Patent Application: US-82011692-A

Abstract:
the invention relates to proteobacteria that show unusually high level resistance to a wide range of metal oxides and oxyanions and to methods using selected subgroups for efficient reduction of certain metal oxides and oxyanions to the free metal . high level resistance was shown to be affected by growth conditions , and was observed in facultative photoheterotrophs such as rhodobacter sphaeroides grown either chemoheterotrophically or photoheterotrophically .

Description:
the invention generally relates to proteobacteria having the ability under certain conditions to efficiently reduce metal oxides and oxyanions to the free metal , and to their ability to survive in the presence of a wide range of metal oxides and oxyanions . the several examples following illustrate free metal deposition in a species of r . sphaeroides and its growth in the presence of toxic metal oxides and oxyanions . selective growth conditions conducive to high level resistance of the microorganisms are also described . the following examples are intended to illustrate the practice of the present invention and are not intended to be limiting . numerous variations of growth conditions are envisioned which are expected to optimize for different metal oxides and oxyanions . it is also expected that one or more resistance factors , plasmid or chromosomal , identified with resistance will be isolated and sequenced , providing cassettes for transforming various host cells . this example illustrates the intrinsic resistance of several species of proteobacteria to tellurite . intrinsic high - level resistance to tellurite is found in only a few species of these purple non - sulfur bacteria during chemoheterotrophic , anaerobic / dark , and photoheterotrophic growth conditions . several wild - type strains were grown either aerobically , anaerobically in the dark , or photoheterotrophically in minimal media in the presence of teo 3 2 - . table 1 lists the bacterial strains tested for high level resistance to tellurite . table 1__________________________________________________________________________bacterial strains . organism / strain relevant genotype / phenotype . sup . a reference__________________________________________________________________________escherichia colijm83 ara , . sub .-- δ ( lac - proab ), rpsl , messing , thi , . sub .-- φ80dlacz . sub .-- δm15 1979s17 - 1 c600 :: rp - 4 , 2 - simon , et al ., tc :: mu :: km :: tn7 hsdr , hsdm . sup .+, 1983 recarhodobacter capsulatusb10 wild - type weaver , et al ., 1975rhodobacter sphaeroides2 . 4 . 1 wild - type , 5 endogenous plasmids2 . 4 . 1δs 2 . 4 . 1δ ( 42 - kb plasmid ) suwanto2 . 4 . 1 - ga car . sup .- cohen - bazire , 19562 . 4 . 7 wild - type , 2 endogenous plasmidsbc17 2 . 4 . 1 - ga , fbcbc , car . sup .-, km . sup . r yun , 1990cfxa . sup .- cfxa , km . sup . r hallenbeck , et al ., 1990acfxb . sup .- cfxb , sp . sup . r , sm . sup . r hallenbeck , et al ., 1990acfxa . sup .- b . sup .- cfxa , cfxb , km . sup . r , sp . sup . r , sm . sup . r hallenbeck , et al ., 1990acyca1 cyca , km . sup . r donohue , 1988mm1004 2 . 4 . 1 :: tnphoa , dorase . sup .-, km . sup . r moore and kaplan , 1989mm1006 2 . 4 . 1 :: tnphoa , bchl . sup .-, km . sup . r moore and kaplan , 1989prka . sup .- prka , km . sup . r hallenbeck , et al ., 1990bprkb . sup .- prkb , sp . sup . r , sm . sup . r hallenbeck , et al ., 1990bprka . sup .- b . sup .- prka , prkb , km . sup . r , sp . sup . r , sm . sup . r hallenbeck , et al ., 1990bpuc705 - ba pucba , km . sup . r lee , 1989pufb1 pufbalmx , km . sup . r davis , 1988puha1 puha , km . sup . r sockett , 1988rs2 wild - type meinhardt , 1985ws8 wild - type , 1 endogenous sistrom , 1977 plasmidrhodocyclus gelatinosusstr - 1 wild - type uffen , 1976rhodopseudomonas palustris1e5 wild - type firsow , 1977rhodopseudomonas viridisf wild - type drews , 1966rhodospirillum rubrumha wild - type__________________________________________________________________________ . sup . a km . sup . r , sp . sup . r , and sm . sup . r denote resistance to kanamycin , spectinomycin , and streptomycin , respectively . all proteobacteria were grown at 30 ° c . with the exception of e . coli which was cultured at 37 ° c . on a gyrotary shaker . cultures of r . sphaeroides and r . gelatinosus were grown in lb , yp , or smm containing either 0 . 