BACKGROUND
the toxin arsenic in soil and aqueous environments is considered as one of the prominent environmental causes of cancer mortality in the world, especially in bangladesh, india and china.
in recent years, chronic intake of groundwater with high levels of arsenic has caused endemic arsenicosis in several provinces of china and new cases of arsenicosis are continuously emerging  <cit> .
developing efficient and environment-friendly technologies to remove arsenic from soil and water systems is of great importance to many countries including china.
bioremediation of heavy or toxic metal contaminated sites has been often shown to be more efficient than chemical and physical methods, especially when stimulating indigenous microbial communities  <cit> .
bacteria have developed different strategies to transform arsenic including arsenite oxidation, cytoplasmic arsenate reduction, respiratory arsenate reduction, and arsenite methylation  <cit> .
the primary role of some of these transformations is to cope with arsenic toxicity.
arsenite-oxidizing bacteria oxidize arsenite  to arsenate  which in many cases is considered primarily a detoxification metabolism since as is much less toxic than as.
in addition, as is negatively charged and can be easily adsorbed, thus such bacteria have been used in batch reactors together with immobilizing material for removing arsenic from waste water  <cit> .
as oxidation has been identified in various bacteria including pseudomonas  <cit> , alcaligenes  <cit> , thiomonas  <cit> , herminiimonas  <cit> , agrobacterium  <cit> , and thermus  <cit> .
some of these bacteria were able to use as as the sole electron donor and grew as lithotrophs.
however, characterized heterotrophic arsenite-oxidizing bacteria have not been shown to gain energy through arsenite oxidation and probably use as oxidation as a detoxification mechanism.
arsenite oxidation was catalyzed by a periplasmic arsenite oxidase.
this enzyme contains two subunits encoded by the genes aoxa/arob/asob  and aoxb/aroa/asoa  respectively  <cit> .
recently aoxb-like sequences have been widely found in different arsenic contaminated soil and water systems  <cit> .
two families of arsenite transport proteins responsible for as extrusion, arsb and acr3p, have been shown to confer arsenic resistance  <cit> .
the founding member of the arsb family, arsb from e. coli, has been extensively characterized and shown to be a  <dig> kda, inner membrane protein with  <dig> transmembrane helices  <cit> .
either arsb alone or in association with arsa catalyzes the extrusion of arsenite and antimonite from cells  <cit> .
in most cases, arsb is co-transcribed with arsc encoding an arsenate reductase.
it has been suggested that evolution and horizontal gene transfer  of both the arsb and the arsc family may have happened simultaneously in microbial evolution  <cit> .
in many cases, as is taken up by aquaglyceroporins  <cit>  and extruded by arsb  <cit> .
members of acr3p transporters showed a function similar to arsb, but the two proteins have no significant sequence similarity.
even though acr3p is much less characterized, it has been reported to be present in more phylogenetically distant species than arsb.
acr3p could be divided into two subfamilies, acr3p and acr3p, based on their phylogenetic dissimilarities  <cit> .
acr3p appeared to be more specific and transported only arsenite but not antimonite  <cit> , except that acr3p of synechocystis was able to transport both arsenite and antimonite  <cit> .
the arsenite transporter gene arsb, acr <dig> and acr <dig> have been identified in various soil bacteria  <cit> .
however, a correlation between genotype and arsenite resistance level has not been found yet.
the impact of microbial arsenite oxidation and arsenate reduction were reported to influence environmental arsenic cycles  <cit> .
understanding the diversity and distribution of indigenous bacterial species in arsenic-contaminated sites could be important for improvement of arsenic bioremediation.
microbial species with arsenic biotransforming capabilities had so far not been evaluated in soil systems in china.
the objectives of this study were:  study the distribution and diversity of arsenite-resistant and arsenite-oxidizing bacteria in soils with different arsenic-contaminated levels;  investigation of the different arsenite oxidase and arsenite transporter genes and attempt to correlate their presence to the arsenic resistance level of these bacteria.
RESULTS
distribution and diversity of arsenite-resistant bacteria in soils with different levels of arsenic
analysis of microbial species and diversity of arsenite-resistant bacteria were performed in  <dig> soil samples with high , intermediate  and low  levels of arsenic contamination.
a total of  <dig> arsenite-resistant bacteria were obtained and  <dig> of them showed arsenite oxidizing abilities.
based on analyses of colony morphologies and 16s rdna-rflp, a total of  <dig> strains were obtained including  <dig> arsenite-oxidizing bacteria.
nearly full-length 16s rdna sequences were used for bacterial identification.
among the analyzed  <dig> strains,  <dig> showed 100% nucleotide identities,  <dig> had 99% identities,  <dig>  had 98% identities and  <dig>  had 97% identities to sequences deposited in genbank.
phylogenetic analysis divided the  <dig> strains into  <dig> genera belonging to  <dig> major bacterial lineages: α-proteobacteria , β-proteobacteria , γ-proteobacteria , firmicutes  and actinobacteria  .
