Microbiology (1995), 141 721 7 27 Printed in Great Britain Carbon-arsenic bond cleavage by a new ly isolated Gram-negative bacterium, strain ASVZ John P. Quinn and Geoffrey McMullant Author for correspondence: John P. Quinn. Tel: +44 1232 245133 ext. 2287/2250. Fax: +4 4 1232 236505. School of Biology and Biochemistry, Queen's Universit y of Belfast, Medical Biology Centre , 97 Lisburn Roa d, Belfast B T 9 7BL, Northern Ireland Strain ASVZ, an u nide ntified Gram-negat ive bacterium newly isolate d from activated sl ud ge , wa s found t o utilize arsonoacetate at conc entr ati ons up to at least 30 mM as so le carbo n and energy so ur ce , w it h es sentially quan titative extracellular release of arsenate. Cell-f ree con versi on of arsonoacetate could not be obtained, bu t resting-cell stud ies indicated that the carbon- arseni c bond cle avage activity was inducible in the presen ce of arsonoacetate and was of limited substrate specificity, also breaking down arsonochloroacetate. The inorga nic prod uct of t he reaction may be arsenite sin ce an indu cible arsenite- oxidizing activity was als o found i n arsonoacetate-metabolizingcells. This i the f irst report of a micro-orga nism capable of uti lizi ng a co mpound containing the carbon-arseni c bond. Th e result s indi cate th at the ability of bacteria to degrade arsonoace tate is not fortuitous and may be found in environments not previously ex pose d to organoar senicals. Keywords : arsenate, arsenite, organometalloid, organoarsenical , Pseudomonas diminuta INTRODUCTION The metalloid arsenic, 20th most common element in the earth's crust, is chemically similar to phosphorus, also in group 15 of the elements. Thus bioalkylated forms of arsenic are distributed widely (cf. organophosphonates). Such arsenical compounds may arise from the need of organisms to protect themselves from the uncoupling effects of arsenate, which they cannot avoid taking in by their phosphate-concentrating mechanisms. Th e fact that arsenate is much more easily reduced than phosphate may make the observed alkylations possible. Examples of bioalkylated products include methylarsonic and dimethyl- arsinic (cacodylic) acids, which are predominant in terrestrial environments, and more complex species such as arsenobetaine, arsenocholine, and arsenolipids and arsenosugars, which are abundant in marine ecosystems (Andreae, 1986 ; Edmonds & Francesconi, 1987 ; Cullen & Reimer, 1989; Francesconi & Edmonds, 1993). I n addition, xenobiotic compounds containing the C-As bond still enter the environm ent in a variety of agricultu ral applications - some 11 000 tonnes annually in the United States (Abernathy, 1983). Most significant are the con- tinuing uses of arsanilic acid, 3-nitro-4-hydroxyphenyl- arsonic acid and 4-nitrophenylarsonic acid as animal feed additives, and of dimethylarsinic acid, and the mono- and di-sodium salts of methylarsonic acid, as cotto n defoliants ......... .................................................................................................................. ............................ t Presentaddress: School of Biology, University of Ulster, Cromore Road, Coleraine BT52 lSA, Northern Ireland. and post-emergence grass herbicides, respectively (Andreae, 1986 ; Tamaki & Frankenberger, 1992). Although extensive research has been undertaken on the mechanisms, and the extent, of bioalkylation of arsenic (reviewed by Thayer, 1993; Gadd, 1993) much less is known about the environmental fate of organoarsenicals, a nd a lack of information on the mechanisms of C-As bond cleavage constitutes the weakest link in our under- standing of the biogeochemical cycling of the element. The process is attributed exclusively to microbial activity (Andreae, 1986) ; hus the co-metabolism of arsenobetaine by marine sedimentary bacteria has been demonstrated, although the compound did not itself support bacterial growth (Hanaoka e t al., 1987, 1991, 1992). Similarly, the slow mineralization of methylarsonate to arsenate and CO, by a number of genera of soil bacteria has been described, although such demethylation occurred only in a yeast-extract- or acetate-based medium (Von Endt e t al., 1968; Shariatpahani e t al., 1983). To our knowledge, however, in no instance has any micro-organism capable of the utilization of an organoarsenical as sole carbon and energy source yet been reported. No r is any information available on the enzymology of the C-As bond cleavage reaction ; for example, the C-P-bond-cleaving enzymes phosphonoacetaldehyde hydrolase and phosphonoacetate logues (Lacoste e t al., 1992; McMullan & Quinn, 1994). We now describe the utilization of arsonoacetate (H,O,AsCH,COOH), a model com pound containing the 0001-9382 0 1995 SGM 72 1
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The m etalloid arsenic, 20th most comm on element in theearth's crust, is chemically similar to phosp horus, also ingro up 15 of the elements. Thus bioalkylated forms ofarsenic are distributed widely (cf. organophosphonates).Such arsenical compounds may arise from the need oforganisms to protect themselves from the uncouplingeffects of arsenate, which they cannot avoid taking in bytheir phosphate-concentrating mechanisms. Th e fact thatarsenate is muc h mo re easily reduced tha n pho sphate maymake the observed alkylations possible. Examples ofbioalkylated products include m ethylarsonic and dimethyl-arsinic (cacodylic) acids, which are predominant interrestrial environments, and more complex species suchas arsenobetaine, arsenocholine, and arsenolipids andarsenosugars, which are abundant in marine ecosystems(Andreae, 1986; Edmonds & Francesconi, 1987;Cullen
& Reimer, 1989; Francesconi&
Edm onds, 1993).In addition, xenobiotic com pounds containing the C-Asbond still enter the env ironm ent in a variety of agricultu ralapplications - some 11000 tonnes annually in the UnitedStates (Abernathy, 1983). Most significant are the con-tinuing uses of arsanilic acid, 3-nitro-4-hydroxyphenyl-
arsonic acid and 4-nitrophenylarsonic acid as animal feedadditives, and of dimethylarsinic acid, and the mono- anddi-sodium salts of methylarsonic acid, as cotto n defoliants
t Present address:School of Biology, University of Ulster, Cromore Road,
Coleraine BT52 lSA, Northern Ireland.