4 % succinate , 0 . 4 % malate , or 0 . 4 % butyrate as a carbon source . cultures of r . capsulatus were grown in rcvb minimal medium containing 0 . 4 % malate as a carbon source ; r . rubrum was grown in smm containing 0 . 4 % malate , and 0 . 1 % yeast extract . r . palustris and r . viridis were grown in smm containing 0 . 4 % malate , 0 . 1 % yeast extract , and 50 μg / ml each of p - aminobenzoic acid and cyanocobalamin . when necessary , antibiotics were added to growth media at the following final concentrations : kanamycin ( km ), 25 μg / ml ; spectinomycin ( sp ), 50 μg / ml ; and streptomycin ( sm ), 50 μg / ml . anaerobic - dark growth of r . sphaeroides on smm medium containing dmso , and photoheterotrophic growth conditions have been previously reported . under aerobic conditions , both r . sphaeroides and r . capsulatus expressed intrinsic high level resistance to teo 3 2 - while virtually all other strains of bacteria tested showed much lower resistance under the same culture conditions . results are shown in table 2 . as indicated in table 2 , intrinsic high level resistance to tellurite occurred in only a few species of purple non - sulfur bacteria during aerobic and photoheterotrophic growth conditions . moreover , the level of tellurite resistance was strain dependent : the mic of k 2 teo 3 for r . sphaeroides rs2 was approximately two - to three fold lower than the mic for either strain 2 . 4 . 1 , 2 . 4 . 7 or ws8 . with the exception of r . gelatinosus , which exhibited no growth dependent difference in inhibitory teo 3 2 - concentration , mics were approximately 50 % higher when cells were grown table 2__________________________________________________________________________determination of intrinsic hlr to teo . sub . 3 . sup . 2 - by proteobacteria . phylogenetic . sup . a mic k . sub . 2 teo . sub . 3 ( μg / ml ). sup . borganism strain subgroup aerobic photosynthetic . sup . c__________________________________________________________________________rhodospirillum rubrum ha α - 1 20 10rhodopseudomonas palustris 1e5 α - 2 200 100rhodopseudomonas viridis f α - 2 80 50rhodobacter sphaeroides 2 . 4 . 1 α - 3 900 600 ws8 α - 3 800 600 2 . 4 . 7 α - 3 800 500 rs2 α - 3 400 250rhodobacter capsulatus b10 α - 3 800 500rhodocyclus gelatinosus str - 1 β - 1 5 10escherichia coli jm83 γ - 3 & lt ; 5 . sup . na . sup . d s17 - 1 γ - 3 & lt ; 5 na__________________________________________________________________________ . sup . a based on the classification of woese et al . . sup . b mics were determined in the appropriate minimal synthetic medium a 30 ° c . . sup . c incident light intensity , 10 w / m . sup . 2 . . sup . d na , not applicable . aerobically , regardless of the strain or species . the jm83 and s17 - 1 strains of e . coli failed to grow in minimal medium containing 5 μg / ml k . sub . 2 teo . sub . 3 . the ability of r . sphaeroides to grow in the presence of selenium , tellurium and rhodium oxyanions is demonstrated in the following example . growth of r . sphaeroides in the presence of te , se or rh - containing oxyanions cells of r . sphaeroides 2 . 4 . 