precipitate, as observed for the m and m strains used as negative controls. indeed, these strains carry a mutation in aoxa or aoxb genes coding for the small and the large subunit of arsenite oxidase, respectively. genes disrupted by transposon insertions were identified in these new mutants. as expected, four of the mutants showed insertions in the aoxab operon. more interestingly, six mutants carried a transposon insertion outside the aoxab operon. two mutants were found to be affected in the aoxrs two-component signal transduction system located upstream of the aoxab operon in h. arsenicoxydans. these results further support our transcriptomic data suggesting that these two genes play a role in arsenic response. two transposon insertions were shown to disrupt genes of the modeabc operon coding for a molybdenum high-affinity transport system, i.e. modc encoding an atp-binding cassette transport protein and modb encoding a molybdenum transport system permease. remarkably, transposon insertions were also located in dnaj encoding a heat shock protein, and in rpon encoding the alternative nitrogen sigma factor of rna polymerase. two additional experiments were performed to complete the physiological characterization of these mutants with respect to arsenite oxidation. first, arsenic species were quantified by hplc-icp-aes on filtered culture supernatants. h. arsenicoxydans was grown in liquid medium supplemented with mm arsenite and showed 100% transformation of as into as after h, whereas m and m mutants used as controls were not able to transform as into as. the same loss of arsenite oxidase activity was measured in ha, ha, ha and ha mutants. in contrast to the results obtained on agar plates, ha and ha strains showed 100% transformation of arsenite. previous studies have demonstrated that the bioavailability of metals or trace elements considerably varies according to the type of matrix used for microbial growth. we therefore assumed that mo might be partly sequestred on cdm agar medium, resulting in a lack of arsenite oxidase activity on plate. to test this hypothesis, as oxidase tests were performed on cdm agar plates supplemented with μm mo. the addition of mo to the solid medium restored as oxidase activity in both ha and ha mutants while it had no effect on other mutant strains. determined by hplc-icp-aes after h growth in cdm medium containing mg.liter of arsenite. b second, we have previously demonstrated that the polar flagellum-dependent motility of h. arsenicoxydans is increased in the presence of as, suggesting that arsenite oxidation may result in a gain of energy. the motility of mutant strains was therefore tested on plates containing different concentrations of as, i. e. mm, mm and mm. the diameter of the swarming rings was measured after h. as shown in figure the disruption of aoxa, aoxb, aoxr, aoxs or rpon genes abolished the improvement of swarming performances in the presence of as. unlike those mutants, a disruption in dnaj completely abolished the motility of h. arsenicoxydans in the presence or the absence of as. dnaj is known to be essential for the expression of the flhdc flagellar master operon in escherichia coli. the lack of motility observed in the dnaj mutant suggests the existence of a similar flhdc-dependent regulation of flagellar genes in h. arsenicoxydans. more importantly, the dnaj mutant was affected in both the motility and arsenite oxidation, suggesting that these two mechanisms are co-regulated. effect of aoxr, aoxs, rpon and dnaj on arsenite oxidase synthesis to get further insight into the involvement of aoxr, aoxs, rpon and dnaj in arsenite oxidase activity, western immunoblotting experiments were performed using antibodies raised against aoxb. the abundance of this protein was evaluated from total protein extracts of h. arsenicoxydans wild-type and mutant strains grown in the presence or not of as. aoxb was detected as a single band corresponding to a molecular mass of kda in as-challenged h. arsenicoxydans strain. this single band was not observed in the various mutant strains. furthermore, arsenite oxidase activity on native gel was only detected in as-challenged wild type total extract. taken together these results suggest that the lack of activity in the mutant strains is due to the absence of aoxb protein, which may result from an effect of aoxr, aoxs, rpon and dnaj on aoxab expression. effect of aoxr, aoxs, rpon and dnaj on the control of arsenite oxidase operon expression to determine the involvement of aoxr, aoxs, dnaj and rpon on aoxab transcription, we performed quantitative rt-pcr experiments. for each strain, changes in aoxb transcript abundance were compared to two internal controls, i.e. the putative rna methyltransferase gene and the peptide deformylase gene, in cultures challenged or not by as. the expression of aoxb mrna was