CA2051071A1 - Ammonia production - Google Patents
Ammonia productionInfo
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- CA2051071A1 CA2051071A1 CA 2051071 CA2051071A CA2051071A1 CA 2051071 A1 CA2051071 A1 CA 2051071A1 CA 2051071 CA2051071 CA 2051071 CA 2051071 A CA2051071 A CA 2051071A CA 2051071 A1 CA2051071 A1 CA 2051071A1
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- gene
- nifl
- mutant
- bacteria
- bacterium
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Abstract
ABSTRACT
AMMONIA PRODUCTION
A mutant nitrogen fixing bacterium wherein nitrogenase activity is controlled by a nifA or niA-like gene which is in turn regulated solely by a nifL or nifL-like gene characterised by the modification that it has a mutation in the nifL or nifL-like gene, but a functional nifA or nifA-like gene.
AMMONIA PRODUCTION
A mutant nitrogen fixing bacterium wherein nitrogenase activity is controlled by a nifA or niA-like gene which is in turn regulated solely by a nifL or nifL-like gene characterised by the modification that it has a mutation in the nifL or nifL-like gene, but a functional nifA or nifA-like gene.
Description
~ITT8H TECHNOLOGY GROUP TEL: 44-71-403-7586 9.Sep.91 12:59 No.004 P 02 2~5:~07~
, E1eld of the lnv~ntlon Thls lnventlon ls ln the ~lell~ of ammonla productlon by nitrogen flxlng bact0rla.
~escrl~lon of th~ prlor ~rt 05 Many mltroorgan1sms are able to asslml~ate the ma~or bloelements (l.a. carbon, n1trogen, sulphur, hydrogen and oxy~en) ln an lnorganlc form. The ablllty to use N2 ~s a nltrogen source 1~ restr~cted to prokaryotes, and Is relatlvely rare ev~n among thls group. The enzyme system responslble for N2 Flxatlon ls o called nltr~genase. Blosynthesls o~ the ~omponents ot the nltrogenase system ls de-termlned by 15 to ZO dlfferent nlf genes.
Free-llvlng nltrogen ~lxlng bacter1a flx an amount of atmospherlc nltrogen sufflclent for thelr own needs. Evldence for th~s ls ~hat slgnlflcant amounts of ammonla are rarely found ln the culture medlu~ ~ln lAboratory cultures) or envlronment (1n the soll) of nltrogen flxlng organlsms. Re~ulatorY mechanlsms controlllng nlf gene transcrlptlon andlor nltrogena~e ~ctlYlty ensure that eellular energy ls not wasted by the ~lxatlon of m~re nltrogen than 15 necessary to meet the demands o~ bact~rlal cell growth and vlablllty. ~n partlcular, ~xcess e~vlr~nmental ammonla or oxygen prevents expr~sslon of nlf genes ln free 11vlng d1azotrophs ~nltrogen ~lxln3 bacterla). Ammonla makes nltrogenase unnecessary and o~yge~ lnactl~tss the ~nzyme. Slnce these bacterla produce ~mmonla for asslmllatlon ~y plan-ts ln thQ
form o~ ammonlum catlons ~NH4-~, the term am~onlum ls used herelnafter for breYltY.
Attempts to lnduce ammonlum excretlon have up to now center~d on physlo10glcal suppresslon of g~netlc manlpulatlon o~ the enzymes lnvolved ln ammonlum asslml1atlon. Tr~atment of cyanobacterla, wlth L-methlon1ne-DL-sulfoxlmlne ~SX), an lnhlbitor of glutamlne synthet~se ~GS), resulted ln the excretlon of 0.3 to 7 mM NH4~ lnto the growth medlum, ~Musgrove ~ al., 1982, Blotech. Letters, 4, 647-652, Ne~-ton Q~ al., 1985, Blochlm, B~ophys. Acta, ~Q~, 44-50 ~nd R~os ~ ~1., 1984, Appl.
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~RIT~SH~TECHNOLOGY GRnUP TEL: 44-71-403-7586 9.Sep,91 12:59 No.fJ04 P.03 Envlron. Mlcroblol. g~ 114-118). Mutants o~ Anabaena resls~ant to MSX or the NH4~ ~nalogue ethylene dlamlne excreted up to 1.6 mM NH4+ (Po1uklna et al. 198Z Mlcrobl~logy 51 90-95 Splller et al. 19~6 J. Bacterlol. 1~5 412-419 and Thomas et ~1.. 7990 05 Appl. ~nvlron Mlcroblol. ~ 34~-3S04). Amon~ eubacter1a ammon1um excret~on was repor~ed to occur ln Gln- or Gln~ ~s~L-l mutants of Klebslella Dne~monlae ~Anderson et ~1.. 1977 J.Gen.
M1croblol. lQ~. 107-122 and Shanmugam et al.~ 1975 Proc. Natl.
Acad. Sc1. USA. 7~ 136-139) ln ~utants of ~hq~oba~Q~
c~sulatus and Azosplrlllum brasllensQ altered ln the productlon of GS (~all et ~1- 1979 J. Bacterlol 1~1. 1459-1463~. and ln methylamlne reslstan~ mutants o~ Azotobacter lnelandl~ (Gordon et ~1.. 1983 Can. J. Mlcroblol ~ 973-978~. Although slgnlflcant ammontum excretlon has been observed these chemlcally treated or mutated organlsms requlre supple~entatlon of larg~ amounts of the amlno acld g~utamlne for growth.