1 were grown in liquid medium as in example 1 when medium contained teo 3 or teo 4 2 - , cells settled to the bottom of culture tubes over the course of the growth phase due to the intracellular accumulation of a dense metal deposit . copious gas evolution was observed concomitant with cell growth . centrifugation of broth cultures at 10000 × g resulted in a black cell pellet and a clear supernatant . colonies of r . sphaeroides which formed on agar medium containing teo 3 2 - produced a black deposit which did not diffuse into the medium . cells remained viable despite the accumulation of intracellular deposits : black colonies streaked onto agar medium containing no teo 3 2 - gave rise to normally pigmented colonies apparently through the dilution of metal complexes in the membranes of progeny cells . similar results were obtained for selenium and rhodium containing compounds : when culture media contained seo 3 2 - or seo 4 2 - , the cells became bright red in color ; in rhodium sesquioxide - containing media , the cells appeared grayish bronze . the relative toxicity of these five compounds to r . sphaeroides was seo 4 2 - & gt ; teo 4 2 - & gt ; teo 3 2 - & gt ; seo 3 2 - & gt ; rh 2 o 3 . 5h 2 o . the effect of culture conditions and medium composition on the high - level resistance of r . sphaeroides to heavy - metal oxides was examined . significant differences in resistance were found depending on the nature of the carbon source , incident light intensity and the presence of oxygen . effect of culture conditions and medium composition on high level resistance to teo 3 2 - r . sphaeroides 2 . 4 . 1 was grown either in complex or defined medium as indicated in table 3 . while cultures of r . sphaeroides 2 . 4 . 1 grown in smm expressed hlr to teo 3 2 - , cells grown in rich media such as lb , yp or proteose - peptone were sensitive to very low levels of the oxyanion , table 3 . this was true for cultures grown aerobically or anaerobically . likewise , a thirty - to forty - fold reduction in teo 3 2 - resistance was observed when smm was supplemented with either peptone , casamino acids , tryptone , or yeast extract . to determine if there was a single common component present in these supplements which was affecting hlr , smm containing 0 . 4 % succinate was supplemented individually with each of the twenty amino acids . this analysis indicated that a single amino acid , l - cysteine , was solely responsible for the increased sensitivity to teo 3 2 - . neither cystine , glutathione nor thioglycollate , however , decreased hlr to teo 3 2 - when added to smm , nor did the presence of alternate electron acceptors , such as trimethylamine - n - oxide or dmso , table 3 . the fact that the addition of l - methionine to smm had no affect on hlr to teo 3 2 - contrasted with previously studies with e . coli which demonstrated that exogenously supplied l - methionine enhanced teo 3 2 resistance some two - fold ( scala and williams , 1962 ; 1963 ). a similar inhibition of hlr by l - cysteine was also observed for teo 4 2 - , seo 3 2 - , and seo 4 2 - . likewise , hlr to none of these compounds was enhanced by the addition of exogenous methionine . table 3__________________________________________________________________________effects of medium composition and growth conditions on hlr toteo . sub . 3 . sup . 2 -. mic k . sub . 2 teo . sub . 3 ( μg / ml ) photosynthetic anaerobic - medium . sup . a supplement . sup . b aerobic 10 w / m . sup . 2 3 w / m . sup . 2 dark . sup . d__________________________________________________________________________complex : luria - bertani -- 20 80 40 & lt ; 10yeast extract / peptone -- 20 20 10 & lt ; 10proteose - peptone -- 10 20 10 10defined : smm + butyrate -- 1000 700 500 200smm + succinate -- 900 600 300 150smm + malate -- 800 400 150 100smm + succinate 30 mm tmao 850 650 400 150smm + succinate 1 mm l - methionine 800 500 200 100smm + succinate 1 mm cystine 500 550 200 100smm + succinate 1 mm glutathione 550 550 250 150smm + succinate 1 mm thioglycollate 500 500 250 100smm + succinate 0 . 3 % peptone 30 20 20 20smm + succinate 0 . 3 % yeast extract 30 40 10 10smm + succinate 0 . 3 % tryptone 20 30 10 20smm + succinate 0 . 3 % casamino acids 20 30 10 20smm + succinate 1 mm l - cysteine 20 40 20 10__________________________________________________________________________ . sup . a smm contained 0 . 