increased by a fold factor after as exposure in the h. arsenicoxydans wild-type strain. in contrast, aoxb expression was not increased in ha, ha, ha and ha mutant strains, suggesting that the corresponding proteins play a crucial role in aoxab operon expression. strain ratio error expression ratios of aoxb in h. arsenicoxydans wild-type and mutant strains without as versus an as hours induction, as measured by quantitative rt-pcr. expression of each gene was normalized to the expression of the two housekeeping genes hear and hear coding for a peptide deformylase and a putative rna methyltransferase, respectively. standard errors were calculated from the data of two quantitative pcr replicates obtained from two independent biological replicates. bold text indicates statistically significant induction. molecular mechanisms of arsenite oxidase transcription the aoxr and aoxs genes encode a two-component system while rpon encodes a sigma factor which recognizes a particular promoter with a specific -12/- binding site. these three proteins may therefore play a role in the initiation of aoxab transcription. to get further insight into the molecular interactions between those regulators and the aoxab promoter, we mapped the transcriptional start site of this operon by the amplification of aoxab cdna ends and 5'race. messenger rnas were extracted from induced ) and non induced h. arsenicoxydans wild-type strain cultures. a single transcriptional start site was identified from induced cells at - bp relative to the translation start codon, while no transcriptional start site was identified from non induced cells. in agreement with this, a tggcacgcagtttgc putative -12/- σ54-dependent promoter motif was identified upstream of the aoxab transcriptional start site. in addition, multiple alignment of aoxab promoter sequences present in databases revealed a similarity to promoters recognized by σ in a. tumefaciens, thiomonas sp., rhizobium sp. nt- achromobacter sp., rhodoferax ferrireducens, ochrobactrum tritici. in contrast, no such σ54-dependent promoter motif was found in several strains containing the aoxab operon but lacking the two-component transduction system aoxrs operon, such as chloroflexus aurantiacus, chlorobium limicola, thermus thermophilus, burkholderia multivorans, roseobacter litoralis, pseudomonas sp.ts chlorobium phaeobacteroides and chloroflexus aggregans. remarkably, a multiple alignment of amino acid sequences revealed that aoxr shares significant homology with a number of σ rna polymerase transcriptional activators, i.e. % identity with zrar and % identity with atoc from e. coli k aoxr contains three conserved domains shared by most enhancer binding proteins, namely a n-terminal response regulator receiver domain, a central σ interaction domain common to all σ dependent ebps and a c-terminal dna binding helix-turn-helix domain enable to bind to specific upstream activation sequences. aoxr shares similarities with several ebps of σ essential for the formation of an open complex formation during σ54-dependent transcriptional initiation, in particular the σ activator sequence gaftga loop which directly binds to σ conserved region iii. taken together, these observations strongly suggest that aoxr interacts directly with rpon to initiate the transcription of aoxab operon in h. arsenicoxydans. discussion despite many works devoted to arsenic metabolism in microorganisms, little is known about the regulation of arsenite oxidase activity. in the present study, the combination of transcriptomic, genetic and molecular data provided a comprehensive view of the role of various proteins in the control of arsenite oxidation in h. arsenicoxydans. we showed that some proteins play an indirect role in this process, as their presence is not essential for aoxab synthesis. in this respect, the aoxb large subunit contains a mo site required in arsenite oxidase enzymatic activity. ha and ha mutations were located in the molybdenum high-affinity transport system operon, which further support the key role of this element in enzyme activity. in addition, the recovery of as oxidase activity in these two mutants in the presence of an excess molybdenum suggests that mo may also be transported through an alternative uptake system in mod mutants, e.g. a low-affinity uptake system involving non specific permeases such as hear hear hear or hear or a sulfate transport system, as described in e. coli mod mutants. more importantly, our results suggest that aoxr and rpon constitute a transcriptional complex that play a major role in the initiation of aoxab operon transcription. three mutants, i.e. ha, ha and ha, were affected in this process. the amino acids sequence analysis of h. arsenicoxydans aoxr and aoxs revealed the existence in these proteins of structural features common to partners of two-component