among the  <dig> strains,  <dig> were isolated from the highly arsenic-contaminated soil ,  <dig> were from the intermediate arsenic-contaminated soil  and  <dig> from the low arsenic-contaminated soils  .
acinetobacter, delftia, arthrobacter, microbacterium, aeromonas, acidovorax, ensifer, janibacter, janthinobacterium, klebsiella, kocuria, micrococcus, shewanella and thauera were identified from the highly arsenic-contaminated ts soil only.
achromobacter and williamsia were specific for the sy site.
exiguobacterium particularly existed in the ly/yc sites.
comamonas, pseudomonas and stenotrophomonas were identified from both the ts and sy sites.
agrobacterium, rhodococcus and bacillus were identified from both the ts and ly/yc sites.
acinetobacter, comamonas, pseudomonas, stenotrophomonas, delftia, agrobacterium and bacillus were the major arsenite-resistant bacteria in all the analyzed soil samples .
arsenite-oxidizing bacteria were phylogentically distant
five arsenite-oxidizing bacteria were identified including achromobacter sp.
sy <dig> , agrobacterium spp.
ts <dig>  ts <dig>  ly <dig> , and pseudomonas sp.
ts <dig>  .
all of them were heterotrophic since they could not use co <dig> as the sole carbon source and also could not grow with arsenite as the sole electron donor .
the 16s rdna identities of these strains were analysed and compared to the following related arsenite-oxidizing bacteria.
achromobacter sp.
sy <dig> shared 98% 16s rdna identity to achromobacter sp.
nt <dig>   <cit> .
agrobacterium spp.
ts <dig>  ts <dig>  ly <dig> showed 99%, 98% and 99% 16s rdna identities to agrobacterium sp.
5a   <cit>  respectively.
the 16s rdna identity between pseudomonas sp.
ts <dig> and pseudomonas putida strain os- <dig> was 97%   <cit> .
arsenite resistance levels of arsenite-resistant bacteria vary greatly
the mic range for arsenite of the  <dig> strains was from  <dig> mm to  <dig> mm .
the numbers of the strains with mic values ≥  <dig> mm, ≥  <dig> mm, ≥ <dig> mm, ≥ <dig> mm, ≥  <dig> mm, ≥  <dig> mm and ≥  <dig> mm were  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> respectively.
certain correlations were found between the arsenite resistance levels, the bacterial species and soils with different arsenic-contaminated levels:  all of the  <dig> strains belonging to firmicutes showed a very low mics ;  among the strains belonging to pseudomonas or agrobacterium, the mics of arsenite oxidizers were higher than the non-arsenite oxidizers ;  the average mic of the  <dig> arsenite oxidizers  was higher than the  <dig> non-arsenite oxidizers ;  a total of  <dig> highly arsenite-resistant bacteria  whose mics exceeded  <dig> mm were only obtained from highly arsenic-contaminated ts soil;  only intermediate  and low  mic levels of arsenite-resistant bacteria were identified from the intermediate and low arsenic-contaminated soils;  strains of the highly arsenic-contaminated ts site had higher arsenite resistance levels  than the strains of the intermediate  and low arsenic soils .
identification and distribution of the arsenite oxidase gene aoxb and arsenite transporter genes arsb, acr <dig> and acr3
a total of  <dig> arsenite oxidase genes and  <dig> arsenite transporter genes were successfully amplified from  <dig> strains using pcr with degenerate primers.
the acr <dig> and acr <dig> were amplified in strains of the high and intermediate arsenic-contaminated soils only.
in contrast, aoxb and arsb were found in strains isolated from all three kinds of arsenic-contaminated environments .
strains containing both an arsenite oxidase gene  and an arsenite transporter gene  showed a higher average arsenite resistance level than the strains possessing arsenite transporter genes only .
the aoxb sequences were amplified from all of the five arsenite oxidizers.
no aoxb sequences were amplified from any of the  <dig> non-arsenite oxidizers.
the deduced amino acid sequence of aoxb from achromobacter sp.
sy <dig> showed 95% identity to aoxb from achromobacter sp.
nt- <dig>   <cit> .
the deduced aoxbs from agrobacterium spp.
ts <dig>  ts <dig>  and ly <dig> displayed 95%, 96%, and 95% identities to aoxb of agrobacterium tumefaciens 5a   <cit> , and 94%, 96%, and 95% identities to aoxb from rhizobium sp.
nt <dig>   <cit>  respectively.
the identity of aoxbs between pseudomonas sp.
ts <dig> and alcaligenes faecalis ncib  <dig> was only 61% .
comparison between 16s rdna and deduced aoxb phylogenetic trees indicated that their evolutionary relationship was similar.
analyses of arsenite transporter genes using the blastx algorithm showed proteins with 71%–80% aa identities that contained  <dig> arsb and  <dig> acr3p, 81%–90% aa identities had  <dig> arsb,  <dig> acr3p and  <dig> acr3p, aa identities over 90% included  <dig> arsb,  <dig> acr3p and  <dig> acr3p.
the arsb, acr <dig>  and acr <dig> were amplified from both the arsenite oxidizers and non-arsenite oxidizers.