4 % of the carbon source listed . . sup . b supplement was added to culture medium to the final concentration listed . . sup . c incident light intensity . . sup . d supplemented with 60 mm dmso . regardless of medium composition , the mic of k 2 teo 3 for r . sphaeroides 2 . 4 . 1 was always two - to three - fold higher in aerobically - vs . photosynthetically - grown cultures . this was consistent with results obtained earlier for cells grown in succinate - containing smm ( table 2 ). analyses also demonstrated that hlr to teo 3 2 - in photosynthetically - grown cultures was directly proportional to incident light intensity ; in all the growth media examined , mics were at least two - fold higher for cultures grown at 10 w / m 2 than for those grown at 3 w / m 2 , see table 3 . a final observation with respect to medium composition concerned the effect the oxidation state of the carbon source had on the level of teo 3 2 - resistance in r . sphaeroides 2 . 4 . 1 . while the mic of k 2 teo 3 for cells grown aerobically in smm containing malate as the carbon source was 800 μg / ml , when more reduced carbon sources such as succinate or butyrate were substituted the mics increased to 900 , and 1000 μg / ml , respectively . similar results were also observed when cells were grown anaerobically in the light ( photosynthetically ) or anaerobically in the dark ( in smm containing dmso ), see table 3 . these data suggested that the toxicity of teo 3 2 - was inversely related to the oxidation state of the carbon source : the more reduced the carbon source , the higher the mic of teo 3 2 - . this example illustrates the remarkable resistance of r . sphaeroides to a wide variety of oxides and oxyanions . the example is illustrated with strain 2 . 4 . 1 but similar resistance has been obtained with related strains such as rhodobacter sphaeroides 2 . 4 . 1as , 2 . 4 . 7 , ws8 , rs2 , 2 . 4 . 1 - ga ; rhodobacter capsulatus b10 ; rhodopseudomonas palustris le5 ; rhodopseudomonas viridis f . mutant r . sphaeroides having intrinsic high level resistance to oxides and oxyanions have been deposited with the american type culture collection , 12301 parklawn drive , rockville , md . 20852 under the budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure . all restrictions on the availability to the public of the material so deposited are irrevocably removed upon the granting of a patent . atcc designations for the deposits are : atcc 49848 ( r . sphaeroides 2 . 4 . 1δs ), deposited on jan . 24 , 1992 , atcc 55398 ( r . sphaeroides 2 . 4 . 1 ), deposited on mar . 9 , 1993 ,; atcc 17028 ( r . sphaeroides 2 . 4 . 7 ), deposited on feb . 12 , 1965 ,; and atcc 33303 ( rhodobacter capsulatus b10 ), deposited on feb . 26 , 1987 . a preferred strain used in some of the examples of the invention is rhodobacter sphaeroides 2 . 4 . 1 . this strain differs from the american type culture collection strains ( atcc 17023 , atcc 11167 , atcc 14690 , ncib 8253 and ncib827 ) which are also named as 2 . 4 . 1 . the 2 . 4 . 1 strain used herein was originally provided by dr . w . r . sistrom over 20 years ago . it is believed that he received this strain from the laboratory of dr . r . y . stanier , who in turn received it from dr . c . b . vanniel . it is unclear how the discrepancies in nomenclature between the 2 . 4 . 1 strain obtained from dr . sistrom and the atcc strains arose . the rhodobacter sphaeroides 2 . 4 . 1 used herein may be obtained from dr . samuel kaplan , the university of health science center at houston , department of microbiology and molecular genetics , p . o . box 20708 , houston , tex ., usa 77225 . an equally preferred strain is rhodobacter sphaeroides 2 . 4 . 1δs , which has ben deposited with the american type culture collection , rockville , md . 