The regulatory pathwayS and m~chanlsms lnvolve~ ln the represslon of nl~ genes by ammonlum have been extenslvely characterlzed ln ~he free llvlng dlazotrophs Klebsl&1~ pneumont~e and Azotobact~L
vlne!~ndll. Of central lmportance ln both vrganlsms ~and other gram nega~lve nltrogen flxlng bacterla) 1~ that a posltlvQly-act1n~ re~ulatory proteln ~IFA 15 requlred to act1vate transcr1pt1On of th3 other M~f gen~s whlch are necessary ~or nltr~qenase structure and act1vlty. In K. ~ç~mQnl~ ammon1um z5 represses nltrogenase synthesls by preventlng elther th~ actlvlty or synthesls o~ NIFA whlch occ~rs by t~o separate mechan~sms The nlf~ gene ls adJacent to and downstr~am of nlfL ~thus formln~
the nlfLA operon) and these two genas are co-expressed. The NIF~
proteln blnds to and lnactlvates NIFA lf ammonlum ls present even at relatlvely low levels ~> approx. S~M). ht hl~her 1eve1s of ammonlum ~ approx. 200~M) expresslon oF the D~fL~ ~peron does not occur and so the NIFA proteln ls not syntheslz~d.
~ranscr~ptlon of nlf~ requlres phosphorylated NTRC proteln but thls proteln ls dephosphorylated and hence 1nactlve ln cells ~rown wlth ammonlum. Thus ln ~. ~L~Iu~mlae ~epresslon of ~RITISH TECHl~lOLnbY GRO~IP TEL: 44-71-403-7586 9.Sep.91 12:59 No.004 P.04 2~5:~0~ ~
n1trogenase synthesls by ammonlu~n occurs at two levels, lnactlvatlon of NIFA by NIFL and preventlon of nl~ expresslon by dephosphorylated NTRC.
Nltrogen flxatlon ln ~ nel2ndll ls d~ermlned by the 05 presence of three b10chemlcally and genetlcally dlstlnct nltro~enase enzymes, ~ach of whlch ls syntheslzed under dl~f~rent condltlons of metal supply. The ~olybdenum nltro~enase gene whlth ls slmllar to the enzyme purlfled from a number of other nltrogen flx~n~ organlsms, requlres 15 - 20 ~ene produces for lts structure and actlvlty. In ~. vlnQLandll as ln ~. ~n~umQnl~c Gxpresslon of the ~ enes requlres actlve NIFA produ~t. Unllke ln ~. ~nQ onlae ho~ever, HTRC ls not requlr~d for nltrogen flxatlon whlch lmpl1es that NTRC ls not necessary for ~lf~
expresslon.
~NA sequenclng of the ~lf~ reglon of ~ ~I eL~ndli revealed a gene wh~se translatlon product was slmllar to NIFA of K.
pneumonla~. The DNA sequence of 200bp upstream o~ DlfA revealed a partlal open rea~lng fr~me (ORF) whlch predlct~d ~ protcln wlth homology to the C-termlnal reg1~ns of the plfL and n~_~ gene products o~ K. pneumonlae. ~Bennett ~ al., 1986, Mol.
Mlc~oblol., ~, 315-321~.
It ~ould be d~slrable to be abla to lnduce ammonlum productlon ~n these nltrogen flxln~ bac-terla ~or a ~arlety o~
purposes. Attempts to de-regulate nltro0enase synthesls ln ~ Lslella pneumonlae by mutatlng elther th~ ~lfL or ntrC genes wQrQ unsuc~essful, ~he resultlng n1~ mutant was only weakly abl~ to escape ammon1um represslon, and the n~ mutant was nlfA~, whlch means th~t nltrogenass synth~s1s does not occur and the organlsm ls unable to flx nltrogen at ~11.
The problem ls that attempts to produce ammonla from nltro~en flxlng bacterla by de-regulatlon of nltrogenase productlon have been unsuccessful and althQugh attempts to produce ammonla by genetlcally manlpulatlng ammonlum as~lm11atlon enzymes have been succes~ful, th~ organlsm ltselP ls unable to ~row normally.
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E~RITISH TECH1~1nLClG`~ GROUP TEL: 44-71-403-7586 g.Sep.91 12:59 1~10.004 P.05 ~3~ ~ ~7;l Summary o~ the Inven~lon It has surprlslngly been foundl ~hat ln organlsms whereln nltrogen~se synthesls ls ~ontrolled by nl~ or nlfA-llke genes whlch are in turn re~ulated only by nl~ or ~EL-l1ke genes, 05 mutat~ons ln the nlfl or nlfL-llke genes regult ln mutant stralns o~ ~acterla whlch produce and excrete s~gnlflcant amounts of ammonlum. Surpr1slngly, these mutants grow as w~ s the parental stralns.
pe~s~1ptlon of thc pr.ef~LL~d embQdlment_ ~hls lnventlon ls appllcabls to any specles of nltrogen flxlng bacterla ~hlch contaln nlfL or nl~.~-llke yenes as part o~
thelr nltrogenase enzymQ system, sald nl~L or nlfL-llke genes solely controlllng the transcrlptlon of nlfA or nlfA-llke ~enes whlch ln turn regulate transcrlptlon of other nlf. genes necessary for nltrogenase stru~t~re and actlvlty. Fo~ convenlence herelnafter, n~f~ or nlfL-llke genes wlll be referred to slmply as nlfL and llkewlse nlfA or nlf~-llke gen0s wlll be ref~rr~d to as nlf~-Accordlng to a flrst aspect of the lnventlon there ls provlded a mutant of a nltrogen flxlng bacterlum as descrlbedabove, characterlsed ln ~hat lt contalns a mutant nlfL gene and a functlonal ~LE~ gene.
