BACKGROUND
antimony  is a metal belonging to group v in the periodic table.
it is present in aquatic systems and soil, with stibnite  representing the most common mineral form  <cit> .
antimonials have been used to treat leishmaniasis for over 60 years  <cit> .
however, the u.s. environmental protection agency recognized sb as a priority pollutant  <cit> , and the who has proposed 5 μg/l to be the highest acceptable sb concentration in potable water  <cit> .
although sb pollution has gained increased attention in recent decades , the exact mechanism of sb toxicity in both mammals and microorganism remains unclear  <cit> .
a comparison of two common inorganic forms of sb revealed that antimonite  was more toxic than antimonate   <cit> .
the abiotic sb oxidation in the natural environment is extremely slow  <cit> .
in contrast, sb-oxidizing bacteria can oxidize sb at a relatively high rate.
thus, sb oxidation was proposed to be a microbial detoxification process that could be useful for environmental sb bioremediation .
a few sb-oxidizing bacteria had been reported in earlier literature  <cit> .
recently, dozens of sb-oxidizing bacteria have been isolated and identified both by our group and by others  <cit> .
the arsenite oxidase aioab in agrobacterium tumefaciens was also found to be able to oxidize sb  <cit> .
in addition, the oxidoreductase anoa was shown to be responsible for bacterial sb oxidation  <cit> .
however, the disruption of both of these genes did not result in a complete loss of sb oxidation, indicating the existence of other mechanisms responsible for bacterial sb oxidation.
in general, aerobic respiration is inevitably accompanied by the production of reactive oxygen species .
ros include superoxide , hydroxyl , hydroperoxyl , and peroxyl  radicals and other oxidizing agents, such as hydrogen peroxide   <cit> .
among the various ros, o2
•- is the primary radical that results from the univalent reduction of molecular oxygen  via the bacterial respiratory chain.
subsequently, o2
•- can be converted into h2o <dig> and o <dig> by superoxide dismutase .
both o2
•- and h2o <dig> can generate highly reactive hydroxyl radicals via the haber-weiss reaction or the fenton reaction  <cit> .
ros can damage different types of macromolecules; thus, bacteria have evolved defense mechanisms against ros and induce specific genes in response to oxidative stress  <cit> .
glutathione  is the central substrate involved in cellular protection against ros and their toxic products in eukaryotic cells  <cit> .
in bacteria gsh plays multiple role in protection against environmental stresses such as osmotic shock and acidity, as well as against toxins and oxidative stress.
indeed, gsh represents the primary low molecular weight thiol in many gram-negative bacteria  <cit> .
two enzymes  catalyze the de novo synthesis of gsh.
these two enzymes are encoded by distinct genes  in most bacteria  <cit> .
previously, the aerobic, arsenite-oxidizing bacterium pseudomonas stutzeri ts <dig> was isolated from arsenic-contaminated soil  <cit>  and the genome sequence was published .
in this study, we showed that strain ts <dig> is able to oxidize sb to sb.
therefore, in an effort to determine the molecular mechanism underlying bacterial sb oxidation, more than  <dig> tn <dig> transposon insertion mutants were generated and screened.
we isolated a mutant of gsha  that showed an increase in sb oxidation rate.
the sb oxidation rates of the wild type, mutant and complemented strain, the gene transcription of gsha, sod and kate, as well as the cellular contents of ros, gsh and h2o <dig> were analyzed since gsha is responsible for the biosynthesis of gsh and therefore involved in the cellular stress defense system.
methods
bacterial strains, plasmids, primers and culture conditions
the strains, plasmids and primers used in this study are listed in table  <dig> and additional file 1: table s <dig>  the pseudomonas stutzeri strains were grown in a chemically defined medium   <cit>  containing  <dig>  mmol/l phosphate  and shaken under aerobic conditions at 28 °c.
the escherichia coli strains were cultured at 37 °c in luria-bertani medium  <cit> .
rifampin , kanamycin , tetracycline  or chloromycetin  were added when needed.table  <dig> the strains and plasmids used in this study
pseudomonas stutzeri
ts44-gsha540
ts44-gsha540
ts44-gsha540
ts44-gsha540
escherichia coli
pct-zori-gsha
plsp-pgsha
plsp-psodb
plsp-psodc
isolation of a sb sensitive transposon mutant and complementation of the mutant strain
plasmid prl27-kan was transferred into p. stutzeri ts <dig> by conjugation from e. coli strain s17- <dig> carrying tn <dig> as described previously  <cit> .
to obtain sb sensitive mutants, the colonies of transformants from the mating plates were spread onto lb agar plates containing 50 μg/ml kan and 50 μg/ml rif, in the presence or absence of  <dig>  mmol/l c8h4k2o12sb2s <dig> .
the mutants did not grow in the presence of the indicated concentration of sb.
the insertion sequence of the mutant was determined using a plasmid rescue strategy as described by zheng et al.
<cit> .
the sequences were compared to the draft genome sequence of p. stutzeri ts <dig> using blast  <cit>  and compared to the protein sequence database at genbank using the blastx algorithm  <cit> .
finally, the sb-sensitive tn5-insertion mutant ts44-gsha <dig> was isolated.
the construction of the gsha complementary strain ts44-gsha-c was accomplished using the high-copy broad host range plasmid pct-zori containing a kan resistance determinant .
briefly, a 2023 bp dna fragment containing the complete gsha coding region along with 156 bp upstream and 289 bp downstream sequence was pcr-amplified using the gsha-f/gsha-r primers  and subsequently cloned into hindiii + bamhi-digested pct-zori.
the resulting plasmid pct-zori-gsha was transformed into e. coli s17- <dig> and conjugated into the mutant ts44-gsha <dig>  yielding the complementary strain ts44-gsha-c. the integrity of the mutant and the complementary strain was confirmed by pcr amplification and subsequent dna sequencing.
growth and sb oxidation tests
overnight cultures of p. stutzeri strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c  were inoculated into 100 ml of cdm medium with or without  <dig>  mmol/l sb and incubated at 28 °c for 48 h with shaking at 170 rpm.
culture samples were collected to determine the od <dig> value using a uv spectrophotometer  at the designated time points.
sb/sb concentrations were monitored using hydride-generation atomic fluorescence spectroscopy combining hplc  according to the method described by li et al.