the strains containing  <dig> arsenite transporter genes simultaneously were pseudomonas sp.
sy <dig> , shewanella sp.
ts <dig>  delftia spp.
ts <dig>  ts <dig>  ts <dig>  ts <dig> and pseudomonas sp.
sy <dig> , stenotrophomonas spp.
ts <dig>  sy <dig>  sy <dig>  aeromonas spp.
ts <dig>  ts <dig> and pseudomonas sp.
ts <dig>  .
phylogenetic analysis of arsb, acr3p and acr3p showed a clear separation of the acr3p and arsb clusters .
the acr3p cluster was further divided into two phylogenetic groups, acr3p and acr3p.
the arsb cluster was formed by  <dig> sequences from β-, γ-proteobacteria and firmicutes; the acr3p group had  <dig> sequences from γ-proteobacteria and actinobacteria; the acr3p group contained  <dig> sequences from α-, β-, and γ-proteobacteria .
horizontal transfer of arsenite transporter genes may have occurred with acr <dig> and arsb
the arsenite oxidase gene aoxb appeared to be vertically transferred when comparing the phylogeny of 16s rrna genes with those encoding aoxb.
in contrast, certain inconsistency occurred when comparing phylogenetic trees based on 16s rrna genes and arsenite transporter genes.
phylogenetic discrepancies could be detected in  <dig> acr <dig> and  <dig> arsb :  aeromonas spp.
ts <dig>  ts <dig> belonging to γ-proteobacteria based on 16s rdna analysis were assigned to the β-proteobacteria based on acr3p sequences;  stenotrophomonas spp.
ts <dig>  sy <dig>  sy <dig> belonging to γ-proteobacteria using 16s rdna analysis were assigned to α-proteobacteria based on acr3p sequences;  comamonas sp.
ts <dig>  ts <dig> and delftia sp.
ts <dig> were shown to belong to β-proteobacteria, but were assigned to the γ-proteobacteria clade using acr3p sequences;  ly <dig> belonged to α-proteobacteria based on the 16s rrna gene, but its arsb was in γ-proteobacteria clade .
the phylogenetic discrepancies exhibited that these  <dig> arsenite transporter genes were probably acquired by horizontal gene transfer .
furthermore,  <dig> of these horizontally transferred acr <dig> genes were from the strains isolated from the highly arsenic-contaminated ts soil.
discussion
the first goal of this study was to determine the distribution and diversity of arsenite-resistant bacteria from soils with different levels of arsenic contamination.
in addition, the ability to oxidize arsenite was further analyzed.
since the soils were collected from the surface and subsurface zones, only aerobic conditions were used in bacterial isolation.
thus, only aerobic/facultative aerobic bacteria were obtained in this study.
we identified acinetobacter, comamonas, pseudomonas, stenotrophomonas, delftia, agrobacterium, and bacillus as the major genera of arsenite-resistant bacteria in the four arsenic-contaminated soils.
among them, acinetobacter, agrobacterium, bacillus, and pseudomonas species were commonly found at other arsenic-contaminated sites  <cit> .
to our knowledge, janibacter, micrococcus, thauera, and williamsia were novel arsenite-resistant bacteria isolated in this study.
we found that the high arsenic ts site revealed a much higher diversity of arsenite-resistant bacteria and the resistance levels observed were also much higher than in isolates found in the intermediate and low arsenic-contaminated sites.
it is a limitation that only one medium  was used for bacterial isolation which could result in the observed differences between sites.
the  <dig> strains with arsenite mics >  <dig> mm were all obtained from the high arsenic soil.
generally, it has been proposed that high arsenic contamination is likely to exert a strong selective pressure leading to low microbial diversity  <cit> .
however, the ts site used in our study had several hundred years of smelting history  <cit>  which may result in the evolution of more bacterial species that were already well adapted at elevated arsenic concentrations.
moreover, pennanen et al.
<cit>  reported that at long-term field sites, soil microbial communities have had time to adapt to metal and/or metalloid stress.
turpeinen et al.
<cit>  also found that the diversity of arsenic-resistant bacteria in higher arsenic-, chromium- and copper-contaminated soil was higher than that in less contaminated soil.
these results suggested that microorganisms had been adapted to high arsenic stress and maintained their diversity in ts site after a long-term exposure to arsenic.
the aoxb genes were detected in all of the five arsenite oxidizers but not in the non-arsenite oxidizers.
this indicates that aoxb may be specific for most of the aerobic arsenite-oxidizing bacteria and useful for detecting arsenite-oxidizing microorganisms in the environment.
inskeep et al.
<cit>  reported that arsenite oxidase genes are widely present in different arsenite oxidizers and widespread in soil-water systems.
we have enriched pristine soils with arsenite to isolate arsenite-oxidizing bacteria from non-contaminated soils but without success.
to our knowledge, all of the cultured arsenite oxidizers obtained so far were isolated from arsenic-contaminated sites.
inskeep et al.
