Patent Application: US-25674894-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 proteobacteria 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 . the disclosed methods are adaptable to the production of hydrogen from cultures of peoteobacteria grown in the presence of tellurite class oxyanions with carbon dioxide and nitrogen as carbon and nitrogen sources . the methods have commercial application for efficient bioremediation of carbon dioxide and nitrogen .

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 . rhodobacter sphaeroides 2 . 4 . 1δs , atcc accession number 49848 has been deposited with the american type culture collection ( atcc ) 12301 parklawn rd ., rockville , md . 20852 under the budapest convention . 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______________________________________escherichiacolijm83 ara , δ ( lac - proab ), rpsl , thi , messing , 1979 φ80dlacz δm15s17 - 1 c600 :: rp - 4 , 2 - tc :: mu :: km :: tn7 simon , et al ., 1983 hsdr , hsdm . sup .+, recarhodobactercapsulatusb10 wild - type weaver , et al ., 1975rhodobactersphaeroides2 . 4 . 1 wild - type , 5 endogenous plasmids van neil , 19442 . 4 . 1 . increment . s 2 . 4 . 1δ ( 42 - kb plasmid ) suwanto2 . 4 . 1 - ga car . sup .- cohen - bazire , 19562 . 4 . 7 wild - type , 2 endogenous plasmids van neil , 1944bc17 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 , 19882 . 4 . 9 wild - type van neil , 1944mm1004 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 , 1988rdxa1 rdxa , sp . sup . r , sm . sup . r neidle and kaplan , 1992rs630 wild - type sistrom , 1977si4 wild - type drews , 1966swl wild - type fornari and kaplan , 198228 / 5 wild - typers2 wild - type meinhardt , 1985ws8 wild - type , 1 endogenous plasmid sistrom , 1977rhodocyclusgelatinosusstr - 1 wild - type uffen , 1976rhodo - pseudomonaspalustris1e5 wild - type firsow , 1977rhodo - pseudomonasviridisf wild - type drews , 1966rhodo - spirillumrubrumha 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 , 2 . 4 . 9 , si4 , swl 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 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 2 teo 3 . table 2______________________________________determination of intrinsic hlr to teo . sub . 3 . sup . 2 - by proteobacteria . mic k . sub . 2 teo . sub . 3 ( μg / ml ). sup . b pho - to - syn - phylogenetic . sup . a the - organism strain subgroup aerobic tic . sup . c______________________________________rhodospirillum rubrum ha α - 1 20 10rhodospirillum fuluum wild - α - 1 ng 20 typerhodopseudomonas palustris 1e5 α - 2 200 100agrobacterium tumefaciens a136 α - 2 75 narhodopseudomonas viridis f α - 2 80 50rhobacter sphaeroides 2 . 4 . 1 α - 3 900 600 ws8 α - 3 800 600 2 . 4 . 7 α - 3 800 500 rs2 α - 3 400 250 rs630 α - 3 700 600 swl α - 3 600 600 si4 α - 3 300 400 2 . 4 . 9 α - 3 250 400rhodobacter capsulatus b10 α - 3 800 500paracoccus denitrificans wild - α - 3 200 na typerhodocyclus gelatinosus str - 1 β - 1 5 10acinetobacter calcoaceticus adp - 1 γ - 3 25 naescherichia coli jm83 γ - 3 . sup . na . sup . d s17 - 1 γ - 3 . sup . na5______________________________________ . 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 . the ability of r . sphaeroides to grow in the presence of selenium , tellurium , europium and rhodium oxyanions is demonstrated in the following example . growth of r . sphaeroides in the presence of te , se , eu 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 , europium 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 . in europium oxide - containing media , the cells were grayish - white in appearance . 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 & gt ; eu 2 o 3 . 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 ; 10protease - peptone -- 10 20 10 10defined : smm + butyrate -- 1000 700 500 200smm + succinate -- 900 600 300 150smm + malate -- 800 400 150 100smm + tartrate -- 300 1009 50 50smm + glycerol -- 150 75 25 15smm + acetate -- 50 40 20 25smm + ethanol -- 25 25 & lt ; 5 & lt ; 5smm + 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 rare earth 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 . 1δs , 2 . 4 . 7 , 2 . 4 . 9 , si4 , swl , ws8 , rs2 , rs630 , 2 . 4 . 1 - ga ; rhodobacter capsulatus b10 ; rhodopseudomonas palustris le5 ; rhodopseudomonas viridis f . 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 , nc1b 8253 and nc1b827 ) 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 . van niel . 