signal transduction systems, which are composed of a sensor kinase and a response regulator. moreover, the comparison of aoxs and aoxr protein sequences with those of a. tumefaciens revealed similarities. indeed, the aoxs protein sequence contains short blocks of conserved motifs that are consistent with a role of sensor histidine kinase, e.g. the "h", the "g2" and the "n" domains. in addition, four highly invariant residues playing a central role in phosphorylation correspond to asp asp asp and lys in the h. arsenicoxydans aoxr protein. the three asparagine residues are known to interact together to form an "acid pocket", characteristic of orthodox receivers, into which the side chain of lys protrudes. finally, a transmembrane region and a amino acids residue cluster possibly exposed to the periplasm are present in aoxs and could serve as a signal receptor in the presence of as in the medium. the detection of as would then lead to aoxs autophosphorylation at a histidine residue via atp hydrolysis and phosphotransfer to an aspartate residue in the response regulator aoxr, as recently proposed in a. tumefaciens. remarkably, our results demonstrated for the first time that the alternative n sigma factor is essential for the initiation of arsenite oxidase transcription. indeed, a mutation in the corresponding gene led to a complete loss of as oxidation and aoxb transcription in ha. σ is one of the alternative sigma subunits of rna polymerase responsible for specific binding to dna. the core rna polymerase complexed with σ is usually associated with nitrogen assimilation and fixation, but is also known to play a role in various physiological processes, e.g. flagellar synthesis, carbon source utilization or bacterial virulence. to date, only one report has shown that σ participates in the transcription of genes possibly involved in metal tolerance, i.e. the zrar/s genes that code for a zinc and lead responsive two-component regulatory system in e. coli. rna polymerase together with σ binds to a specific promoter site, with the consensus dna sequence ytggcacgnnnnttgcwnnw, forming a transcriptionally inactive closed complex. such a characteristic -12/- σ54-dependent promoter motif, i.e. tggcacgcagtttgc, was identified pb upstream of the transcriptional initiation codon of aoxab with respect to the + transcriptional start site, which confirmed the need for rpon in the initiation of aoxab transcription. changes in the conformation of σ54-rna polymerase are nucleotide dependent. indeed, the dna melting step absolutely requires the interaction with a transcriptional activator protein. most of these σ54-dependent activators share three domains found in aoxr, i.e. a c-terminal dna binding domain that binds to upstream activation sequences, a conserved central domain belonging to the aaa+ protein family to proceed with initiation of transcription and a n-terminal receiver domain that regulates its own aaa+ domain. a multiple alignment of the central domain revealed a conservation of a common architecture between aoxr and σ ebps. indeed, seven highly conserved sequence motifs corresponding to a σ interaction domain of aoxr further support the direct interaction of aoxr with rpon to stimulate the transcription of aoxab operon in h. arsenicoxydans. this central σ interaction domain has been already used to identify new σ ebps. it contains the highly conserved signature sequence gaftga loop proved to be required for direct interaction with σ, the walker a and b atpase motifs necessary for the open complex formation. both multiple sequences alignment and genetic environment analysis of aox promoters suggest the existence of a wide diversity in the transcriptional control of the aox operon. indeed, as in h. arsenicoxydans, a σ54-dependent promoter signature was identified in bacteria possessing a two-component transduction system aoxrs operon downstream of the aoxab operon, e.g. a. tumefaciens and o. tritici. in contrast, no σ54-dependent promoter motif and no aoxr homologous gene were found in other bacteria, e.g. c. aurantiacus or c. aggregans. these observations suggest that the transcription of the aox operon in these bacteria may involve other regulatory proteins and that aoxr may represent a specific co-activator of rpon in the initiation of the aox operon transcription. finally, our results provide evidence that the dnaj co-chaperone is required for as oxidation. dnaj is part of the dnak-dnaj-grpe hsp machinery. hsp chaperones represent one of the most potent defence cellular mechanism against environmental insults as dnak-dnaj-grpe are known to assist protein folding or to be involved in mrna stability. in the present study we showed that there is no induction of aoxab transcription in the dnaj mutant, resulting in a loss of aoxab synthesis. several possible mechanisms involving dnaj in the