<cit> .
the measured data were analyzed with single factor analysis of variance  method in excel program.
gsha activity and determination of gsh content
to detect gsha activity and gsh content, p. stutzeri strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c were cultured under aerobic condition with shaking in 100 ml of liquid cdm medium at 28 °c.
sb was added at a final concentration of  <dig>  mmol/l when the od <dig> reached  <dig> .
cultures without the addition of sb were used as controls.
one mililiter of cells was centrifuged at  <dig>  × g at 4 °c after 30 min of cultivation.
the pellet was washed twice using phosphate-buffered saline , then resuspended in 1 ml of pbs and sonicated on ice to dissolve the cell membranes.
the total protein content of the sonicate was measured by the coomassie brilliant blue g-250-staining method  <cit> .
bovine serum albumin was used as the standard.
the gsha activity and gsh content were determined using  <dig>  3-naphthalenedicarboxyaldehyde   as described previously  <cit> .
to measure gsha activity, an equal volume of cell lysate was mixed with the gsha reaction buffer and substrate solution .
fifty microliter of reaction terminator  was added after 45 min of incubation at 28 °c.
the mixture was incubated on ice for 20 min and centrifuged at 8000 × g for 5 min.
then, 20 μl of the supernatants were transferred to wells of black microlon 96-well plates .
the samples were mixed with 180 μl of nda derivatization solution .
fluorescence was measured  with an envision® multimode plate reader  after 60 min of incubation at 37 °c and converted to the γ-glutamylcysteine concentration using appropriate calibration standards.
the procedure for measuring gsh content was almost the same as the procedure to detect gsha activity, except that the reaction terminator was added immediately when the equal volume of cell lysate was mixed with the gsha reaction buffer and substrate solution .
determination of the ros content
the cellular ros content was tested with the fluorescent probe  <dig>  7-dichlorofluorescin diacetate  .
the cells were cultivated and collected for processing as described above.
cells from  <dig>  ml cultures were washed two times with pbs buffer and resuspended in  <dig>  ml of pbs buffer.
then, a  <dig>  ml cell suspension was mixed with 5 μl of  <dig>  mmol/l dcfh-da and incubated at 37 °c for 30 min to develop the fluorescent product dcf.
the cells were harvested by centrifugation for 3 min at  <dig>  × g and washed two times using pbs buffer  to remove background fluorescence.
the fluorescence was measured  on the envision® multimode plate reader   <cit> .
the ros contents were normalized to the total protein content as described above.
quantification of gene expression using a lacz reporter gene fusion and qrt-pcr
quantitative reverse transcription pcr  was employed to test the transcription of gsha, two superoxide dismutase -encoding genes  and the catalase kate.
the strains were inoculated into 100 ml of cdm medium.
<dig>  mmol/l of sb was added  when the od <dig> reached  <dig> .
the cells were harvested after 30 min.
total rna was extracted using the trizol® reagent  and treated with dnasei following the manufacturer’s instructions.
the synthesis of cdna from 300 ng of total rna was performed using the revertaid first strand cdna synthesis kit   <cit> .
the resulting cdna was used as a template for qrt-pcr with the sybr-green® qpcr master mix .
primers rt-gsha-f/rt-gsha-r, rt-sodb-f/rt-sodb-r, rt-sodc-f/rt-sodc-r and rt-kate-f/rt-kate-r were used to test the expression of gsha, sodb, sodc and kate, respectively.
the rt-pcrs were performed using the ab viia  <dig> rt-pcr system  following the manufacturer’s recommended protocol.
the annealing temperature for gsha and sodb was 55 °c, while for sodc and kate it was 48 °c.
for the lacz reporter fusion analysis, plasmid plsp-kt2lacz was used to construct the lacz fusions.
dna fragments containing the predicted promoter regions of gsha, sodb and sodc were amplified by pcr using the primers pgsha-f/pgsha-r, psodb-f/psodb-r and psodc-f/psodc-r, respectively.
the dna fragments were digested using ecori and bamhi and ligated into the double-digested plsp-kt2lacz.
the resulting plasmids plsp-pgsha, plsp-psodb, and plsp-psodc were separately transferred into strains ts <dig> and ts44-gsha <dig> by conjugation employing e. coli s17- <dig>  the resulting strains containing the above constructs were inoculated into 100 ml of cdm medium and cultivated with shaking at 28 °c.
<dig>  mmol/l of sb was added when the od <dig> reached  <dig> .
the samples were collected and the β-galactosidase activity was tested according to the method described by miller  <cit>  after 30 min of incubation.
determination of the linear correlation between h2o <dig> and sb oxidation rate
to test the h2o <dig> content, strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c were incubated as described above.
to eliminate the h2o <dig> content difference caused by the different amount of cell collection, sb was added when the od <dig> reached  <dig> , and then 2 ml of cells were harvested after a 30 min incubation with sb.
the cells were resuspended in 1 ml of k3po <dig>  after washing twice with 50 mmol/l k3po <dig>  and sonicated on ice.
then, the sonicated cell lysates were centrifuged at  <dig>  × g at 4 °c, and  <dig>  ml of the supernatant was transferred to wells of black microlon 96-well plates .
the samples were mixed with 50 μl of amplex red   and 50 μl of horseradish peroxidase  , then incubated at 37 °c for 15 min.
fluorescence was measured  as described above using the envision® multimode plate reader  and converted to the h2o <dig> concentration using appropriate calibration standards  <cit> .
to detect the dynamic variation of the h2o <dig> and sb contents, strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c were inoculated into 100 ml of cdm medium supplemented with  <dig>  mmol/l sb.
the cells were harvested and sonicated as described above at the designated time points.
the sb contents were monitored using hplc-hg-afs as described above, while the h2o <dig> contents were determined by the ar/hrp method as described above.
a boiled cdm culture of strain ts44-gsha <dig> was used as a control.
to test the effect of h2o <dig> on bacterial growth, strain ts <dig> was inoculated onto cdm plates containing different concentrations of h2o <dig> and cultivated at 28 °c for 48 h.
in vitro oxidation of sb by h2o <dig> was performed using an un-inoculated liquid cdm medium containing  <dig>  mmol/l of sb and the media with the addition of  <dig>   <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  mmol/l of h2o <dig> and reacted within 10 min.
RESULTS
a sb-sensitive mutant could be generated by transposon mutagenesis
more than  <dig> transposon insertions were isolated and screened for loss of sb resistance with one mutant displaying lower sb resistance.
this strain, ts44-gsha <dig>  was selected for further characterization .
the tn <dig> transposon had inserted into gsha encoding a putative glutamate-cysteine ligase at nucleotide  <dig> .
the gsha encoded glutamate-cysteine ligase is the rate-limiting enzyme in de novo gsh biosynthesis and therefore is involved in conferring resistance to ros and their toxic products  <cit> .
additionally, a strain complementing the gsha insertion was constructed and named ts44-gsha-c. diagnostic pcrs were used to confirm the transposon mutation and the complementation .
an insertion in gsha led to an increased sb oxidation rate
cells of p. stutzeri ts <dig>  ts44-gsha <dig> and ts44-gsha-c were incubated in cdm medium lacking sb supplementation after thorough washing.
the growth of strain ts44-gsha <dig> was slightly slower compared to the wild type strain .
the growth of strain ts44-gsha <dig> was further delayed by supplementation with  <dig>  mmol/l sb, causing strain ts44-gsha <dig> to need an extra 24 h to reach the lag phase .
the gsha complementing strain ts44-gsha-c showed no difference in growth compared to the wild type strain regardless of whether sb was added or not.
although the growth of ts44-gsha <dig> was delayed, strain ts44-gsha <dig> oxidized 88% sb to sb in 48 h .
in contrast, strains ts <dig> and ts44-gsha-c both oxidized only 48% sb to sb in 48 h .
the sb-oxidation rate of ts44-gsha <dig> significantly increased by 83% in the monitored 48 h time frame  .fig.