20852 . strains of rhodobacter are readily isolated from soil or pond water samples . strain ws8 , like other strains of rhodobacter , may be readily identified as an r . sphaeroides related strain on the basis of the following properties . the cells are very short , small , gram - negative , motile rods , frequently occurring in pairs but seldom in longer chains . gelatin is not liquified ; growth is vigorous on lactate , realate or glucose , less vigorous on mannitol , tartrate , or propionate , and absent on glycerol or citrate ; anaerobic ( phototropic ) cultures are brownish yellow ; semi - aerobic cultures are reddish - orange . except for the lack of growth on glycerol , these are characteristics of r . sphaeroides . additionally , other strains of photosynthetic proteobacteria exist which are likely to effect for metaloxide and oxyanion reduction , for example , other strains of r . sphaeroides commonly referred to as &# 34 ; 2 . 4 . 1 &# 34 ; ( e . g ., atcc 11167 , atcc 14690 , atcc 17023 , ncib 8253 , and ncib 8287 ) that , while genetically distinct from 2 . 4 . 1 should be expected to carry out oxide and oxyanion reduction in a manner similar to 2 . 4 . 1 . a total of twenty oxides and oxyanions were assayed for toxicity to r . sphaeroides 2 . 4 . 1 . results are shown in table 4 . twenty oxides and oxyanions , listed in table 4 , were assayed for toxicity to r . sphaeroides 2 . 4 . 1 . of those examined , only cro 3 , cro 4 2 - , and mno 4 - had mics & lt ; 20 μg / ml ; the others had mics in smm & gt ; 100 μg / ml under all growth conditions examined . oxides having limited solubilities in smm ( e . g ., moo 3 , table 4__________________________________________________________________________quantitation of oxide andoxyanion resistance in r . sphaeroides 2 . 4 . 1 . intracellular gas mic ( μg / ml ). sup . acompound deposition evolution standard medium low - po . sub . 4 . sup . 3 - medium . sup . c__________________________________________________________________________moo . sub . 3 -- -- . sup . sat &# 39 ; d sol &# 39 ; n . sup . b sat &# 39 ; d sol &# 39 ; nnh . sub . 4 vo . sub . 3 -- -- sat &# 39 ; d sol &# 39 ; n sat &# 39 ; d sol &# 39 ; nsb . sub . 2 o . sub . 3 -- -- sat &# 39 ; d sol &# 39 ; n sat , d sol &# 39 ; nsno . sub . 2 -- -- sat &# 39 ; d sol &# 39 ; n sat &# 39 ; d sol &# 39 ; nrh . sub . 2 o . sub . 3 . 5h . sub . 2 o +++ +++ sat &# 39 ; d sol &# 39 ; n sat &# 39 ; d sol &# 39 ; nna . sub . 2 seo . sub . 4 ++ + 150 100na . sub . 2 seo . sub . 3 ++++ +++ 800 500k . sub . 2 teo . sub . 4 +++ ++ 500 500k . sub . 2 teo . sub . 3 ++++ +++ + 600 600nasio . sub . 4 -- -- 400 150na . sub . 2 sio . sub . 3 -- -- 400 100na . sub . 2 sio . sub . 4 -- -- 300 100na . sub . 2 haso . sub . 4 -- -- 1500 1600na . sub . 2 moo . sub . 4 -- -- 1400 1500na . sub . 2 wo . sub . 4 -- -- 1600 1600na . sub . 2 sno . sub . 4 -- -- 800 800na . sub . 2 so . sub . 3 -- -- 600 500na . sub . 2 cro . sub . 4 -- -- 10 20kmno . sub . 4 -- -- 20 & lt ; 10cro . sub . 3 -- -- 20 & lt ; 10__________________________________________________________________________ . sup . a photoheterotrophic growth in smm containing succinate ( 10 w / m . sup . incident light intensity . . sup . b compounds with solubilities & lt ; 10 μg / ml did not inhibit growth in saturated solution . . sup . c medium contains 2 mm po . sub . 4 . sup . 3 -, 10fold lower than that of th standard formulation . nh . sub . 4 vo . sub . 3 , rh . sub . 2 o . sub . 3 . 5h . sub . 2 o , sb . sub . 2 o . sub . 3 , and sno . sub . 2 ) did not affect cell growth when present in growth media as saturated solutions . only cultures grown in the presence of te -, se -, or rh - containing oxyanions evolved gas and accumulated intracellular deposits , table 4 . hlr to these five compounds was unaffected by extracellular po . sub . 4 . sup . 3 -, which suggested hlr to these compounds in r . sphaeroides 2 . 4 . 1 was not mediated by components of the phosphate - transport system . this would preclude any similarity between the mechanism of intrinsic hlr in r . sphaeroides and that encoded by the incpα plasmid determinants , tela and telb ( walter et al ., 1991 ). r . sphaeroides was also highly resistant to a second class of oxyanions , the &# 34 ; periodate class &# 34 ;, but the resistance mechanism to this class differed significantly from that of the &# 34 ; tellurite class .