The preferred bacterla are t~ose whlch normally have stron~
plant assoclat~ons, elther by be1ng present ln the rhlzosphere or by belng attached to plant roots or llvlng lnsld~ plant roots or stems (so called systemlc andophyt~s)~ or all bacterla from the genus A7o~0b~~Qr wh~ther plant assoclated or not. Partlcularly preferred bacterla are from the genus Azotobacter, especlally A~otQbacter Ylnelandl1. The most preferred straln ls the subJect o~ a patent depos1t, dQposlted ln accordance wlth the prov1slons of the Budapest Treaty at the Na~lonal Collectlon o~ Industrlal and Marlne Bacterla Ltd., (NCIMB~, 23 St. Machen Drlve, Aberdeen, AB2 lRY, Scotland, Unlted Klngdom, o~ 30 August l9gl and glven the accesslon n~mber 40438, or ~ mutant or var~nt thereof havlng the deslred functlons of growth and productlon of ~mmonl~.
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BRITISH TECHl~l[lLDGY GROUP TEL: 44-71-403-7586 9.Sep.91 1~:5~ 1'10.004 P.O~
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Accordlng to a second ~spect of the lnventlon, there ls provlded a process ~or the 1solatlon of the nl.fL gene by, for example, standard heterologous hybrldlsatlon technlques, from the nltrogen flxlng bacterla descrlbed hlere1nbefore, the constructlon 05 of the mutatlons ln the n~ gene, and the relntroductlon of nl~L
mutatlons lnto th~ chromosome of the bacterla to r~place the wlld-type nlFL gene. It ls nec~ssary that mutat10ns lnactlvate the functlon of the ~lfL ~ene produst bu~ do not lnter~ere wlth expresslon of the downstream nlfA gene. Examples are presented whlch show that nl~L ~utants contlnue to produce nltrogenase and express nltrogenase genes (nlfHDK) in the presence of hlgh levels of ammon~um ~n the medlu~.
It wlll be apparent to those skllled ln the art that mutatlons may be carrled out ln a Yar~ety ~f w~ys. rhe mutatlons ~ay be carrled out by the addltlon of extra gen~tlc materlal lnto the n~fL gene ~so called "lnsertlon mutants"), deletlon of part of the nlfL g2na (so called "deletlon ~utants") or exchange o~
nuclelc acld sequences renderlng the proteln non-~unctlonal ~or those renderlng the proteln ~unctl~nal. Such exchan~es may be as large as the nlfL gene ltself, or an e~change of a slngle base ln the ~NA codlng ~or the gene ~so called "po~nt mutatlon") may bQ
sufflclent. Technlques for carrylng out the mutatlons wlll be apparant to those skllled ln the art.
In the bacterla of the ln~entlon, ths ~fL ~nd nlfA genes are normally f~und ln an operon, under the ~ontrol of one promoter, wlth nl~L transcr1p~on occurrlng prlor t~ nlfA transcrlptlon.
The ~unctlonallty of the nlf~ gene must be preserved 1n the mutant. That ls, the nlf~ gene must be retalned as ln the parent organl5m or, lf altered, the alterat~on must be non-lnter~erlng.
It ls lmportant that the mutatlon does not prevent n~
productlon, ~or example, by 1nsert10n of a polnt mutatlon 1nto nlf~ wh1ch lntroduces a term1natlon slgnal or by lntroductlon oF
a polar mutatlon. The mutatlon must therefore be one whlch does not ~nter~ere w~th mRNA productlon. The nlfA gene has lts own Shlne-Dalgarno sequence and hence the ~utatlon ln nlfL need not , , , ~ :
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~RITI~H TECHNnLQCY GRnup TEL: 44-71-403-7586 9.Sep.91 12:59 No.004 P.07 2~071 be 1n frame w~th the readlng frame of nlfA, s1ncQ transcrlptlon of ntfA wlll contlnue as normal, desplte mutatton of nlfL. It ls known that the n~f~l~ operon promoter ~5 ~uch that nl~A ls produced at levels wh~ch the bacter~a flnd physlologlcally 05 tolerable. Over-productlon of nlf~ by, for examplQ, mutatlons wh~ch result Sn the productlon of a strong promoter, ls detrlmenta~ to the cell. ~herefore mutatlons ln the nlfL ~ene must not result ln replacement o~ thQ natural gene promoter wlth a stronger promotar. The promoter of the nlfl~ operon must not be stron~er than the natural promoter found ln the wlld type bacterlum and ~referably ls the natural promoter.
When carrylng out the lnventlon ln the deposlted straln of eas~obacter ylnelandll lt ~s therefore preferable, ln order to achleve normal expresslon of ~lf~ ln the nl~L ~tant, to retaln a 200 base palr r~glon betwee~ the BglII ~n~ ~aI sltes upstream of the nlfL codlng reglon. Thls reglon carrles the promotQr of the ~l~LA operon ln the A. vlnelandll straln.
According to a th~rd aspect of the lnventlon there ls al50 proYlded a mutated nlfL gene whlch may be lnserted lnto wlld type ~0 bacterla tn order to render them nlfL-. Pr~erably the mutant gene ~5 from ~z ~ C~ ~ and ls lnserted lnto other ~ oto~a~ter specles. More preferably, the gene ls from the deposlted straln Of 2ZQ~ er vln~landll.
Accordlng to a fourth aspect of the 1nventlon there ls provlded a method of contro111ng the ~evel o~ ammonlum productlon by bacterla descrlb~d here1nbe~ore. It ls ~nown th~t tho lncrease ln pH of the cultur~ medlum whlch occur~ c~ncomltantly wlth ammonlum excretlon, llmlts the amount of ammonlum excreted because the hlgh pH lnactlvates nltrog~nase. Control of p~l, elther by lncreased buffer capaclty of the medlum or by removal o~ the ammonlum as lt ls excretecl, can be utl11zed to glve hlgher levels of ammonium productlon, or lower levels as requ1red, for exa~ple ln the use o~ ~lfL mutants of ~. vlnelandll, or other nltrogen ~lxlng organlsms descrlbed herelnb~fore to produce ammonlum whllst cells are tmmoblllzed on calctum alglnate BRITI~H TECHNOLQGY GROUP TEL: 4~l-71-403-7586 9.Sep.91 12:59 l~lo.004 P,Og 2 ~ 7 1 part1cles or beads.