<cit>  detected aoxb-like sequences from arsenic-contaminated environments but not from pristine soils indicating that arsenite oxidation is a major process in arsenic-contaminated environments.
the expression level of aoxb could probably be applied to monitor environmental arsenic-contaminated levels.
a phylogenetic analysis of the  <dig> arsenite oxidizers based on the 16s rrna genes and the aoxb genes showed a similar phylogeny indicating genomic stability of the aoxb genes.
the mics of the arsenite oxidizers displayed a maximal arsenite resistance level in both pseudomonas and agrobacterium.
furthermore, strains containing both the arsenite oxidase and any type of transporter gene showed a higher arsenite resistance level.
these results suggest that bacteria capable of both arsenite oxidation and arsenite efflux mechanisms have an elevated arsenite resistance level.
we also found that arsenite can be fully oxidized even at concentrations close to the mic in arsenite oxidizers sy <dig> and ts <dig> .
recently, we have amplified and sequenced the arsc/acr <dig> operon  in the adjacent downstream region of aoxb in pseudomonas.
sp.
ts <dig> .
kashyap et al.
<cit>  found that in agrobacterium tumefaciens strain 5a, disruption of aoxr caused a loss in the ability to oxidize arsenite and furthermore resulted in an apparent reducing phenotype probably due to the action of cytosolic arsc and subsequent pumping out of as.
it is noteworthy to point out that there are two processes of as reduction in the environment.
one is the use of as as a terminal electron acceptor under anaerobic conditions.
the other is the intracellular reduction of as to as under aerobic conditions due to the arsc-dependent cytoplasmic arsenate reduction as part of the arsenic resistance system .
since as is the species being pumped out of cell , the presence of as in the environments can also be detected under aerobic condition.
one of the main purposes in this research was to determine the correlation among the bacterial arsenite resistance level, bacterial distribution in the environment and the different types of arsenite transporter gene families.
we found that the acr <dig> genotypes were predominant over arsb  in our samples which was in agreement with a report by achour et al.
<cit> .
in addition, we found any two types of arsenite transporter genes can coexist in the same strain .
related reports also found the presence of multiple sets of arsenic resistance genes and operons in one strain, especially the arsenite transporter genes.
pseudomonas putida kt <dig> contains two operon clusters  for arsenic resistance  <cit> .
acidithiobacillus caldus has three sets of arsenic resistance determinants, one located on the chromosome and the other two exist on the transposon  <cit> .
corynebacterium glutamicum has two typical arsenic-resistant operons and additional arsb and arsc genes, of which two arsenite transporter genes belonged to the acr <dig> group  <cit> .
the genome of herminiimonas arsenicoxydans revealed the presence of four arsenic resistance operons including two arsb genes and one acr <dig>  <cit> .
multiple sets of arsenic resistance determinants were also reported in b. subtilis  <cit>  and desulfovibrio desulfuricans g <dig>  <cit> .
these findings indicate that microorganisms have acquired multiple resistance determinants via chromosomal duplication or horizontal gene transfer to cope with arsenic toxicity.
in this study, acr <dig> and acr <dig> appeared to be mostly associated with high arsenite resistance since they were only identified from the high and intermediate arsenic-contaminated sites, while arsb was found in all three sites.
one explanation is that acr <dig> may have a higher affinity and velocity to extrude arsenite than arsb and thus seems to be more effective.
heavy metal contaminated environments were shown to provide a strong selective pressure for transfer of related resistance genes within soil systems  <cit> .
in this study, aoxb and acr <dig> appeared to be more stable than acr <dig> and arsb since phylogenetic discrepancies between 16s rrna genes and acr3/arsb were found which supported hgt events of acr <dig> and arsb.
most of the hgt occurred in strains identified from the highly arsenic-contaminated ts soil .
this indicates that arsenite transporter genes may be horizontally transferred and increasingly present in a microbial population under conditions of long-term elevated arsenic stress.
it is important to note that hgt occurred in somewhat closely related species in this study, however, this does not detract from the suggestion of hgt and it is likely that the hgt events occurred between these closely related species.
martinez et al.
<cit>  reported that pib-type atpases  were broadly transferred in arthrobacter and bacillus in radionuclide and metal contaminated soils.
jackson and dugas  <cit>  also suggested that horizontally transferred arsc resulted in the diversities and complexities of arsenate reductase during its evolution.
excluding arsc, other genes related to arsenic resistance  had not been reported as being transferred by hgt.
to our knowledge, this is the first study to report widespread horizontal transfer of arsenite transporter genes.
the hgt event and subsequent maintenance may have occurred increasingly under the high arsenic pressure  <cit>  and resulted in plastic changes in microbial diversity.
CONCLUSIONS
this work investigates the distribution and diversity of microbial arsenite-resistant species in soils representing three different levels of arsenic contamination, and further studies the arsenite resistance and arsenic transforming genes of these species.
our research provides valuable information of microbial species and genes responsible for arsenite oxidation and resistance, and increases knowledge of the diversity and distribution of the indigenous bacteria that may be stimulated for successful bioremediation of arsenic contamination.