<dig> growth curves of p. stutzeri strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c without  or with  the addition of  <dig>  mmol/l sb.
error bars correspond to the standard deviations of the means from three independent experiments
fig.
<dig> sb oxidation in p. stutzeri strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c with the addition of  <dig>  mmol/l sb.
the amounts of sb and sb in culture fluids were calculated based on culture volume to normalize for the total amount of antimony added.
sb and sb concentrations in the culture fluids were measured using hplc-hg-afs.
error bars correspond to the standard deviations of the means from three independent experiments
an insertion in gsha eliminated gsha activity and influenced cellular contents of gsh and ros
we investigated both gsha activity and the gsh and ros content to elucidate how the presence of gsha affected sb oxidation.
the gsha activity in strains ts <dig> and ts44-gsha-c was slightly increased following the addition of sb; however, gsha activity was undetectable in the insertional mutant strain ts44-gsha <dig> , indicating that the transposon insertion functionally disrupted gsha in strain ts <dig>  consistent with this result, the gsh content was decreased by approximately 66% in the mutant compared to strains ts <dig> and ts44-gsha-c regardless of whether sb was provided .fig.
<dig>
gsha insertion affected gsh content, gsha activity and ros content.
a, gsha activity, , gsh content, , ros content and  ros content in strain ts44-gsha <dig>  data symbols shown in panels ,  and  are the same.
data are expressed as the mean ± sd, n =  <dig>  **indicates a significant difference from the control
as a next step, cellular ros content was determined because gsh is the central substrate for cellular protection against ros and their toxic products  <cit> .
the cellular ros content in strain ts44-gsha <dig> was increased by approximately three-fold compared to strains ts <dig> and ts44-gsha-c regardless of whether sb was provided or not.
the cellular ros content was significantly decreased in strain ts44-gsha <dig> after 30 min of incubation with sb compared to no sb addition , despite the fact that gsha was mutated and the gsh content was obviously decreased .
after addition of sb, the ros content changed quickly in 30 min, and decreased significantly in the strain ts44-gsha <dig> .
in such a short time, the oxidation rate of sb did not significantly change.
the sb oxidation mainly occurred in the 36–48 h time frame .
sb affected transcription of gsha, sodb, sodc and kate differently
the expression of gsha, sodb, sodc and kate was monitored, because gsha, sodb, sodc and kate are all involved in changes of gsh, ros or h2o <dig>  there are two distinct genes  encoding superoxide dismutase  on the genome of p. stutzeri ts <dig> that encode for fe sod and cu-zn sod, respectively.
these two enzymes shared over 83% and 58% amino acid sequence similarity with the respective enzymes of other species of pseudomonas, respectively, and can convert o2
•- to h2o <dig> and o <dig>  in the lacz reporter fusion analysis, we only transformed the lacz fusions designated plsp-pgsha, plsp-psodb and plsp-psodc into strains ts <dig> and ts44-gsha <dig> because the gsha complementary strain ts44-gsha-c carried a plasmid that might be incompatible with the lacz fusion.
the transcription levels of gsha, sodb, sodc and kate were tested by qrt-pcr in all three strains.
sb had no effect on the transcription of gsha in strains ts <dig>  ts44-gsha <dig> or ts44-gsha-c .
the gsha::lacz fusion only showed a low level of background expression levels in the gsha insertional mutation strain ts44-gsha <dig>  , which indicated a very low promoter activity of gsha, while qrt-pcr analysis showed no transcription of gsha in strain ts44-gsha <dig> .
moreover, lacz fusion and qrt-pcr both showed that the sodb gene was strongly induced by sb in all three strains .
sb only induced transcription of sodc and kate in the mutant strain ts44-gsha <dig>  but not in strains ts <dig> and ts44-gsha-c .fig.
<dig> the effect of sb to the transcription of gsha, sodb, sodc and kate.
the transcription of gsha, sodb and sodc was tested by a lacz reporter fusion and qrt-pcr, while the kate transcription was only tested by qrt-pcr.
for lacz reporter fusion , data are expressed as the mean ± sd, n =  <dig>  **indicates a significant difference from the control .
for qrt-pcr, error bars correspond to the standard deviations of the means from three biological replicates.
gene expression was normalized to the 16s rrna gene.
the results are presented as the mean gene expression normalized to mrna levels in sb-free cdm.
data symbols shown in all panels are the same
fig.
<dig> the h2o <dig> content is correlated with bacterial sb oxidation.
the p. stutzeri strains were cultured as described above.
a the h2o <dig> concentration was tested after 30 min of incubation with sb.
**indicates a significant difference from the control .
b h2o <dig> content and  sb concentration in strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c from  <dig> to 48 h of incubation in cultures supplemented with  <dig>  mmol/l sb.
data are expressed as the mean ± sd, n =  <dig>  d correlation between h2o <dig> and sb concentrations in strain ts44-gsha540
disruption of gsh synthesis allowed accumulation of h2o2
in order to examine whether the ros-protection system is involved in h2o <dig> production, we tested the h2o <dig> content in the presence and absence of sb in all strains.
the h2o <dig> content was slightly higher in gsha mutant strain ts44-gsha <dig> without addition of sb compared to strains ts <dig> and ts44-gsha-c .
notably, after a 30 min incubation with  <dig>  mmol/l sb, the h2o <dig> content was increased in the mutant strain ts44-gsha <dig>  but slightly decreased in strains ts <dig> and ts44-gsha-c , indicating that the disruption of gsh synthesis is related to the accumulation of h2o <dig>  in addition, it appears the conversion of ros by sod was more efficient than the h2o <dig> conversion by kate and thus would both sod and kate were induced by sb.
h2o <dig> is responsible for sb oxidation
h2o <dig> was shown to be an efficient sb oxidant in vitro  as the un-inoculated control showed no sb oxidation .
a control with dead cells after boiling the culture also showed no oxidation of sb .
as the efficiency of sb oxidation in the mutant strain ts44-gsha <dig> was shown to be highest from  <dig> to 48 h , the h2o <dig> and sb content in strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c were also measured from  <dig> to 48 h. interestingly, the h2o <dig> content in strain ts44-gsha <dig> was significantly decreased from  <dig>  to  <dig>  nmol/mg protein, while the h2o <dig> content was stable at a low level in strains ts <dig> and ts44-gsha-c .
correspondingly, sb was oxidized to sb concomitant with a decrease in h2o <dig> content, while the sb concentration was almost stable in strains ts <dig> and ts44-gsha-c .
the consumed h2o <dig> content and the oxidized sb showed a linear correlation with a correlation coefficient of  <dig>  , indicating that h2o <dig> is responsible for sb oxidation.
in addition, we could show that growth was inhibited with increasing concentration of h2o <dig> , thus sb oxidation may contribute to the bacterial detoxification of h2o <dig> and sb.
discussion
at present, several sb-oxidizing strains have been reported  <cit> ; some of these strains were also able to oxidize as  <cit> .
the arsenite oxidase aioab was demonstrated to be capable of oxidizing sb, but the aioab kinetic rate of the reaction was orders of magnitude higher for as than for sb  <cit> .