The lnYantlon 1s partlcularly use~ul ~or those nltrogsn flxlng bacterla whlch are normally abundant ln the rhlzosphere or found assoclated wlth speclflc plants ~lthor by beln~ bound tOr 05 g1ycoprotelns such as lectln on plant roots or actually llvlng lnslde plant roots and ste~s ~systemlc endophyt~s).
Accordlng to a flfth aspect cf the lnventlon there ls provlded a method o~ prov1dlng plants ~lth ~ sourcs o~ ftxed nltrogen by lntroductlon o~ mutant nttrogen flxln~-bacterla as descrlbed herelnbefore lnto nltrogen-flxlng assoclatlon wlth plant ~Issue ~lncludlng parts of plants seed whole plants etc.). Nltrogen-flxlng assoclatlon wlth plants can manlfest ltsel~ ln several ways. Once lntroduced lnto n~trogen-flxlng assoclatlon ~lth plants the bacterla may bs pr~sent ln the rhl~ospher~ or be assoclated wlth p~ant~ as d~scrlbQd aboYe.
Th~ mutant n~trogen flxlng bacterla may be lntroduced to the plant tlss~e ln a varlety of ways. They c~n be applled to the plants themselves before or after plan-tlng the 5011 before or after plantlng has occurred or to sQeds. Convenlently the bacterla are applled as an aerosol or ln a l~quld or solld form.
The mutant bacterla may be ~f a totally dlf~erent ~amlly genus or specles to the wlld t~YpQ bacterla normally ~ound ln nltrogQn-flxlng assoclat~on w~th plants.
The mutatlons ln the nl1 gene may be lntroduced by mutatlng the wlld type bacterlum ltself by methods herelnb~for~ descrlbed or may be tntroduced by gens replacement. The bacterla may be mutants of the wlld type bact0rla whlch are normall~ found assoclat~d wlth plants. Gene replacement 1nvolves the repla~ement of a gene from one orsanlsm wlth in thls case a mutant nl~L gene from another organtsm. Pre~erablY~ the two organl sms are of the same fam11y or genus mor~ pre~srab1y of the same specles or straln.
The lnventlon also provldes a way o~ lntroduclng a source o~
flxed nltro~en to those plants to whlch there may be no nltrogen flxlng bacterlum notmally assoclated. Preferably the mutant , ~ : ~ :` :
BRITJ~H TEcHNnLnG~ GRnup TEL: 44-71-403-7586 ~.Sep.91 12:59 No.004 P.09 7 ~ ~
bacterla provldln~ the sourc~ o~ ammonl~ to these plants ls ~Q~o~a~hL vlnelandll, whlch ls able to blnd to lectlns present on plant roots~ Such Azotobac~er. Yi~Q1~ndll capable o~ lectln bindlng ls constructed accordlng to the teachlng descrl~ed ~y 05 Blshop et ~1., 1977 Sclence, l9~, 938-939 and Dlaz ~
Nature, 1989, ~, 579-5~l. These papers deserlbe the constructlon of ~. ylnelandll stralns that hlnd to lectlns produced by two specl0s of legumlnous plants, 7rlfollum repens and PlsumLsatlva. The ~enes lmportant for provldln~ the ablllty of le~tln blndln~ wlll be transferred ~rom ~hlzoh9um tr~QLl~m and ~ um~nosarum to ~. Yl,~,elandll nlfl mutant stralns. The gene for lectln productlon from Plsum s~,tlya wlll be transform~d lnto plant cells of a varlety of specles as descrlbed ln Blshop et al., and Dlaz Q~ . above. The p~oductlon of ~. satlva lectln receptors ~n ~. ~I,nelandli wlll allow lt to blnd to roots or other tlssues of plants carrylng genes that determlne lectln blndlng ~rom Rhlzoblum legu,m,l,nosarum. The blndln~ o~ a . y~lnel,and~l nlfL mu~ants to plant roots or other tlssues may allow the provlslon of flxed nltrogen ~rom bacte~la dlr~ctly to plants.
Ç9~s.rlpt.iQn o~ the drawln~s Elgure 1 shvws a restrlctlon map of the Az~obac~
vlne~andli nlEL~ operon, and constructlon of plasmlds.
E~gu~ _2 shows the levels of ammonla productlon by mutant and wlld type Azotoba~ter ~,ne,landll.
Fmbodlments of ~he lnventlon wlll be descrlbed by way of Example only.
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~RITI~H TECHNOLnGY GROUP TEL: 44-7l-403-7586 9.Sep.91 12:59 No.004 P.10 2~ 0~
EXAMPLE l . Growth oF A. ~lnelan~ll ~ tralns of ~. Ylnelandll were grown aeroblcally at 30~C ln ~urk`s sucrose medlum as descrlbed prevlously by To~kdarlan ~ 1., 1986, Embo J~, 5, 399-407. Llcluld 25ml cultures, contalned 05 ln 125ml ~lasks, were lncubated on a rotary shaker (lBOrpm~, Competence medlum ~CM) was Burk's sucrose m~dlum prQpared wlthout the addltlon of Fe and Mo salts. L~ medlum was used for growlng E. ~Ql~ Antlb10tlcs for selectlon of reslstan~e genes on plasmlds or ln genomlc transformants wsre added at concent~atlons prevlously reported ~Santero Q~ ~1-, 1988, Mol. Mlcroblol., ~, 303-314).