&# 34 ; neither io 4 - , sio 3 2 - , nor sio 4 2 - was reduced to its elemental state , and no gas evolution was observed . in sharp contrast to the &# 34 ; tellurite class ,&# 34 ; resistance to these oxyanions decreased three - to four - fold when the extracellular phosphate was reduced ten - fold . this suggested that resistance in r . sphaeroides 2 . 4 . 1 occurred as a result of reduced transport or increased efflux via a phosphate - transport system - mediated mechanism . it is interesting to note , however , that intrinsic resistance to these compounds in r . sphaeroides was still some twenty - fold greater than that of the γ - 3 proteobacteria ( summers and silver , 1978 ). a third class of oxyanions to which r . sphaeroides was highly resistant , the &# 34 ; arsenate class ,&# 34 ; was also examined . this group included arsenate , molybdate , stannate , sulfite , and tungstate . similar to the &# 34 ; tellurite class &# 34 ; oxyanions , resistance to these compounds was unaffected by extracellular phosphate levels . in contrast , however , hlr to &# 34 ; arsenate class &# 34 ; compounds did not result in oxyanion reduction or intracellular metal sequestration . like the &# 34 ; periodate class &# 34 ; oxyanions , these compounds were not reduced to their elemental states , and no gas was evolved . these data supported the existence of a third and distinctly different mechanism to effect hlr to &# 34 ; arsenate - class &# 34 ; oxyanions . the ability of r . sphaeroides to concentrate tellurium metal in the cytoplasmic membrane is shown in this example . the dense metal deposit was shown to be localized to the cytoplasmic membrane after a sucrose gradient isolation , leaving the intracytoplasmic ( or photosynthetic ) membrane unaffected . two one - liter cultures of r . sphaeroides 2 . 4 . 1 were grown photoheterotrophically ( 10 w / m 2 incident light intensity ) in smm containing 0 . 4 % succinate to a cell density of approximately 1 . 5 1 . 5 × 10 9 cells / ml . prior to inoculation one flask was supplemented with 275 mg k 2 teo 3 ( equivalent to 138 . 3 mg te iv ) to give a final medium concentration of 250 μg / ml k 2 teo 3 . following subcellular fractionation , the dense black deposit which accumulated within cells grown in teo 3 2 - - containing medium was localized to the cytoplasmic membrane via centrifugation through a discontinuous sucrose gradient . no metallic material was observed in the enriched chromatophore fraction ( consisting of photosynthetic or intracytoplasmic membrane ) at the 20 : 40 % interface . 850 mg of crude membrane - metal complex was isolated . after purification and extraction with acetone : methanol and ethanol , 203 mg of a finely - divided metallic material resulted . analysis of a 50 . 4 mg sample of this material identified 23 . 2 mg of te o ( a minimum net purity of 46 %). the minimum te o deposited in the one - liter culture , therefore , was 93 mg ( 203 mg × 0 . 46 ) or 0 . 7333 mmole . since the growth medium initially contained 138 . 3 mg of te iv , a minimum te iv to te o conversion of 67 % was obtained . assay of the membrane fraction of cells grown in the absence of teo 3 2 - revealed no te o , nor was any detected in the cytoplasmic or periplasmic fractions of either culture by this method . these results demonstrated conclusively that r . sphaeroides 2 . 4 . 1 could effect the intracellular reduction of te iv , which resulted in the deposition of metallic te o in the cytoplasmic , but not intracytoplasmic , membrane . although no gas was evolved from cells grown in the absence of teo 3 2 - , 208 ml of water was displaced from the gas collection vessel over the culture grown in the presence of teo 3 2 - . this corresponded to 8 . 