moreover, anoa belonging to the sdr superfamily was reported to be able to catalyze sb oxidation using nadp+ as a cofactor  <cit> .
however, disruption of aioa and anoa in agrobacterium tumefaciens caused a decrease in sb oxidation of only about 25%  <cit>  and 27%  <cit> , respectively, indicating the existence of other bacterial sb oxidation mechanisms.
after discovering that the presence of gsha affected the sb oxidation rate by transposon mutagenesis in strain ts44-gsha <dig>  we proposed that some abiotic cellular components, such as gsh, ros or h2o <dig>  may have a role in the bacterial oxidation of sb.
subsequently, we conducted a comprehensive analysis.
first, we found that a disruption of gsha caused a decrease in the cellular gsh amount; second, it is conceivable that the gsha mutant strain resulted in an increase in cellular ros content compared to the wild type; third, we could also show a linear correlation between the decrease of h2o <dig> content and the increase in sb oxidation rate, indicating that sb oxidation consumed h2o <dig> and acts as a detoxification mechanism to counter this cellular stressor.
in addition, it appeared that sb directly caused a disruption of the ros-protection system in strain ts44-gsha <dig> and allowed the accumulation of h2o <dig> instead of affecting gsh levels, since neither the activity of gsha nor the gsh content were influenced by sb.
previously, it was reported that sb may consume residual gsh  <dig> complex) in red blood cells  <cit> .
however, we did not observe significant changes in gsh content when sb was added to wild type strain ts <dig> and the gsha complemented strain ts44-gsha-c, indicating little of this complex was formed and sb was mainly oxidized to sb by h2o <dig>  we therefore propose a new model for sb oxidation in p. stutzeri ts <dig>  i) the addition of sb would trigger the ros-protective system by inducing the transcription of sodb, sodc and kate, with sodb and sodc catalyzing the conversion of ros to h2o <dig>  and kate responsible for the depletion of excessive h2o2; ii) the increased cellular h2o <dig> content enhanced the sb oxidation rate; and iii) the addition of sb played a selection role on the characterization of the gsha insertion ; iv) in addition, the accumulated h2o <dig> was partially consumed by kate , since kate was induced by sb , and similar result was described recently with transcription of the peroxidase-encoding gene kata being induced by both sb and h2o <dig>  <cit> .fig.
<dig> the proposed model for sb bacterial oxidation in p. steutzeri ts <dig>  in this study, i) the addition of sb would trigger the ros-protective system by inducing the transcription of sodb, sodc and kate, with sodb and sodc catalyzing the conversion of ros to h2o <dig>  while kate responsible for the degradation of excessive h2o <dig> ; ii) the increased cellular h2o <dig> content enhanced the sb oxidation rate; and iii) the addition of sb played a selection role on the characterization of gsha insertion; iv) in addition, the accumulated h2o <dig> is partially cosumed by the upregulated catalase kate
several chemical substances  have been reported to be capable of mediating sb oxidation in vitro .
oh• was the oxidant in acidic solutions and h2o <dig> was the main oxidant in neutral and alkaline solutions involved in sb oxidation, with a ph range of  <dig>  to  <dig>   <cit> .
sb was reported to cause cellular oxidative stress in lymphoid tumoral cells  <cit> .
h2o <dig> is a substantial component of cellular oxidative stress  <cit>  and can inhibit cellular growth  <cit> .
in this study, the ph of the cultures changed from the initial  <dig>  to approximately  <dig>  following exposure to sb, indicating that h2o <dig> may correlate with an increase in the sb oxidation rate.
in addition, the in vitro experiment provided direct evidence that h2o <dig> can oxidize sb to sb  and a correlation between the concentrations of sb and h2o <dig> was found in vivo .
as an moderatedly reactive intermediate, o2
•- may also have an effect on sb oxidation.
however, in the presence of high level of sod, the steady-state of o2
•- would be no more than  <dig>  nmol/l and thus the effect of o2
•- is negligible  <cit> .
thus, we conclude that h2o <dig> oxidized sb to sb.
CONCLUSIONS
this study proposed a novel mechanism for microbial antimonite oxidation involving changes in cellular content of components related to oxidative stress .
although h2o <dig> was reported to be capable of oxidizing sb chemically as early as  <dig>  <cit> , this is the first study to demonstrate that h2o <dig> is responsible for sb-oxidation in a microbe.
the results showed that a disruption of bacterial gsh-dependent ros-protection mechanism allowed h2o <dig> to build up and thus promote the oxidation of sb to sb.
in addition, the oxidation of sb to sb is a detoxification process against the cellular stressor h2o <dig>  we do not exclude the possibility of an additional enzymatic process responsible for sb oxidation in strain ts <dig>  since genes encoding a putative arsenite oxidase aioba were found on its genome  <cit>  which may function as a sb oxidase.
our data and other findings  <cit>  could show that microbial antimonite oxidation contains both biotic and abiotic components.
additional files
additional file 1: table s <dig>  primers used in this study.
additional file 2: figure s <dig>  the gene cluster containing gsha in strain ts <dig> and diagnostic pcr confirming the transposon mutation to create mutant strain ts44-gsha <dig> and complementation to create ts44-gsha540-c. , the transposon insertion site of the gsha mutant is shown by the vertical arrow.
, pcr used primers r6k-f/r6k-r and tnpa-f/tnpa-r  using genomic dna of strain ts44-gsha <dig> as template.
lane  <dig> is the amplification of r6k fragment, while line  <dig> represents the tnpa fragment.
, pcr used primers gsha-f/gsha-f .
lane  <dig>  strain ts <dig>  lane  <dig>  gsha gene insertional inactivation strain ts44-gsha <dig> and lane  <dig>  the complemented strain ts44-gsha540-c. m, the molecular weight marker .
amplicon identities were confirmed by dna sequencing.
additional file 3: figure s <dig>  in vitro oxidation of sb by h2o <dig>   sb was added into uninoculated liquid cdm medium to a final concentration of  <dig>  mmol/l.
cdm containing  <dig>  mmol/l of sb and with the addition of  <dig>   <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  mmol/l of h2o <dig>  respectively.
additional file 4: figure s <dig>  the tolerance of p. stutzeri strains ts <dig>  ts44-gsha <dig> and ts44-gsha-c to h2o <dig>  the strains were grown in liquid cdm medium until reaching an od <dig> of  <dig>  and then serially diluted 10-fold.
the  <dig>  10− <dig>  10− <dig>  and 10− <dig> dilutions were spotted onto agar plates with different h2o <dig> concentrations.
not applicable.
funding
the present study was supported by the national natural science foundation of china  to gw.
the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
availability of data and materials
all data generated or analyzed during this study are included in this published article and its supplementary information files.
authors’ contributions
dw performed the experiments and wrote the draft of the manuscript, fz designed and performed the experiments and helped to draft the manuscript.
qw and cr revised the draft of the manuscript.
py and jg performed the experiments.
gw designed the study and revised the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval and consent to participate
not applicable.