~MPLE 2 : Isolatlon o.f.an L ~ og~ G~IC~ Ir~.gmen~
carrylng the re~lon 1151~mL~f_DlE~
About 300bp of th~ 3'-end of A gene ~ncodlng a protetn wlth partlal sequence homology to the C-termlnl of the nlfL and n~r~
gene products o~ ~. 4ne~mQnl~Q had been cloned ln the plasmld pDB150 and sequenced by Bsnnett ~ al., ls~8, Mol. Mlcroblol., ~, 315-321. In order to lsolate and clone the entlre upstream gene, plaques rrom a lambda llbrary of ~. vlnelandll genomlc DNA were ~0 screened fvr hybrldlzatlon to a DNA probe labelled w1th 32P-dcTp~ Th~s probe was a 1.2Kb ~ nI ~ra~ment from pDB150 wh1ch cont21ns the 3'-end of the nlflln~R-llke ~ne and the 5'-~nd of nlfA (ses Flg. 1) ~Bennett Q~ al., 1988, Mol.
Mlcroblol., ~, 315-321). An ~pproxlmately 12.5~b EcoR~ ~ragment was ldentlfled ln the lns~rt DNA prepared ~rom tha progeny of one hybrldlzlng plaque; )t was subcloned lnto pBR~25 to glve pAB21.
~Thls 1~.5Kb fragment corresponded ln slze tv a ~enomlc fragment prevlo~sly reported to hybrld ke to a nl~B probe; a nlfA::Tn5 mutant, MV3, conta1ned a new nlfA-hybrldlzlng fragment of about l9Kb ln slze ~Santero ~t ~1., 1~88 Mol. M~croblol, 2, 303-814).
A restr1ctlon map o~ the lnsert tn pAB21 showed that one end oF
the 12.5KB ~QRl fragment had restrlctlon sltes correspondlng to those report~d by Bennett ~ al. (see a~ove) ~or pVBlSO. Part o~
thls fragment and the n1~/n.lfB re~lon, and subclones der1ved for thls work, are sho~n ln Flg. 1.
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Methods ~or blottlng and screenlng the lambd~ llbrary and ~or clonlng DNA fragments were standard procedures d~scrlbed ln Sambrook ~ al., 1989, Molecular Clonlng, Cold Sprlng Harbor Laboratory Press. Cold Sprln~ Harbor, and by lnstru~tlons 05 provlded by supp11ers of restrlctlon en~ymes.
E~M~LE 3 : Constructlon of a D1~9-l~cZ fuslon ae.nq The transposon Tn5-B21 carryln~ and ~ gene encodln~
tetracycllnQ reslst~nce ~Tcr) was lntroduced lnto E. coll carrylng pAB~, (a 5maI-~hI ~ragment of pA~5 ln pACYC177), (see ~19. 1) by lnfectlon wlth ~ ::Tn5-B21, ~Slmonet Q~ al. 19~9, Gene, ~Q, 161-169). Plasm1d derlvatl~s c~rrylng Tn5-B21 lnsertlons were lsolated and characterlzed as descrlbed prevlously. (Thomas ~ ~1-, Appl. Envl~on. Mlcroblol., ~, 3499-~S04~.
XAMPLE 4_ ; .Transf~r~ o~lL~ D~l eLll Cornpetent cells were prepared by a slmpllf1eatlon of a ~ethod descrlbed prevlously (Page et ~1., 197B, Can. J. Mlcroblol., 24 1~90-1594): ~hstead of growlng c~l~s 1n l~quld eompatence medlum (CM) prlor to transformatl~n, cells were resuspended dlrectly from the second round o~ growth on CM agar lnto lml of llquld CM
~6mM MgS04 to a dens1ty of ab~ut 108 cells ml~l. Approxlmately 50~1 of resuspended cells wera spotted onto a CM agar plate ~nd 0.1-l~g of plasm1d ~NA was mlxed ~1th the c~lls. After ~ncubatlon at 30 for two days, approxlmately S x 107 cells wsra ~5 transferred to ~electlY~ medlum contalnlng ~pproprlate antlbl~tlcs. Cells not mlxed wlth DNA were plated as controls.
Transformants arose at 104 - 105 (~g DNA)-l.
EXAMPLE S :. Isolatlon of tlle KlXX C~ ..t~
The reglon upstream of nlfA ln ~ n~l3ngl~ was ldentlfled and clon~d as descrlbed ln Example 1. The KIXX cassette, conta~nlng the KMr ~ene ~ h) from TnS, was 1solated as a 1.5Kb ~m~I fragment ~From the plasmld pUC4-KIXX ~Pharmacla ~td., UK).
~hls fra~ment was ll~ated wlth pAB26, whl~h had been dlgested wlth ~I ànd ~m~I; the S~lI overhang was blunt-ended uslng Klenow polymerase and dfloxynuclaotldas ~Fl~. 1), ThQ r~sultlng : .
. ~: . , , ~ITT~ ECHNQLOGY GROIIP TEL: 44-71-403-7586 ~.Sep.~1 12:59 No.004 P.12 2~5~)71 plasmlds pABzs ~nd pAB30, wlth th~ KIXX cassette 1nserted In opposlte orlentatlons, ~re transformed as dQscrlbed ln Example 4 lnto two stralns o- ~. Y.inelandll~ wlld type UW136 and the nlf~-lacZ fuslon straln M~101. Kmr transformants were screened 05 ~or reslstance to carbenlclllln ~Cbr); CBs derlYatlves were assumed to have arlsen from a double erossover recomblnatlon event ln whlch the wlld-type n1~LJ~L~ ke gene was replaced by th~ KIXX-contalnlng DNA. Kmr CB5 tr~nsformants o~ UW136 werQ
obtalned w~th pAB29 but not wlth pAB30. The pAB29-der1ved straln, MV376, was Nlf~. Southern blots o~ DNA from MV376, MV378, and ~lV380, d~gested w~th ~lI, were hybrldlz~d to a 32P-labelled probe from thQ mutat~d reglon (the 0.3Kb ~AlI-S~hI
fragment from pAB31; see Flg. 1). Southern hybrldl2atlon experlments showed that ln all three mutants, the 3.5K~ wlld-type 5~1I fragment was absent and replaced by a 6.2Kb hybrldl~lng fragment.