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 was deposited with the american type culture collection on jan . 24 , 1992 under the terms of the budapest treaty and accorded the accession number 55398 . it 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 , on mar . 9 , 1993 , and accorded the accession number 49848 . r . sphaeroides rs630 and swl were also deposited with the american type culture collection on mar . 9 , 1993 under the terms of the budapest treaty and accorded the accession numbers atcc55399 and actt55400 , respectively . 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 , nc1b 8253 , and nc1b 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 - seven oxides and oxyanions were assayed for toxicity to r . sphaeroides 2 . 4 . 1 . results are shown in table 4 . twenty - seven oxides and oxyanions , listed in table 4 , were assayed for toxicity to r . sphaeroides 2 . 4 . 1 . of those examined , only cro 3 , kreo 4 , naruo 4 , koso 4 , 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 , pbo , pb 2 o 3 , pbo 2 , eu 2 o 3 , nh 4 vo 3 , rh 2 o 3  5h 2 o , sb 2 o 3 , and sno 2 ) did not affect cell growth when present in growth media as saturated solutions . only cultures grown in the presence of te -, se -, eu or rh - containing oxyanions evolved gas and accumulated intracellular deposits , table 4 . hlr to these six compounds was unaffected by extracellular po 4 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 ). table 4______________________________________quantitation of oxide andoxyanion resistance in r . sphaeroides 2 . 4 . 1 . mic ( μg / ml ). sup . a intracellular gas standard low - po . sub . 4 . sup . 3 - compound deposition evolution medium medium . sup . c______________________________________moo . sub . 3 - - 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 &# 39 ; 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 of the standard formulation . 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 0 ( a minimum net purity of 46 %). the minimum te 0 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 0 conversion of 67 % was obtained . assay of the membrane fraction of cells grown in the absence of teo 3 2 - revealed no te 0 , 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 0 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 0 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 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 1 complex ( bc17 ) were sensitive to 10 μg / ml k 2 teo 3 under all growth conditions . table 5__________________________________________________________________________analysis or intrinsic hlr to tellurile in r . sphaeroides mutants . relevant photo - mic k . sub . 2 teo . sub . 3 ( μg / ml ). sup . a genotype / synthetic photosynthetic . sup . b anaerobic - 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 plasmid ) + 850 600 1502 . 4 . 1 - ga car . sup .- + 600 450 100mm1004 dorase . sup .- + 800 550 ngpuc705ba 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 .- - ( 400 ). sup . d ng . sup . e & lt ; 10cyca1 cyt c . sub . 2 . sup .- - ( 400 ) ng & lt ; 10pufb1 puf . sup .- - ( 400 ) ng & lt ; 10rdxa1 rdxa . sup .- + ( 400 ) 150 40cfxa . 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 mics 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 . 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 0 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 0 . 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 . 1 δs 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 the phenotype associated with oxyanion or metal oxide metabolism is not associated with the &# 34 ; s &# 34 ; factor and this strain may be used in conjugal genetic studies involving orit mediated chromosome transfer . 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 ability of r . sphaeroides to evolve molecular hydrogen and deposit metallic selenium in the cytoplasmic membrane under photoheterotrophic growth conditions is shown in this example . evolution of hydrogen gas from cultures of r . sphaeroides 2 . 4 . 1 grown photoheterotrophically with butyrate as a carbon source . a ten - liter culture of r . sphaeroides 2 . 4 . 1 is grown photoheterotrophically at ambient temperatures of between 15 ° and 35 ° c . 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