and post-emergence grass herbicides, respectively(Andreae, 1986;Tamaki & Frankenberger, 1992).
Alth oug h extensive research has been undertaken on themechanisms, and the extent, of bioalkylation of arsenic
(reviewed by Thayer, 1993; Gadd, 1993) much less isknow n abo ut the environmental fate of organoarsenicals,and a lack of information on the mechanisms of C-Asbond cleavage constitutes the weakest link in our under-standing of the biogeochemical cycling of the element.Th e process is attribute d exclusively to microbial activity(Andreae, 1986); hus the co-metabolism of arsenobetaineby marine sedimentary bacteria has been demonstrated,although the compound did not i tself support bacterialgrowth (Hanaoka e t al., 1987, 1991, 1992). Similarly, theslow mineralization of methylarsonate to arsenate andCO, by a number of genera of soil bacteria has beendescribed, althou gh such demethylation occu rred only ina yeast-extract- or acetate-based medium (Von En dt e t al.,
1968; Shariatpahani e t al., 1983). To our knowledge,however, i n no instance has any micro-organism capableof the utilization of an organoarsenical as sole carbon andenergy source yet been reported. Nor is any informationavailable on th e enzymology of the C-As bond cleavagereaction ; for example, the C-P-bond-cleaving enzymesphosphonoacetaldehyde hydrolase and phosphonoacetatehydrolase show no activity towards their arsonate ana-logues (Lacoste e t al., 1992; McMullan & Qu inn, 1994).
We now describe the utilization of arsonoacetate(H,O,AsCH,COOH ), a model com poun d containing the
CH,AsC10,. C,H,,N requires C, 30.7 YO H, 6.3 YO N ,5.1 Y O . he electrophoret ic mobil ity at pH 2 was 0.37 thatof arsenate (0.15 that o f arsono(ch1oro)acetic a cid).
Arsonoacetic acid (HOOC-CH,-AsO,H,)
This was m ade by the Meyer (1883) react ion of t reat ing
chloroa cetic acid with alkaline arsenite (Pa lmer, 1925),
and the barium sal t was converted into the free acid asdescribed by Rozovskaya e t al. (1984).
Dibromomethylarsonicacid (Br,CH-AsO,H,)
Arsonoacetic acid (4.5 g, 24 mmol) was dissolved in amixture of 20 ml water and 20 ml acetic acid. Bromine(2-6 ml, 50 mm ol Br,) was added and dissolved in the
mixture . After a lag it largely deco lorized and the solu tioneffervesced. After the mixture had s tood o vernight , pa perelectrophoresis showed the presence of an arsenate-
positive sp ot of greater mo bility at pH 2 than the s tart ingcompound. The so lu t ion was added to a co lumn of
29 cm x 2.6 cm of the s t ron gly basic resin Dow ex 1x8 inthe acetate form, which was washed with 100ml water,and 500 ml port ions of 5 M formic acid and 10 M formic
acid. Th e last of these was evaporated to dryness and theproduct crystallized. The solid was dissolved in ethyl
acetate, and the solut ion was dried over sodium sulphate,filtered, and evaporated to dryness. The resul t ing sol id
was dissolved in ethyl acetate and crystallized on addingl ight petroleum. Yield: 5-2 g (6 3 %). M.p. 142-144 "C.
Elemental analysis gave C, 4-01YO; H . 1.00%.
CH,AsBr,O, requires C, 4-03% ; H, 1-02YO. i t rat ion ofa por t ion in water showed one pK to be below 3, and the
other about 6.7. A solut ion of the acid in aqueous
amm onia (densi ty 0.88 g ml-l) was unchange d, as judgedby pap er electrophoresis , after 3 d at 20 "C ; this unreact-
ivity is similar to that of chloromethylphosphonic acidreported by Schwarzenbach e t al . (1949, p. 1185).
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Received 25 July 1994; revised 7 November 1994; accepted 22 November