Although the KIXX lnsertlon ln both or1entatlons ln the ~lf~
gene of ~. vlnela~dll resulted ln a Nlf+ phenotype, l~sertlons o~
Tn5 or the n (transcrlptlon termlnator) cassette ln A. vlnelao~11 nlfL resu1ted ln A Nlf- phenotype. Therefore, the nlfLA genes are probably cotranscrlbed ln ~. vlnelan.~ as ln ~. ~n?~m~nl3Q.
Although the KIXX ~h cartrldye ~nserted upstre~m to oppos~
transc~lptlon can lnhlblt axpresslon of downstream g~nQs ln ~- Ylnelandll the nlfA sene ln the nlfL (KlXX) mutants MV376 and MV378 ls obvlously express~d. Thls could ar1se from unexp~cted promoter ac-tlvlty ln the cassette or from promoter-llke sequences generated by the KIXX lnsert10n ln th~ nlf~ r~glon.
Insert~on of KIXX ln the orlentatlon where expresslon of ~4h was ln the same dlrectlon as nlf~ resulted ln somewhat hlgher constltutlve levels of ~-galactosldase (MV380) than when lnserted ln the opposlte dlrectlon ~MV37~). However, lt was not posslble to construct a Nlf~ ~lfL2-KIXX derlYatlvQ ln the wlld-type ~qulvalent to MV380, whlch, as lt carrles a ntg~-lacZ fusion, 15 Nlf-. There~or~ excesslve expresslon ~f n.lfA dr1ven by th~ very strong ~4h promoter ls probably lethal ln a Nlf+ ~. v~nelandll ~ ' , ~ .'' .
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, ~RITJ-~h' TECHNOLQGY GROl1P TEL: 4'l-71-403-7586 9.Sep.~1 12:59 No.004 P.13 2~1 07~
_ 12 -background, posslbly dua to dlverslon o~ too much ATP towards nltrogenase synthesls ~nd actlvlty or to NH4~ toxlclty.
EXAMPLE 6 : .En~yme assays Nltrogenase and ~-galactosldase actl~ltles were mea5ured as 05 descrlbed prevlously. (Sambr~ok qt ~1.. 198~, "Molecular Clonlng". Cold Sprlng Harbor Laboratory Press, Cold Sprlny Harbor, ~nd ~almslsy ~ 9l, Appl. Envlron. Mlçroblol., 57, 522-524).
The results of ~hese ~ssays are shown 1n Table l below:- :
IA~L~
Nltrogenase and ~-galactosldase act~vltles ~n wlld-type and nlf~ ~utants of ~. Ylnelandll Straln Genotype ~N +NH4 UWl3S wlld-type 4~ 0 ~V376 nlf~l :KIXX ~8 4~
MVl01 nlf~ acz l$~4lb 432 MV378 ~lf~ ~ ll9G7 377Z
n1~ KIXX
MV380 ~lf~ ç~ lS384 6474 ~lf~2:KI~
lO -N N free med1um, a Nltrogenase actlvl~y ln UWl36 and MV376 me~sured as acetylene reductl~n ~nmol ethylene produced ~ln~l ~mg proteln)~~).
b ~-galactosldase act~vlty ln MVlOl, MV378, ~V380: measured as Mlller unlts (26~.
Each value ls the mean from 2 - 3 lndependent determlnatlons;
varlatlon was < lOX ~rom ~he mean.
~RITI~H ~ECHNOLOBY ~Rnup TEL: 44-71-403-7586 ~.Sep.91 12:5g No.004 ~.14 2 ~ 7 1 The data ln Tabl~ 1 show that MV376 was Nlf~ and ~xpressed wlld-type acetylene reductlon actlYlty both ln N-free medlum and ln medlum wltl~ ammon~um at 15mM ~ concentratlon that repressed nltrogenase ~ctlvlty ln the ~lld-type straln UW136. Sl~llarly 05 both MV378 and MV3~0 expr~ssed ~-galactosldas~ actlvlty ln the absence or presenc~ of ammonlum whl~r~as actlv1ty ln thc nlfL~
straln M~101 was repressed by lSmM ammon3um.
Other actl~tles r~pressed by ammonlum ln ~. vlng]andll are those of nltrate reductase and nltrlte reductase. (LuquQ et al.
l9B7 Arch. Mlcroblol. 148 231-235) the ~xpresslon o~ whlch requlres both the n~r~ and rpoN gene products (Luque ~ al.
Supra Santero ~ al. 1986 3. Bacterlol. 1~6 541-544 and Toukdarlan et ~. 1986 EMBO J. ~ 39g-407) The KIXX
lnsertlon mutat~on had no eff~ct on normal re~ulatlon of n1trate reductase; thls actlv7ty was prssent ln N03~-~rown but not NH4~ N03~)-grown cultures of both ~lld-type UW136 and mutant MV376 Sdata not shown~.
Th~se rssults demonstrate that ~he gene upstr~am o~ n1fA ln ~. vlnel~dl~ medlates ammonlum represslon o~ nlf gene expresslon but not o~ at least one other gene under ammonlum control. Thls phenotype 1s slmllar to that of PlfL mutants o~ ~. pneumoni~
where nlfl ls locatQd l~medlately upstream o~ nlfA. translatlon of the entlra ~. ~lnelandll upstream gene sequentQ tapproxlmately 1.4kb) sho~s a prote1n wlth greater homology to ~. ~n~umorl~
nlfL than to NT~B (31X vs. Z4% ldentlty).