 the toxin arsenic in soil and aqueous environments is considered as one of the prominent environmental causes of cancer mortality in the world, especially in bangladesh, india and china. in recent years, chronic intake of groundwater with high levels of arsenic has caused endemic arsenicosis in several provinces of china and new cases of arsenicosis are continuously emerging. developing efficient and environment-friendly technologies to remove arsenic from soil and water systems is of great importance to many countries including china. bioremediation of heavy or toxic metal contaminated sites has been often shown to be more efficient than chemical and physical methods, especially when stimulating indigenous microbial communities. bacteria have developed different strategies to transform arsenic including arsenite oxidation, cytoplasmic arsenate reduction, respiratory arsenate reduction, and arsenite methylation. the primary role of some of these transformations is to cope with arsenic toxicity. arsenite-oxidizing bacteria oxidize arsenite to arsenate which in many cases is considered primarily a detoxification metabolism since as is much less toxic than as. in addition, as is negatively charged and can be easily adsorbed, thus such bacteria have been used in batch reactors together with immobilizing material for removing arsenic from waste water. as oxidation has been identified in various bacteria including pseudomonas, alcaligenes, thiomonas, herminiimonas, agrobacterium, and thermus. some of these bacteria were able to use as as the sole electron donor and grew as lithotrophs. however, characterized heterotrophic arsenite-oxidizing bacteria have not been shown to gain energy through arsenite oxidation and probably use as oxidation as a detoxification mechanism. arsenite oxidation was catalyzed by a periplasmic arsenite oxidase. this enzyme contains two subunits encoded by the genes aoxa/arob/asob and aoxb/aroa/asoa respectively. recently aoxb-like sequences have been widely found in different arsenic contaminated soil and water systems. two families of arsenite transport proteins responsible for as extrusion, arsb and acr3p, have been shown to confer arsenic resistance. the founding member of the arsb family, arsb from e. coli, has been extensively characterized and shown to be a kda, inner membrane protein with transmembrane helices. either arsb alone or in association with arsa catalyzes the extrusion of arsenite and antimonite from cells. in most cases, arsb is co-transcribed with arsc encoding an arsenate reductase. it has been suggested that evolution and horizontal gene transfer of both the arsb and the arsc family may have happened simultaneously in microbial evolution. in many cases, as is taken up by aquaglyceroporins and extruded by arsb. members of acr3p transporters showed a function similar to arsb, but the two proteins have no significant sequence similarity. even though acr3p is much less characterized, it has been reported to be present in more phylogenetically distant species than arsb. acr3p could be divided into two subfamilies, acr3p and acr3p, based on their phylogenetic dissimilarities. acr3p appeared to be more specific and transported only arsenite but not antimonite, except that acr3p of synechocystis was able to transport both arsenite and antimonite. the arsenite transporter gene arsb, acr and acr have been identified in various soil bacteria. however, a correlation between genotype and arsenite resistance level has not been found yet. the impact of microbial arsenite oxidation and arsenate reduction were reported to influence environmental arsenic cycles. understanding the diversity and distribution of indigenous bacterial species in arsenic-contaminated sites could be important for improvement of arsenic bioremediation. microbial species with arsenic biotransforming capabilities had so far not been evaluated in soil systems in china. the objectives of this study were: study the distribution and diversity of arsenite-resistant and arsenite-oxidizing bacteria in soils with different arsenic-contaminated levels; investigation of the different arsenite oxidase and arsenite transporter genes and attempt to correlate their presence to the arsenic resistance level of these bacteria.  distribution and diversity of arsenite-resistant bacteria in soils with different levels of arsenic analysis of microbial species and diversity of arsenite-resistant bacteria were performed in soil samples with high, intermediate and low levels of arsenic contamination. a total of arsenite-resistant bacteria were obtained and of them showed arsenite oxidizing abilities. based on analyses of colony morphologies and 16s rdna-rflp, a total of strains were obtained including arsenite-oxidizing bacteria. nearly full-length 16s rdna sequences were used for bacterial identification. among the analyzed strains, showed 100% nucleotide identities, had 99% identities, had 98% identities and had 97% identities to sequences deposited in genbank. phylogenetic analysis divided the strains into genera belonging to major bacterial lineages: α-proteobacteria, β-proteobacteria, γ-proteobacteria, firmicutes and actinobacteria. among the strains, were isolated from the highly arsenic-contaminated soil, were from the intermediate arsenic-contaminated soil and from the low arsenic-contaminated soils. acinetobacter, delftia, arthrobacter, microbacterium, aeromonas, acidovorax, ensifer, janibacter, janthinobacterium, klebsiella, kocuria, micrococcus, shewanella and thauera were identified from the highly arsenic-contaminated ts soil only. achromobacter and williamsia were specific for the sy site. exiguobacterium particularly existed in the ly/yc sites. comamonas, pseudomonas and stenotrophomonas were identified from both the ts and sy sites. agrobacterium, rhodococcus and bacillus were identified