regulation of arsenite oxidase can be hypothesized. dnaj may be required for the proper folding or activity of the aoxr regulator. such a function has been demonstrated for the positive regulator crp in a dnaj deletion mutant in e. coli. similarly, a post-transcriptional regulation of the arsenite oxidase itself can not be excluded. moreover, a tat signal has been detected in the aoxa sequence of h. arsenicoxydans. proteins secreted to the periplasm via a tat protein export pathway are known to require a folding by hsp chaperones before their secretion. dnaj could be one of these chaperones. another possible target of dnaj may be the rpon sigma factor, as this chaperone has been demonstrated to play a role in the regulation of σs in various species. alternatively, several mechanisms are known to be involved in the stability of messenger rna. for example, in e. coli, a long 5' untranslated region has been observed upstream of the transcriptional start site of the flhdc flagellum master operon. this region plays a crucial role in the stability of the mrna controlled by csra. in the present report, the aoxab transcriptional start site was located bp upstream of the translational start codon, providing evidence that such a long 5'utr does not exist upstream of the aox operon. however, this does not rule out a role of dnaj on the stability of aoxab mrna, as hsp proteins are known to function as mrna stabilizers and to protect them against nuclease degradation. further experiments will therefore be required to fully elucidate the molecular mechanisms of arsenite oxidase regulation in h. arsenicoxydans.  taken together, our observations provide evidence that multiple proteins play a role in the control of arsenite oxidation in h. arsenicoxydans. the following regulatory model is proposed: aoxs responds to the presence of as in the environment and autophosphorylates. the phosphate is then transferred to aoxr, which acts as a positive regulator of the aox operon and activates the initiation of the transcription in association with rpon. in addition, dnaj acts on the expression or the stability of both arsenite oxidation and motility genes, demonstrating that these two functions are strongly linked. our results include the role of rpon and dnaj in arsenite oxidase synthesis, which provide further insight into the molecular mechanisms used by h. arsenicoxydans to cope with the most toxic form of arsenic in its environment. 
transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxr and aoxs genes, showing that the aox operon transcription is regulated by the aoxrs two-component system. remarkably, transposon insertions were also identified in rpon coding for the alternative n sigma factor of rna polymerase and in dnaj coding for the hsp co-chaperone. in the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of h. arsenicoxydans to as. to get insight into the molecular mechanisms of this enzyme activity, a tn transposon mutagenesis was performed. these results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in h. arsenicoxydans. western blotting with anti-aoxb antibodies and quantitative rt-pcr experiments allowed us to demonstrate that the rpon and dnaj gene products are involved in the control of arsenite oxidase gene expression. finally, the transcriptional start site of the aoxab operon was determined using rapid amplification of cdna ends and a putative -12/- σ54-dependent promoter motif was identified upstream of aoxab coding sequences. these transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water.
 both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. these transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of as to as as a detoxification mechanism.  in the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of h. arsenicoxydans to as. to get insight into the molecular mechanisms of this enzyme activity, a tn transposon mutagenesis was performed. transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxr and aoxs genes, showing that the aox operon transcription is regulated by the aoxrs two-component system. remarkably, transposon insertions were also identified in rpon coding for the alternative n sigma factor of rna polymerase and in dnaj coding for the hsp co-chaperone. western blotting with anti-aoxb antibodies and quantitative rt-pcr experiments allowed us to demonstrate that the rpon and dnaj gene products are involved in the control of arsenite oxidase gene expression. finally, the transcriptional start site of the aoxab operon was determined using rapid amplification of cdna ends and a putative -12/- σ54-dependent promoter motif was identified upstream of aoxab coding sequences.  these results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in h. arsenicoxydans. these data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to as in this microorganism. 