EXAMPLE 7 :. Ammonlum estl~a~lQn Culture supernatants of ~. ~lnelan~ll wlld-type straln UW136 and mutant straln MV376 grown on nltrogen werQ tested for thQ
present o~ ammonlum.
Samples of cultur~s WQr~ taken at dl~ferent tlmes and centrlfu3ed. 0.5ml of supernatant or flltrate was tested for the presence of ammonlum by the lndophenol me~l-od ~Bergersen 1980 Methods for evaluat1ng blologlcal nltrogen flxa~lon John Wlley & Sons Ltd. London). ~hls conslsted of the addltlon ln order of a) O.Sml phenol-~odlum nltroprussld~ solut10n (phenol 509 1~
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~ sodlum n1troprusslde, 0.25g 1-1); b)O.5ml of sodlum hypochlorlte solut~on ~O.lm); and c~ 2ml of d~st~lled water. The mlxture was lncubated for 30 mln at room temperature. Absorbance at 625nm was measured and ammonlum conc~ntratlon estlmated from a 05 standard curve obtalned wlth ammonlum solu~lons at varlous concentratlons assayed uslny the same reagent solutlon.
The rssults are drawn ln Flgure 2, whereln ~ denotes U~136 and O denotes MY376.
In contrast to UW136, M~376 excreted ammonium rather suddenly towards the end of ~ponentla1 growth. F~nal amounts measured ln MV376 statlonary phasQ cultures wer~ 5 - 10 mM. Excretlon was not llmlted by carbon sourcQ avallablllty because half of the supplled sucrose remalned at lts cessatlon.
~AMPLE 8 : Constructlon of nlfL deletlon mut~t of ~- vln~
The plasmld pAB27 carrylng tha nlfL reglon was dlgsstQd wlth lII and llgated to a 3.8 Kb ~mHI fragment from pJ017 whlch carrlQs the gene cartrldge, sac ~Hynes ~ al., Gene, 198g 1~, 111-120. The ~3~ gene confers a sensltlvlty to sucrose when transferred to certaln gl~am negatlve bacterla, l.e. organlsms carrylng the sac 0ens cannot grow on sucroso-conta~n1ng medla.
The resul~lng plasmld, pLA5:Sac-Km, c~rrles the sac cartrldg~
whlch replaces a 585 base palr ~g~II fragment, dsletlng the S -end of the ~ene and 20 basQ palrs upstream from thQ
translatlon start ~odon. In order to transfer the nlpl:
mutatlon from pLA5:Sac-Km lnto the chromosome, the plasmld ~as transformed lnto ~. Ylnelandl1 U~135 ~nd kanamycln-reslstant colonles were selected. These were found to be unabl~ to ~row on sucrose-contalnlng med~um but could grow normally on ~lucose as carbon sourc8. One colonles was purlfled several tlmes an~ had stable s~crose-sensltlve, kanamycln-reslstant phenotype Thls mutant was MV399. MV399 had a Nlf mlnus phenotype because the ~c cartrldge lnsert prevented expresslon of the downstr~am nlf~
gene whlch ls essentlal for expresslon of n~f genes.
In order to replace the nl~.L~ mutatlon wlth a nlfL
deletlon mutatlon unlnterrupted by ~ DNA, pAB27 was d!~ested .
~RITT~H T~:CHI~IOLOGY GROUP TEL: 4~1-71-403-75~6 9.Sep.91 1~:59 No.004 P.16 ~51~71 wlth ~lI1 and ligated to ttself. Plasmld pLA4 was lsolated and found to contaln tha 585 base palr ~lII deletlon wlthtn the nlfL
gene. MY399 was transformed wlth pLA4 and colonles growln~ on sucrose-contalnlng am~onlum-free medlum were selected, These Nlf 05 plus colonles were also kanamycln-sensltlvo lndlcatlng that the Km cartrldge had been rep1aced. One such colony was purlfled and tested for ammonlum excretlon, Thls straln, MV440, was found to excrete up to lOmM ammonlum lnto the growth medlum and the pH
of cultures lncreased to about 8.5 ~hen no further ammonlum was excreted. Thus a deletlon o~ the ~'-end o~ the nlfL gene, whlch does not affect expresslon of the downstream nlfA gene, leads to a mutant stratn of ~. Y1neland~ wh~ch excretes large amounts of ammontum s~mllar to the phenotype o~ the nl~L:KIXX mutant MV376 descrlbed prevlously.
. .
~ " , ..
~, , .
. ~:
, E1eld of the lnv~ntlon Thls lnventlon ls ln the ~lell~ of ammonla productlon by nitrogen flxlng bact0rla.
~escrl~lon of th~ prlor ~rt 05 Many mltroorgan1sms are able to asslml~ate the ma~or bloelements (l.a. carbon, n1trogen, sulphur, hydrogen and oxy~en) ln an lnorganlc form. The ablllty to use N2 ~s a nltrogen source 1~ restr~cted to prokaryotes, and Is relatlvely rare ev~n among thls group. The enzyme system responslble for N2 Flxatlon ls o called nltr~genase. Blosynthesls o~ the ~omponents ot the nltrogenase system ls de-termlned by 15 to ZO dlfferent nlf genes.