from both the ts and ly/yc sites. acinetobacter, comamonas, pseudomonas, stenotrophomonas, delftia, agrobacterium and bacillus were the major arsenite-resistant bacteria in all the analyzed soil samples. arsenite-oxidizing bacteria were phylogentically distant five arsenite-oxidizing bacteria were identified including achromobacter sp. sy, agrobacterium spp. ts ts ly, and pseudomonas sp. ts. all of them were heterotrophic since they could not use co as the sole carbon source and also could not grow with arsenite as the sole electron donor. the 16s rdna identities of these strains were analysed and compared to the following related arsenite-oxidizing bacteria. achromobacter sp. sy shared 98% 16s rdna identity to achromobacter sp. nt. agrobacterium spp. ts ts ly showed 99%, 98% and 99% 16s rdna identities to agrobacterium sp. 5a respectively. the 16s rdna identity between pseudomonas sp. ts and pseudomonas putida strain os- was 97%. arsenite resistance levels of arsenite-resistant bacteria vary greatly the mic range for arsenite of the strains was from mm to mm. the numbers of the strains with mic values ≥ mm, ≥ mm, ≥ mm, ≥ mm, ≥ mm, ≥ mm and ≥ mm were and respectively. certain correlations were found between the arsenite resistance levels, the bacterial species and soils with different arsenic-contaminated levels: all of the strains belonging to firmicutes showed a very low mics; among the strains belonging to pseudomonas or agrobacterium, the mics of arsenite oxidizers were higher than the non-arsenite oxidizers; the average mic of the arsenite oxidizers was higher than the non-arsenite oxidizers; a total of highly arsenite-resistant bacteria whose mics exceeded mm were only obtained from highly arsenic-contaminated ts soil; only intermediate and low mic levels of arsenite-resistant bacteria were identified from the intermediate and low arsenic-contaminated soils; strains of the highly arsenic-contaminated ts site had higher arsenite resistance levels than the strains of the intermediate and low arsenic soils. identification and distribution of the arsenite oxidase gene aoxb and arsenite transporter genes arsb, acr and acr3 a total of arsenite oxidase genes and arsenite transporter genes were successfully amplified from strains using pcr with degenerate primers. the acr and acr were amplified in strains of the high and intermediate arsenic-contaminated soils only. in contrast, aoxb and arsb were found in strains isolated from all three kinds of arsenic-contaminated environments. strains containing both an arsenite oxidase gene and an arsenite transporter gene showed a higher average arsenite resistance level than the strains possessing arsenite transporter genes only. the aoxb sequences were amplified from all of the five arsenite oxidizers. no aoxb sequences were amplified from any of the non-arsenite oxidizers. the deduced amino acid sequence of aoxb from achromobacter sp. sy showed 95% identity to aoxb from achromobacter sp. nt-. the deduced aoxbs from agrobacterium spp. ts ts and ly displayed 95%, 96%, and 95% identities to aoxb of agrobacterium tumefaciens 5a, and 94%, 96%, and 95% identities to aoxb from rhizobium sp. nt respectively. the identity of aoxbs between pseudomonas sp. ts and alcaligenes faecalis ncib was only 61%. comparison between 16s rdna and deduced aoxb phylogenetic trees indicated that their evolutionary relationship was similar. analyses of arsenite transporter genes using the blastx algorithm showed proteins with 71%–80% aa identities that contained arsb and acr3p, 81%–90% aa identities had arsb, acr3p and acr3p, aa identities over 90% included arsb, acr3p and acr3p. the arsb, acr and acr were amplified from both the arsenite oxidizers and non-arsenite oxidizers. the strains containing arsenite transporter genes simultaneously were pseudomonas sp. sy, shewanella sp. ts delftia spp. ts ts ts ts and pseudomonas sp. sy, stenotrophomonas spp. ts sy sy aeromonas spp. ts ts and pseudomonas sp. ts. phylogenetic analysis of arsb, acr3p and acr3p showed a clear separation of the acr3p and arsb clusters. the acr3p cluster was further divided into two phylogenetic groups, acr3p and acr3p. the arsb cluster was formed by sequences from β-, γ-proteobacteria and firmicutes; the acr3p group had sequences from γ-proteobacteria and actinobacteria; the acr3p group contained sequences from α-, β-, and γ-proteobacteria. horizontal transfer of arsenite transporter genes may have occurred with acr and arsb the arsenite oxidase gene aoxb appeared to be vertically transferred when comparing the phylogeny of 16s rrna genes with those encoding aoxb. in contrast, certain inconsistency occurred when comparing phylogenetic trees based on 16s rrna genes and arsenite transporter genes. phylogenetic discrepancies could be detected in acr and arsb: aeromonas spp. ts ts belonging to γ-proteobacteria based on 16s rdna analysis were assigned to the β-proteobacteria based on acr3p sequences; stenotrophomonas spp. ts sy sy belonging to γ-proteobacteria using 16s rdna analysis were assigned to α-proteobacteria based on acr3p sequences; comamonas sp. ts ts and delftia sp. ts were shown to belong to β-proteobacteria, but were assigned to the γ-proteobacteria clade using acr3p sequences; ly belonged to α-proteobacteria based on the 16s rrna gene, but its arsb was in γ-proteobacteria clade. the phylogenetic discrepancies exhibited that these arsenite transporter genes were probably acquired by horizontal gene transfer. furthermore, of these horizontally transferred acr genes were from the strains isolated from the highly arsenic-contaminated ts soil. discussion the first goal of this study was to determine the distribution and diversity of arsenite-resistant bacteria from soils with different levels of arsenic contamination. in addition, the ability to oxidize arsenite was further analyzed. since the