37 mmole of gas ( 1 atm , 303 ° k . ), the major component of which was subsequently identified as h 2 by mass spectroscopy , fig1 b . while ionization products of h 2 o , n 2 , and co 2 were detected in both samples , no h 2 was detected in the headspace over the control culture , fig1 a . the trace amounts of argon recorded in each spectra resulted from its use as a carrier in the analyses . combined with earlier results , these data suggested approximately 11 . 5 mmoles of h 2 were evolved per mmole of te o deposited . the requirements for tellurite reduction in r . sphaeroides in vivo were determined by assaying tellurite resistance in a number of mutant strains . mechanism of teo 3 2 - high level resistance in r sphaeroides 2 . 4 . 1 several growth conditions were examined to determine requirements for tellurite reduction in vivo . table 5 indicates the growth conditions tested . as indicated in table 5 , neither the dmso reductase , the b800 - 850 spectral complex , nor the b875 spectral complex was required to effect hlr to teo 3 2 - under any growth condition examined . deletion of the 42 - kb endogenous plasmid of r . sphaeroides 2 . 4 . 1 did not diminish hlr to teo 3 2 - , although we did observe a 20 % increase in teo 3 2 - sensitivity in the carotenoid - deficient strain , 2 . 4 . 1 - ga . a single mutation in either prkb or cfxb diminished hlr to teo 3 2 - 10 - 20 %, whereas strains deleted for either of their homologues , prka or cfxa , were two - fold more sensitive to teo 3 2 - under aerobic growth conditions , and at least three - to five - fold more sensitive under photosynthetic and anaerobic - dark / dmso growth conditions . analyses of additional r . sphaeroides mutants determined the obligate requirement for an intact photosynthetic reaction center ( rc ) and a functional electron transport system for hlr to teo 3 2 - when metabolic activities are carried out photosynthetically . these analyses also demonstrated that certain mutants , while unable to facilitate tellurite reduction , were resistant to intermediate concentrations of tellurite : viz a bchl - mutant ( mm1006 ), a puf - mutant ( pufb1 ), and a strain deleted for cytochrome c 2 ( cyca1 ) were inhibited by 10 μg / ml k 2 teo 3 under anaerobic - dark / dmso growth conditions , but were unaffected by the addition of tellurite under aerobic growth . likewise , the photosynthetically - incompetent double -- deletion strains , cfxa - b - and prka - b - , while unable to effect teo 3 2 reduction either table 5__________________________________________________________________________analysis of intrinsic hlr to tellurite in r . sphaeroides mutants . relevant photo - mic k . sub . 2 teo . sub . 3 ( μg / ml ). sup . a genotype / synthetic photosynthetic . sup . b anaerobic . sup .- strain phenotype competence aerobic 10 w / m . sup . 2 dark . sup . c__________________________________________________________________________2 . 4 . 1 wild - type + 900 600 1502 . 4 . 1δs δ ( 42 - kb + 850 600 150 plasmid ) 2 4 . 1 - car . sup .- + 600 450 100gamm1004 dorase . sup .- + 800 550 ngpuc705b b800 - 850 . sup .- + 850 500 150prkb . sup .- prkb . sup .- + 800 500 150cfxb . sup .- cfxb . sup .- + 750 650 150cfxa . sup .- cfxa . sup .- + 400 100 50prka . sup .- prka . sup .- + 350 100 60mm1006 bchl . sup .- - . sup . ( 400 ). sup . d . sup . ng . sup . e & lt ; 10cyca1 cyt c . sub . 2 . sup .- . sup .- ( 400 ) ng & lt ; 10pufb1 puf . sup .- - ( 400 ) ng & lt ; 10cfxa . sup .- b . sup .- cfxa . sup .-, cfxb . sup .- - ( 200 ) ng & lt ; 10prka . sup .- b . sup .- prka . sup .-, prkb . sup .- - ( 150 ) ng & lt ; 10puha1 rc - h . sup .-, b875 . sup .- - & lt ; 10 ng & lt ; 10bc17 car . sup .-, cyt bc . sub . 1 . sup .- - & lt ; 10 ng & lt ; 10__________________________________________________________________________ . sup . a mic were determined in smm containing succinate at 30 ° c . . sup . b incident light intensity . . sup . c supplemented with 60 mm dmso . . sup . d () indicates resistance to teo . sub . 3 . sup . 2 -, but no deposition of te . sup . 