 antimony is a metal belonging to group v in the periodic table. it is present in aquatic systems and soil, with stibnite representing the most common mineral form. antimonials have been used to treat leishmaniasis for over 60 years. however, the u.s. environmental protection agency recognized sb as a priority pollutant, and the who has proposed 5 μg/l to be the highest acceptable sb concentration in potable water. although sb pollution has gained increased attention in recent decades, the exact mechanism of sb toxicity in both mammals and microorganism remains unclear. a comparison of two common inorganic forms of sb revealed that antimonite was more toxic than antimonate. the abiotic sb oxidation in the natural environment is extremely slow. in contrast, sb-oxidizing bacteria can oxidize sb at a relatively high rate. thus, sb oxidation was proposed to be a microbial detoxification process that could be useful for environmental sb bioremediation. a few sb-oxidizing bacteria had been reported in earlier literature. recently, dozens of sb-oxidizing bacteria have been isolated and identified both by our group and by others. the arsenite oxidase aioab in agrobacterium tumefaciens was also found to be able to oxidize sb. in addition, the oxidoreductase anoa was shown to be responsible for bacterial sb oxidation. however, the disruption of both of these genes did not result in a complete loss of sb oxidation, indicating the existence of other mechanisms responsible for bacterial sb oxidation. in general, aerobic respiration is inevitably accompanied by the production of reactive oxygen species. ros include superoxide, hydroxyl, hydroperoxyl, and peroxyl radicals and other oxidizing agents, such as hydrogen peroxide. among the various ros, o2 •- is the primary radical that results from the univalent reduction of molecular oxygen via the bacterial respiratory chain. subsequently, o2 •- can be converted into h2o and o by superoxide dismutase. both o2 •- and h2o can generate highly reactive hydroxyl radicals via the haber-weiss reaction or the fenton reaction. ros can damage different types of macromolecules; thus, bacteria have evolved defense mechanisms against ros and induce specific genes in response to oxidative stress. glutathione is the central substrate involved in cellular protection against ros and their toxic products in eukaryotic cells. in bacteria gsh plays multiple role in protection against environmental stresses such as osmotic shock and acidity, as well as against toxins and oxidative stress. indeed, gsh represents the primary low molecular weight thiol in many gram-negative bacteria. two enzymes catalyze the de novo synthesis of gsh. these two enzymes are encoded by distinct genes in most bacteria. previously, the aerobic, arsenite-oxidizing bacterium pseudomonas stutzeri ts was isolated from arsenic-contaminated soil and the genome sequence was published. in this study, we showed that strain ts is able to oxidize sb to sb. therefore, in an effort to determine the molecular mechanism underlying bacterial sb oxidation, more than tn transposon insertion mutants were generated and screened. we isolated a mutant of gsha that showed an increase in sb oxidation rate. the sb oxidation rates of the wild type, mutant and complemented strain, the gene transcription of gsha, sod and kate, as well as the cellular contents of ros, gsh and h2o were analyzed since gsha is responsible for the biosynthesis of gsh and therefore involved in the cellular stress defense system. methods bacterial strains, plasmids, primers and culture conditions the strains, plasmids and primers used in this study are listed in table  and additional file 1: table s the pseudomonas stutzeri strains were grown in a chemically defined medium containing  mmol/l phosphate and shaken under aerobic conditions at 28 °c. the escherichia coli strains were cultured at 37 °c in luria-bertani medium. rifampin, kanamycin, tetracycline or chloromycetin were added when needed.table the strains and plasmids used in this study pseudomonas stutzeri ts44-gsha540 ts44-gsha540 ts44-gsha540 ts44-gsha540 escherichia coli pct-zori-gsha plsp-pgsha plsp-psodb plsp-psodc isolation of a sb sensitive transposon mutant and complementation of the mutant strain plasmid prl27-kan was transferred into p. stutzeri ts by conjugation from e. coli strain s17- carrying tn as described previously. to obtain sb sensitive mutants, the colonies of transformants from the mating plates were spread onto lb agar plates containing 50 μg/ml kan and 50 μg/ml rif, in the presence or absence of  mmol/l c8h4k2o12sb2s. the mutants did not grow in the presence of the indicated concentration of sb. the insertion sequence of the mutant was determined using a plasmid rescue strategy as described by zheng et al.. the sequences were compared to the draft genome sequence of p. stutzeri ts using blast and compared to the protein sequence database at genbank using the blastx algorithm. finally, the sb-sensitive tn5-insertion mutant ts44-gsha was isolated. the construction of the gsha complementary strain ts44-gsha-c was accomplished using the high-copy broad host range plasmid pct-zori containing a kan resistance determinant. briefly, a 2023 bp dna fragment containing the complete gsha coding region along with 156 bp upstream and 289 bp downstream sequence was pcr-amplified using the gsha-f/gsha-r primers and subsequently cloned into hindiii + bamhi-digested pct-zori. the resulting plasmid pct-zori-gsha was transformed into e. coli s17- and conjugated into the mutant ts44-gsha yielding the complementary strain ts44-gsha-c. the integrity of the mutant and the complementary strain was confirmed by pcr amplification and subsequent dna sequencing. growth and sb oxidation tests overnight cultures of p. stutzeri strains ts ts44-gsha and ts44-gsha-c were inoculated into 100 ml of cdm medium with or without  mmol/l sb and incubated at 28 °c for 48 h with shaking at 170 rpm. culture samples were collected to determine the od value using a uv spectrophotometer at the designated time points. sb/sb concentrations were monitored using hydride-generation atomic fluorescence spectroscopy combining hplc according to the method described by li et al.. the measured data were analyzed with single factor analysis of variance method in excel program. gsha activity and determination of gsh content to detect gsha activity and gsh content, p. stutzeri strains ts ts44-gsha and ts44-gsha-c were cultured under aerobic condition with shaking in 100 ml of liquid cdm medium at 28 °c. sb was added at a final concentration of  mmol/l when the od reached. cultures without the addition of sb were used as controls. one mililiter of cells was centrifuged at  × g at 4 °c after 30 min of cultivation. the pellet was washed twice using phosphate-buffered saline, then resuspended in 1 ml of pbs and sonicated on ice to dissolve the cell membranes. the total protein content of the sonicate was measured by the coomassie brilliant blue g-250-staining method. bovine serum albumin was used as the standard. the gsha activity and gsh content were determined using 3-naphthalenedicarboxyaldehyde as described previously. to measure gsha activity, an equal volume of cell lysate was mixed with the gsha reaction buffer and substrate solution. fifty microliter of reaction terminator was added after 45 min of incubation at 28 °c. the mixture was incubated on ice for 20 min and centrifuged at 8000 × g for 5 min. then, 20 μl of the supernatants were transferred to wells of black microlon 96-well plates. the samples were mixed with 180 μl of nda derivatization solution. fluorescence was measured with an envision® multimode plate reader after 60 min of incubation at 37 °c and converted to the γ-glutamylcysteine concentration using appropriate calibration standards. the procedure for measuring gsh content was almost the same as the procedure to detect gsha activity, except that the reaction terminator was added immediately when the equal volume of cell lysate was mixed with the gsha reaction buffer and substrate solution. determination of the ros content the cellular ros content was tested with the fluorescent probe 7-dichlorofluorescin diacetate. the cells were cultivated and collected for processing as described above. cells from  ml cultures were washed two times with pbs buffer and resuspended in  ml of pbs buffer. then, a  ml cell suspension was mixed with 5 μl of  mmol/l dcfh-da and incubated at 37 °c for 30 min to develop the fluorescent product dcf. the cells were harvested by centrifugation for 3 min at  × g and washed two times using pbs buffer to remove background fluorescence. the fluorescence was measured on the envision® multimode plate reader. the ros contents were normalized to the total protein content as described above. quantification of gene expression using a lacz reporter gene fusion and qrt-pcr quantitative reverse transcription pcr was employed to test the transcription of gsha, two superoxide dismutase -encoding genes and the catalase kate. the strains were inoculated into 100 ml of cdm medium.  