soils were collected from the surface and subsurface zones, only aerobic conditions were used in bacterial isolation. thus, only aerobic/facultative aerobic bacteria were obtained in this study. we identified acinetobacter, comamonas, pseudomonas, stenotrophomonas, delftia, agrobacterium, and bacillus as the major genera of arsenite-resistant bacteria in the four arsenic-contaminated soils. among them, acinetobacter, agrobacterium, bacillus, and pseudomonas species were commonly found at other arsenic-contaminated sites. to our knowledge, janibacter, micrococcus, thauera, and williamsia were novel arsenite-resistant bacteria isolated in this study. we found that the high arsenic ts site revealed a much higher diversity of arsenite-resistant bacteria and the resistance levels observed were also much higher than in isolates found in the intermediate and low arsenic-contaminated sites. it is a limitation that only one medium was used for bacterial isolation which could result in the observed differences between sites. the strains with arsenite mics > mm were all obtained from the high arsenic soil. generally, it has been proposed that high arsenic contamination is likely to exert a strong selective pressure leading to low microbial diversity. however, the ts site used in our study had several hundred years of smelting history which may result in the evolution of more bacterial species that were already well adapted at elevated arsenic concentrations. moreover, pennanen et al. reported that at long-term field sites, soil microbial communities have had time to adapt to metal and/or metalloid stress. turpeinen et al. also found that the diversity of arsenic-resistant bacteria in higher arsenic-, chromium- and copper-contaminated soil was higher than that in less contaminated soil. these results suggested that microorganisms had been adapted to high arsenic stress and maintained their diversity in ts site after a long-term exposure to arsenic. the aoxb genes were detected in all of the five arsenite oxidizers but not in the non-arsenite oxidizers. this indicates that aoxb may be specific for most of the aerobic arsenite-oxidizing bacteria and useful for detecting arsenite-oxidizing microorganisms in the environment. inskeep et al. reported that arsenite oxidase genes are widely present in different arsenite oxidizers and widespread in soil-water systems. we have enriched pristine soils with arsenite to isolate arsenite-oxidizing bacteria from non-contaminated soils but without success. to our knowledge, all of the cultured arsenite oxidizers obtained so far were isolated from arsenic-contaminated sites. inskeep et al. detected aoxb-like sequences from arsenic-contaminated environments but not from pristine soils indicating that arsenite oxidation is a major process in arsenic-contaminated environments. the expression level of aoxb could probably be applied to monitor environmental arsenic-contaminated levels. a phylogenetic analysis of the arsenite oxidizers based on the 16s rrna genes and the aoxb genes showed a similar phylogeny indicating genomic stability of the aoxb genes. the mics of the arsenite oxidizers displayed a maximal arsenite resistance level in both pseudomonas and agrobacterium. furthermore, strains containing both the arsenite oxidase and any type of transporter gene showed a higher arsenite resistance level. these results suggest that bacteria capable of both arsenite oxidation and arsenite efflux mechanisms have an elevated arsenite resistance level. we also found that arsenite can be fully oxidized even at concentrations close to the mic in arsenite oxidizers sy and ts. recently, we have amplified and sequenced the arsc/acr operon in the adjacent downstream region of aoxb in pseudomonas. sp. ts. kashyap et al. found that in agrobacterium tumefaciens strain 5a, disruption of aoxr caused a loss in the ability to oxidize arsenite and furthermore resulted in an apparent reducing phenotype probably due to the action of cytosolic arsc and subsequent pumping out of as. it is noteworthy to point out that there are two processes of as reduction in the environment. one is the use of as as a terminal electron acceptor under anaerobic conditions. the other is the intracellular reduction of as to as under aerobic conditions due to the arsc-dependent cytoplasmic arsenate reduction as part of the arsenic resistance system. since as is the species being pumped out of cell, the presence of as in the environments can also be detected under aerobic condition. one of the main purposes in this research was to determine the correlation among the bacterial arsenite resistance level, bacterial distribution in the environment and the different types of arsenite transporter gene families. we found that the acr genotypes were predominant over arsb in our samples which was in agreement with a report by achour et al.. in addition, we found any two types of arsenite transporter genes can coexist in the same strain. related reports also found the presence of multiple sets of arsenic resistance genes and operons in one strain, especially the arsenite transporter genes. pseudomonas putida kt contains two operon clusters for arsenic resistance. acidithiobacillus caldus has three sets of arsenic resistance determinants, one located on the chromosome and the other two exist on the transposon. corynebacterium glutamicum has two typical arsenic-resistant operons and additional arsb and arsc genes, of which two arsenite transporter genes belonged to the acr group. the genome of herminiimonas arsenicoxydans revealed the presence of four arsenic resistance operons including two arsb genes and one acr. multiple sets of arsenic resistance determinants were also reported in b. subtilis and desulfovibrio desulfuricans g. these findings indicate that microorganisms have acquired multiple resistance determinants