0 . . sup . e ng , no growth . aerobically or anaerobically in the dark ( in the presence of dmso ), were resistant to tellurite at concentrations & lt ; 200 μg / ml under aerobic conditions . in contrast , strains lacking either the rc - h polypeptide ( puha1 ) or the cytochrome bc . sub . 1 complex ( bc17 ) were sensitive to 10 μg / ml k . sub . 2 teo . sub . 3 under all growth conditions . the experiments in this example were aimed at determining the intracellular localization of tellurite reductase activity . subcellular fractions of aerobically grown cells were prepared from wild - type and three mutant strains unable to reduce tellurite . these cells were grown in the absence of teo 3 2 , and were harvested during the mid - exponential phase of growth . this analysis , results of which are shown in table 6 , identified an fadh 2 - dependent teo 3 2 - reductase activity present in the membrane fraction of wild - type r . sphaeroides 2 . 4 . 1 . cells cultured in the presence of teo 3 2 - also expressed similar teo 3 2 - - dependent fadh 2 oxidation activity in vitro . a specific activity of 300 nmole fadh 2 / min per mg protein was detected in the membrane fraction of wild - type cells . an fadh 2 - dependent teo 3 2 - reductase activity was also observed in the photosynthetically - incompetent strain prka - b - , despite this strain &# 39 ; s inability to reduce teo 3 2 - in vivo , see table 5 . this suggested that in addition to an fadh 2 - dependent reductase , at least one other component was required to facilitate complete reduction to te o in vivo . neither bc17 nor puha1 , two mutants which were previously shown to be tellurite sensitive under both aerobic and anaerobic - dark / dmso growth conditions , expressed significant levels of a teo 3 2 - - dependent fadh 2 oxidase activity in vitro . this may explain the inability of either to effect oxyanion reduction and metal sequestration in vivo . negligible reductase activity was observed in the periplasmic and cytoplasmic fractions of all strains , and in separate analyses , a teo 3 2 - - dependent oxidation of nadh or nadph was not detected in subcellular fractions from any of these strains . this would not preclude , however , the participation of a nadh - or nadph - dependent oxidation step in the reduction of an intermediate in the reduction of te iv to te o . this example illustrates the construction of a mutant r . sphaeroides from wild type strain 2 . 4 . 1 . a mutant r . sphaeroides was prepared from wild type strain 2 . 4 . 1 . r . sphaeroides 2 . 4 . 1δs is a derivative of r . sphaeroides 2 . 4 . 1 which has been &# 34 ; cured &# 34 ; of one of its five endogenous plasmids , the 42 - kb plasmid designated e ( fornari et al ., 1984 ) or &# 34 ; s &# 34 ; factor ( suwanto and kaplan , 1989 a ; suwanto and kaplan , 1989 b ; suwanto and kaplan , 1991 ). the plasmid was readily cured by the introduction of either of the incompatibility determinants , inca or incb derived from native &# 34 ; s &# 34 ; factor on a selectable antibiotic resistance containing , unstable plasmid derivative . once &# 34 ; s &# 34 ; was cured the introduced plasmid was readily lost following removal of the antibiotic selection . two important features of 2 . 4 . 1δs are that table 6______________________________________teo . sub . 3 . sup . 2 - - dependent fadh . sub . 2 oxidation in r . sphaeroides2 . 4 . 1 . fadh . sub . 2 oxidation . sup . bstrain . sup . a subcellular fraction ( nmole min . sup .- 1 mg . sup .- 1 ) ______________________________________2 . 4 . 1 periplasm 1 membrane 300 cytoplasm 60bc17 periplasm 2 membrane 51 cytoplasm 20prka . sup .- b . sup .- periplasm 3 membrane 200 cytoplasm 20puha1 periplasm 3 membrane 28 cytoplasm 37______________________________________ . sup . a cells were grown aerobically in smm containing 0 . 4 % succinate . . sup . b 100 μg / ml k . sub . 2 teo . sub . 3 was used in all assays . the phenotype associated with oxyanion or metal oxide metabolism is not associated with the &# 34 ; 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