mmol/l of sb was added when the od reached. the cells were harvested after 30 min. total rna was extracted using the trizol® reagent and treated with dnasei following the manufacturer’s instructions. the synthesis of cdna from 300 ng of total rna was performed using the revertaid first strand cdna synthesis kit. the resulting cdna was used as a template for qrt-pcr with the sybr-green® qpcr master mix. primers rt-gsha-f/rt-gsha-r, rt-sodb-f/rt-sodb-r, rt-sodc-f/rt-sodc-r and rt-kate-f/rt-kate-r were used to test the expression of gsha, sodb, sodc and kate, respectively. the rt-pcrs were performed using the ab viia rt-pcr system following the manufacturer’s recommended protocol. the annealing temperature for gsha and sodb was 55 °c, while for sodc and kate it was 48 °c. for the lacz reporter fusion analysis, plasmid plsp-kt2lacz was used to construct the lacz fusions. dna fragments containing the predicted promoter regions of gsha, sodb and sodc were amplified by pcr using the primers pgsha-f/pgsha-r, psodb-f/psodb-r and psodc-f/psodc-r, respectively. the dna fragments were digested using ecori and bamhi and ligated into the double-digested plsp-kt2lacz. the resulting plasmids plsp-pgsha, plsp-psodb, and plsp-psodc were separately transferred into strains ts and ts44-gsha by conjugation employing e. coli s17- the resulting strains containing the above constructs were inoculated into 100 ml of cdm medium and cultivated with shaking at 28 °c.  mmol/l of sb was added when the od reached. the samples were collected and the β-galactosidase activity was tested according to the method described by miller after 30 min of incubation. determination of the linear correlation between h2o and sb oxidation rate to test the h2o content, strains ts ts44-gsha and ts44-gsha-c were incubated as described above. to eliminate the h2o content difference caused by the different amount of cell collection, sb was added when the od reached, and then 2 ml of cells were harvested after a 30 min incubation with sb. the cells were resuspended in 1 ml of k3po after washing twice with 50 mmol/l k3po and sonicated on ice. then, the sonicated cell lysates were centrifuged at  × g at 4 °c, and  ml of the supernatant was transferred to wells of black microlon 96-well plates. the samples were mixed with 50 μl of amplex red and 50 μl of horseradish peroxidase, then incubated at 37 °c for 15 min. fluorescence was measured as described above using the envision® multimode plate reader and converted to the h2o concentration using appropriate calibration standards. to detect the dynamic variation of the h2o and sb contents, strains ts ts44-gsha and ts44-gsha-c were inoculated into 100 ml of cdm medium supplemented with  mmol/l sb. the cells were harvested and sonicated as described above at the designated time points. the sb contents were monitored using hplc-hg-afs as described above, while the h2o contents were determined by the ar/hrp method as described above. a boiled cdm culture of strain ts44-gsha was used as a control. to test the effect of h2o on bacterial growth, strain ts was inoculated onto cdm plates containing different concentrations of h2o and cultivated at 28 °c for 48 h. in vitro oxidation of sb by h2o was performed using an un-inoculated liquid cdm medium containing  mmol/l of sb and the media with the addition of,,,, and  mmol/l of h2o and reacted within 10 min.  a sb-sensitive mutant could be generated by transposon mutagenesis more than transposon insertions were isolated and screened for loss of sb resistance with one mutant displaying lower sb resistance. this strain, ts44-gsha was selected for further characterization. the tn transposon had inserted into gsha encoding a putative glutamate-cysteine ligase at nucleotide. the gsha encoded glutamate-cysteine ligase is the rate-limiting enzyme in de novo gsh biosynthesis and therefore is involved in conferring resistance to ros and their toxic products. additionally, a strain complementing the gsha insertion was constructed and named ts44-gsha-c. diagnostic pcrs were used to confirm the transposon mutation and the complementation. an insertion in gsha led to an increased sb oxidation rate cells of p. stutzeri ts ts44-gsha and ts44-gsha-c were incubated in cdm medium lacking sb supplementation after thorough washing. the growth of strain ts44-gsha was slightly slower compared to the wild type strain. the growth of strain ts44-gsha was further delayed by supplementation with  mmol/l sb, causing strain ts44-gsha to need an extra 24 h to reach the lag phase. the gsha complementing strain ts44-gsha-c showed no difference in growth compared to the wild type strain regardless of whether sb was added or not. although the growth of ts44-gsha was delayed, strain ts44-gsha oxidized 88% sb to sb in 48 h. in contrast, strains ts and ts44-gsha-c both oxidized only 48% sb to sb in 48 h. the sb-oxidation rate of ts44-gsha significantly increased by 83% in the monitored 48 h time frame.fig. growth curves of p. stutzeri strains ts ts44-gsha and ts44-gsha-c without or with the addition of  mmol/l sb. error bars correspond to the standard deviations of the means from three independent experiments fig. sb oxidation in p. stutzeri strains ts ts44-gsha and ts44-gsha-c with the addition of  mmol/l sb. the amounts of sb and sb in culture fluids were calculated based on culture volume to normalize for the total amount of antimony added. sb and sb concentrations in the culture fluids were measured using hplc-hg-afs. error bars correspond to the standard deviations of the means from three independent experiments an insertion in gsha eliminated gsha activity and influenced cellular contents of gsh and ros we investigated both gsha activity and the gsh and ros content to elucidate how the presence of gsha affected sb oxidation. the gsha activity in strains ts and ts44-gsha-c was slightly increased following the addition of sb; however, gsha activity was undetectable in the insertional mutant strain ts44-gsha, indicating that the transposon insertion functionally disrupted gsha in strain ts consistent with this result, the gsh content was decreased by approximately 66% in the mutant compared to strains ts and ts44-gsha-c regardless of whether sb was provided.fig. gsha insertion affected gsh content, gsha activity and ros content. a, gsha activity,, gsh content,, ros content and ros content in strain ts44-gsha data symbols shown in panels, and are the same. data are expressed as the mean ± sd, n =  **indicates a significant difference from the control as a next step, cellular ros content was determined because gsh is the central substrate for cellular protection against ros and their toxic products. the cellular ros content in strain ts44-gsha was increased by approximately three-fold compared to strains ts and ts44-gsha-c regardless of whether sb was provided or not. the cellular ros content was significantly decreased in strain ts44-gsha after 30 min of incubation with sb compared to no