via chromosomal duplication or horizontal gene transfer to cope with arsenic toxicity. in this study, acr and acr appeared to be mostly associated with high arsenite resistance since they were only identified from the high and intermediate arsenic-contaminated sites, while arsb was found in all three sites. one explanation is that acr may have a higher affinity and velocity to extrude arsenite than arsb and thus seems to be more effective. heavy metal contaminated environments were shown to provide a strong selective pressure for transfer of related resistance genes within soil systems. in this study, aoxb and acr appeared to be more stable than acr and arsb since phylogenetic discrepancies between 16s rrna genes and acr3/arsb were found which supported hgt events of acr and arsb. most of the hgt occurred in strains identified from the highly arsenic-contaminated ts soil. this indicates that arsenite transporter genes may be horizontally transferred and increasingly present in a microbial population under conditions of long-term elevated arsenic stress. it is important to note that hgt occurred in somewhat closely related species in this study, however, this does not detract from the suggestion of hgt and it is likely that the hgt events occurred between these closely related species. martinez et al. reported that pib-type atpases were broadly transferred in arthrobacter and bacillus in radionuclide and metal contaminated soils. jackson and dugas also suggested that horizontally transferred arsc resulted in the diversities and complexities of arsenate reductase during its evolution. excluding arsc, other genes related to arsenic resistance had not been reported as being transferred by hgt. to our knowledge, this is the first study to report widespread horizontal transfer of arsenite transporter genes. the hgt event and subsequent maintenance may have occurred increasingly under the high arsenic pressure and resulted in plastic changes in microbial diversity.  this work investigates the distribution and diversity of microbial arsenite-resistant species in soils representing three different levels of arsenic contamination, and further studies the arsenite resistance and arsenic transforming genes of these species. our research provides valuable information of microbial species and genes responsible for arsenite oxidation and resistance, and increases knowledge of the diversity and distribution of the indigenous bacteria that may be stimulated for successful bioremediation of arsenic contamination. 
soils with long-term arsenic contamination may result in the evolution of highly diverse arsenite-resistant bacteria and such diversity was probably caused in part by horizontal gene transfer events. a total of arsenite-resistant bacteria were identified from soils with three different arsenic-contaminated levels. highly arsenite-resistant bacteria were only isolated from the highly arsenic-contaminated site and belonged to acinetobacter, agrobacterium, arthrobacter, comamonas, rhodococcus, stenotrophomonas and pseudomonas. our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the as resistance levels and related functional genes of these species. bacteria capable of both arsenite oxidation and arsenite efflux mechanisms had an elevated arsenite resistance level. five arsenite-oxidizing bacteria that belonged to achromobacter, agrobacterium and pseudomonas were identified and displayed a higher average arsenite resistance level than the non-arsenite oxidizers. strains containing both an arsenite oxidase gene and an arsenite transporter gene displayed a higher average arsenite resistance level than those possessing an arsenite transporter gene only. aoxb genes encoding arsenite oxidase and arsenite transporter genes were successfully amplified from these strains using pcr with degenerate primers. as is much more toxic and mobile than as, hence microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly influence the human health. the aoxb genes were specific for the arsenite-oxidizing bacteria.
 arsenic is known as a toxic metalloid, which primarily exists in inorganic form and can be transformed by microbial redox processes in the natural environment. as is much more toxic and mobile than as, hence microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly influence the human health. our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the as resistance levels and related functional genes of these species.  a total of arsenite-resistant bacteria were identified from soils with three different arsenic-contaminated levels. highly arsenite-resistant bacteria were only isolated from the highly arsenic-contaminated site and belonged to acinetobacter, agrobacterium, arthrobacter, comamonas, rhodococcus, stenotrophomonas and pseudomonas. five arsenite-oxidizing bacteria that belonged to achromobacter, agrobacterium and pseudomonas were identified and displayed a higher average arsenite resistance level than the non-arsenite oxidizers. aoxb genes encoding arsenite oxidase and arsenite transporter genes were successfully amplified from these strains using pcr with degenerate primers. the aoxb genes were specific for the arsenite-oxidizing bacteria. strains containing both an arsenite oxidase gene and an arsenite transporter gene displayed a higher average arsenite resistance level than those possessing an arsenite transporter gene only. horizontal transfer of acr and arsb appeared to have occurred in strains that were primarily isolated from the highly arsenic-contaminated soil.  soils with long-term arsenic contamination may result in the evolution of highly diverse arsenite-resistant bacteria and such diversity was probably caused in part by horizontal gene transfer events. bacteria capable of both arsenite oxidation and arsenite efflux mechanisms had an elevated arsenite resistance level. 