sb addition, despite the fact that gsha was mutated and the gsh content was obviously decreased. after addition of sb, the ros content changed quickly in 30 min, and decreased significantly in the strain ts44-gsha. in such a short time, the oxidation rate of sb did not significantly change. the sb oxidation mainly occurred in the 36–48 h time frame. sb affected transcription of gsha, sodb, sodc and kate differently the expression of gsha, sodb, sodc and kate was monitored, because gsha, sodb, sodc and kate are all involved in changes of gsh, ros or h2o there are two distinct genes encoding superoxide dismutase on the genome of p. stutzeri ts that encode for fe sod and cu-zn sod, respectively. these two enzymes shared over 83% and 58% amino acid sequence similarity with the respective enzymes of other species of pseudomonas, respectively, and can convert o2 •- to h2o and o in the lacz reporter fusion analysis, we only transformed the lacz fusions designated plsp-pgsha, plsp-psodb and plsp-psodc into strains ts and ts44-gsha because the gsha complementary strain ts44-gsha-c carried a plasmid that might be incompatible with the lacz fusion. the transcription levels of gsha, sodb, sodc and kate were tested by qrt-pcr in all three strains. sb had no effect on the transcription of gsha in strains ts ts44-gsha or ts44-gsha-c. the gsha::lacz fusion only showed a low level of background expression levels in the gsha insertional mutation strain ts44-gsha, which indicated a very low promoter activity of gsha, while qrt-pcr analysis showed no transcription of gsha in strain ts44-gsha. moreover, lacz fusion and qrt-pcr both showed that the sodb gene was strongly induced by sb in all three strains. sb only induced transcription of sodc and kate in the mutant strain ts44-gsha but not in strains ts and ts44-gsha-c.fig. the effect of sb to the transcription of gsha, sodb, sodc and kate. the transcription of gsha, sodb and sodc was tested by a lacz reporter fusion and qrt-pcr, while the kate transcription was only tested by qrt-pcr. for lacz reporter fusion, data are expressed as the mean ± sd, n =  **indicates a significant difference from the control. for qrt-pcr, error bars correspond to the standard deviations of the means from three biological replicates. gene expression was normalized to the 16s rrna gene. the results are presented as the mean gene expression normalized to mrna levels in sb-free cdm. data symbols shown in all panels are the same fig. the h2o content is correlated with bacterial sb oxidation. the p. stutzeri strains were cultured as described above. a the h2o concentration was tested after 30 min of incubation with sb. **indicates a significant difference from the control. b h2o content and sb concentration in strains ts ts44-gsha and ts44-gsha-c from to 48 h of incubation in cultures supplemented with  mmol/l sb. data are expressed as the mean ± sd, n =  d correlation between h2o and sb concentrations in strain ts44-gsha540 disruption of gsh synthesis allowed accumulation of h2o2 in order to examine whether the ros-protection system is involved in h2o production, we tested the h2o content in the presence and absence of sb in all strains. the h2o content was slightly higher in gsha mutant strain ts44-gsha without addition of sb compared to strains ts and ts44-gsha-c. notably, after a 30 min incubation with  mmol/l sb, the h2o content was increased in the mutant strain ts44-gsha but slightly decreased in strains ts and ts44-gsha-c, indicating that the disruption of gsh synthesis is related to the accumulation of h2o in addition, it appears the conversion of ros by sod was more efficient than the h2o conversion by kate and thus would both sod and kate were induced by sb. h2o is responsible for sb oxidation h2o was shown to be an efficient sb oxidant in vitro as the un-inoculated control showed no sb oxidation. a control with dead cells after boiling the culture also showed no oxidation of sb. as the efficiency of sb oxidation in the mutant strain ts44-gsha was shown to be highest from to 48 h, the h2o and sb content in strains ts ts44-gsha and ts44-gsha-c were also measured from to 48 h. interestingly, the h2o content in strain ts44-gsha was significantly decreased from to  nmol/mg protein, while the h2o content was stable at a low level in strains ts and ts44-gsha-c. correspondingly, sb was oxidized to sb concomitant with a decrease in h2o content, while the sb concentration was almost stable in strains ts and ts44-gsha-c. the consumed h2o content and the oxidized sb showed a linear correlation with a correlation coefficient of, indicating that h2o is responsible for sb oxidation. in addition, we could show that growth was inhibited with increasing concentration of h2o, thus sb oxidation may contribute to the bacterial detoxification of h2o and sb. discussion at present, several sb-oxidizing strains have been reported; some of these strains were also able to oxidize as. the arsenite oxidase aioab was demonstrated to be capable of oxidizing sb, but the aioab kinetic rate of the reaction was orders of magnitude higher for as than for sb. moreover, anoa belonging to the sdr superfamily was reported to be able to catalyze sb oxidation using nadp+ as a cofactor. however, disruption of aioa and anoa in agrobacterium tumefaciens caused a decrease in sb oxidation of only about 25% and 27%, respectively, indicating the existence of other bacterial sb oxidation mechanisms. after discovering that the presence of gsha affected the sb oxidation rate by transposon mutagenesis in strain ts44-gsha we proposed that some abiotic cellular components, such as gsh, ros or h2o may have a role in the bacterial oxidation of sb. subsequently, we conducted a comprehensive analysis. first, we found that a disruption of gsha caused a decrease in the cellular gsh amount; second, it is conceivable that the gsha mutant strain resulted in an increase in cellular ros content compared to the wild type; third, we could also show a linear correlation between the decrease of h2o content and the increase in sb oxidation rate, indicating that sb oxidation consumed h2o and acts as a detoxification mechanism to counter this cellular stressor. in addition, it appeared that sb directly caused a disruption of the ros-protection system in strain ts44-gsha and allowed the accumulation of h2o instead of affecting gsh levels, since neither the activity of gsha nor the gsh content were influenced by sb. previously, it was reported that sb may consume residual gsh complex) in red blood cells. however, we did not observe significant changes in gsh content when sb was added to wild type strain ts and the gsha complemented strain ts44-gsha-c, indicating little of this complex was formed and sb was mainly oxidized to sb by h2o we therefore propose a new model for sb oxidation in p. stutzeri ts i) the addition of sb would trigger the ros-protective system by inducing the transcription of sodb, sodc and kate, with sodb and sodc catalyzing the conversion of ros to h2o and kate responsible for the depletion of excessive h2o2; ii) the increased cellular h2o content enhanced the sb oxidation rate; and iii) the addition of sb played a selection role on the characterization of the gsha insertion; iv) in addition, the accumulated h2o was partially consumed by kate, since kate was induced by sb, and similar result was described recently with transcription of the peroxidase-encoding gene kata being induced by both sb and h2o.fig. the proposed model for sb bacterial oxidation in p. steutzeri ts in this study, i) the addition of sb would trigger the ros-protective system by inducing the transcription of sodb, sodc and kate, with sodb and sodc catalyzing the conversion of ros to h2o while kate responsible for the degradation of excessive h2o; ii) the increased cellular h2o content enhanced the sb oxidation rate; and iii) the addition of sb played a selection role on the characterization of gsha insertion; iv) in addition, the accumulated h2o is partially cosumed by the upregulated catalase kate several chemical substances have been reported to be capable of mediating sb oxidation in vitro. oh• was the oxidant in acidic solutions and h2o was the main oxidant in neutral and alkaline solutions involved in sb oxidation, with a ph range of to. sb was reported to cause cellular oxidative stress in lymphoid tumoral cells. h2o is a substantial component of cellular oxidative stress and can inhibit cellular growth. in this study, the ph of the cultures changed from the initial to approximately following exposure to sb, indicating that h2o may correlate with an increase in the sb oxidation rate. in addition, the in vitro experiment provided direct evidence that h2o can oxidize sb to sb and a correlation between the concentrations of sb and h2o was found in vivo. as an moderatedly reactive intermediate, o2 •- may also have an effect on sb oxidation. however, in the presence of high level of sod, the steady-state of o2 •- would be no more than  nmol/l and thus the effect of o2 •- is negligible. thus, we conclude that h2o oxidized sb to sb.  this study proposed a novel mechanism for microbial antimonite oxidation involving changes in cellular content of components related to oxidative stress. although h2o was reported to be capable of oxidizing sb chemically as early as, this is the first study to demonstrate that h2o is responsible for sb-oxidation in a microbe. the results showed that a disruption of bacterial gsh-dependent ros-protection mechanism allowed h2o to build up and thus promote the oxidation of sb to sb. in addition, the oxidation of sb to sb is a detoxification process against the cellular stressor h2o we do not exclude the possibility of an additional enzymatic process responsible for sb oxidation in strain ts since genes encoding a putative arsenite oxidase aioba were found on its genome which may function as a sb oxidase. our data and other findings could show that microbial antimonite oxidation contains both biotic and abiotic components. additional files additional file 1: table s primers used in this study. additional file 2: figure s the gene cluster containing gsha in strain ts and diagnostic pcr confirming the transposon mutation to create mutant strain ts44-gsha and complementation to create ts44-gsha540-c., the transposon insertion site of the gsha mutant is shown by the vertical arrow., pcr used primers r6k-f/r6k-r and tnpa-f/tnpa-r using genomic dna of strain ts44-gsha as template. lane is the amplification of r6k fragment, while line represents the tnpa fragment., pcr used primers gsha-f/gsha-f. lane strain ts lane gsha gene insertional inactivation strain ts44-gsha and lane the complemented strain ts44-gsha540-c. m, the molecular weight marker. amplicon identities were confirmed by dna sequencing. additional file 3: figure s in vitro oxidation of sb by h2o sb was added into uninoculated liquid cdm medium to a final concentration of  mmol/l. cdm containing  mmol/l of sb and with the addition of,,,, and  mmol/l of h2o respectively. additional file 4: figure s the tolerance of p. stutzeri strains ts ts44-gsha and ts44-gsha-c to h2o the strains were grown in liquid cdm medium until reaching an od of and then serially diluted 10-fold. the 10− 10− and 10− dilutions were spotted onto agar plates with different h2o concentrations. not applicable. funding the present study was supported by the national natural science foundation of china to gw. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. availability of data and materials all data generated or analyzed during this study are included in this published article and its supplementary information files. authors’ contributions dw performed the experiments and wrote the draft of the manuscript, fz designed and performed the experiments and helped to draft the manuscript. qw and cr revised the draft of the manuscript. py and jg performed the experiments. gw designed the study and revised the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate not applicable.
an insertion mutation into gsha, encoding a glutamate cysteine ligase involved in glutathione biosynthesis, generated a strain called p. stutzeri ts44-gsha this mutant strain was complemented with a plasmid carrying gsha to generate strain p. stutzeri ts44-gsha-c. the transcription of gsha, the two superoxide dismutase -encoding genes sodb and sodc as well as the catalase-encoding gene kate was monitored because gsha-encoded glutamate cysteine ligase is responsible for the biosynthesis of glutathione and involved in the cellular stress defense system as are superoxide dismutase and catalase responsible for the conversion of ros. compared to the wild type p. stutzeri ts and ts44-gsha-c, the mutant p. stutzeri ts44-gsha had a lower gsh content and exhibited an increased content of total ros and h2o and increased the sb oxidation rate. based on the results, we propose that a disruption of the pathway involved in ros-protection allowed h2o to accumulate. in addition to the previously reported enzyme mediated sb oxidation, the mechanism of bacterial oxidation of sb to sb includes a non-enzymatic mediated step using h2o as the oxidant. in addition, the cellular content of total ros and in particular h2o was analyzed. a positive linear correlation was found between the sb oxidation rate and the h2o content, indicating that the accumulated h2o is correlated to the increased sb oxidation rate. pseudomonas stutzerisb oxidationh2o2transposon mutagenesisgshareactive oxygen species http://dx.doi.org/ /501100001809national natural science foundation of china31170106wang gejiao issue-copyright-statement© the author 2016 currently, little is known about the mechanisms underlying bacterial sb resistance and oxidation. in this study, tn transposon mutagenesis was conducted in the sb-oxidizing strain pseudomonas stutzeri ts to isolate the genes responsible for sb resistance and oxidation.
 microbial antimonite oxidation converts toxic sb into less toxic antimonate and plays an important role in the biogeochemical sb cycle. currently, little is known about the mechanisms underlying bacterial sb resistance and oxidation.  in this study, tn transposon mutagenesis was conducted in the sb-oxidizing strain pseudomonas stutzeri ts to isolate the genes responsible for sb resistance and oxidation. an insertion mutation into gsha, encoding a glutamate cysteine ligase involved in glutathione biosynthesis, generated a strain called p. stutzeri ts44-gsha this mutant strain was complemented with a plasmid carrying gsha to generate strain p. stutzeri ts44-gsha-c. the transcription of gsha, the two superoxide dismutase -encoding genes sodb and sodc as well as the catalase-encoding gene kate was monitored because gsha-encoded glutamate cysteine ligase is responsible for the biosynthesis of glutathione and involved in the cellular stress defense system as are superoxide dismutase and catalase responsible for the conversion of ros. in addition, the cellular content of total ros and in particular h2o was analyzed. compared to the wild type p. stutzeri ts and ts44-gsha-c, the mutant p. stutzeri ts44-gsha had a lower gsh content and exhibited an increased content of total ros and h2o and increased the sb oxidation rate. furthermore, the transcription of sodb, sodc and kate was induced by sb. a positive linear correlation was found between the sb oxidation rate and the h2o content, indicating that the accumulated h2o is correlated to the increased sb oxidation rate.  based on the results, we propose that a disruption of the pathway involved in ros-protection allowed h2o to accumulate. in addition to the previously reported enzyme mediated sb oxidation, the mechanism of bacterial oxidation of sb to sb includes a non-enzymatic mediated step using h2o as the oxidant. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pseudomonas stutzerisb oxidationh2o2transposon mutagenesisgshareactive oxygen species http://dx.doi.org/ /501100001809national natural science foundation of china31170106wang gejiao issue-copyright-statement© the author 2016 