Free-llvlng nltrogen ~lxlng bacter1a flx an amount of atmospherlc nltrogen sufflclent for thelr own needs. Evldence for th~s ls ~hat slgnlflcant amounts of ammonla are rarely found ln the culture medlu~ ~ln lAboratory cultures) or envlronment (1n the soll) of nltrogen flxlng organlsms. Re~ulatorY mechanlsms controlllng nlf gene transcrlptlon andlor nltrogena~e ~ctlYlty ensure that eellular energy ls not wasted by the ~lxatlon of m~re nltrogen than 15 necessary to meet the demands o~ bact~rlal cell growth and vlablllty. ~n partlcular, ~xcess e~vlr~nmental ammonla or oxygen prevents expr~sslon of nlf genes ln free 11vlng d1azotrophs ~nltrogen ~lxln3 bacterla). Ammonla makes nltrogenase unnecessary and o~yge~ lnactl~tss the ~nzyme. Slnce these bacterla produce ~mmonla for asslmllatlon ~y plan-ts ln thQ
form o~ ammonlum catlons ~NH4-~, the term am~onlum ls used herelnafter for breYltY.
Attempts to lnduce ammonlum excretlon have up to now center~d on physlo10glcal suppresslon of g~netlc manlpulatlon o~ the enzymes lnvolved ln ammonlum asslml1atlon. Tr~atment of cyanobacterla, wlth L-methlon1ne-DL-sulfoxlmlne ~SX), an lnhlbitor of glutamlne synthet~se ~GS), resulted ln the excretlon of 0.3 to 7 mM NH4~ lnto the growth medlum, ~Musgrove ~ al., 1982, Blotech. Letters, 4, 647-652, Ne~-ton Q~ al., 1985, Blochlm, B~ophys. Acta, ~Q~, 44-50 ~nd R~os ~ ~1., 1984, Appl.
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~RIT~SH~TECHNOLOGY GRnUP TEL: 44-71-403-7586 9.Sep,91 12:59 No.fJ04 P.03 Envlron. Mlcroblol. g~ 114-118). Mutants o~ Anabaena resls~ant to MSX or the NH4~ ~nalogue ethylene dlamlne excreted up to 1.6 mM NH4+ (Po1uklna et al. 198Z Mlcrobl~logy 51 90-95 Splller et al. 19~6 J. Bacterlol. 1~5 412-419 and Thomas et ~1.. 7990 05 Appl. ~nvlron Mlcroblol. ~ 34~-3S04). Amon~ eubacter1a ammon1um excret~on was repor~ed to occur ln Gln- or Gln~ ~s~L-l mutants of Klebslella Dne~monlae ~Anderson et ~1.. 1977 J.Gen.
M1croblol. lQ~. 107-122 and Shanmugam et al.~ 1975 Proc. Natl.
Acad. Sc1. USA. 7~ 136-139) ln ~utants of ~hq~oba~Q~
c~sulatus and Azosplrlllum brasllensQ altered ln the productlon of GS (~all et ~1- 1979 J. Bacterlol 1~1. 1459-1463~. and ln methylamlne reslstan~ mutants o~ Azotobacter lnelandl~ (Gordon et ~1.. 1983 Can. J. Mlcroblol ~ 973-978~. Although slgnlflcant ammontum excretlon has been observed these chemlcally treated or mutated organlsms requlre supple~entatlon of larg~ amounts of the amlno acld g~utamlne for growth.
The regulatory pathwayS and m~chanlsms lnvolve~ ln the represslon of nl~ genes by ammonlum have been extenslvely characterlzed ln ~he free llvlng dlazotrophs Klebsl&1~ pneumont~e and Azotobact~L
vlne!~ndll. Of central lmportance ln both vrganlsms ~and other gram nega~lve nltrogen flxlng bacterla) 1~ that a posltlvQly-act1n~ re~ulatory proteln ~IFA 15 requlred to act1vate transcr1pt1On of th3 other M~f gen~s whlch are necessary ~or nltr~qenase structure and act1vlty. In K. ~ç~mQnl~ ammon1um z5 represses nltrogenase synthesls by preventlng elther th~ actlvlty or synthesls o~ NIFA whlch occ~rs by t~o separate mechan~sms The nlf~ gene ls adJacent to and downstr~am of nlfL ~thus formln~
the nlfLA operon) and these two genas are co-expressed. The NIF~
proteln blnds to and lnactlvates NIFA lf ammonlum ls present even at relatlvely low levels ~> approx. S~M). ht hl~her 1eve1s of ammonlum ~ approx. 200~M) expresslon oF the D~fL~ ~peron does not occur and so the NIFA proteln ls not syntheslz~d.
~ranscr~ptlon of nlf~ requlres phosphorylated NTRC proteln but thls proteln ls dephosphorylated and hence 1nactlve ln cells ~rown wlth ammonlum. Thus ln ~. ~L~Iu~mlae ~epresslon of ~RITISH TECHl~lOLnbY GRO~IP TEL: 44-71-403-7586 9.Sep.91 12:59 No.004 P.04 2~5:~0~ ~
n1trogenase synthesls by ammonlu~n occurs at two levels, lnactlvatlon of NIFA by NIFL and preventlon of nl~ expresslon by dephosphorylated NTRC.
Nltrogen flxatlon ln ~ nel2ndll ls d~ermlned by the 05 presence of three b10chemlcally and genetlcally dlstlnct nltro~enase enzymes, ~ach of whlch ls syntheslzed under dl~f~rent condltlons of metal supply. The ~olybdenum nltro~enase gene whlth ls slmllar to the enzyme purlfled from a number of other nltrogen flx~n~ organlsms, requlres 15 - 20 ~ene produces for lts structure and actlvlty. In ~. vlnQLandll as ln ~. ~n~umQnl~c Gxpresslon of the ~ enes requlres actlve NIFA produ~t. Unllke ln ~. ~nQ onlae ho~ever, HTRC ls not requlr~d for nltrogen flxatlon whlch lmpl1es that NTRC ls not necessary for ~lf~
expresslon.
~NA sequenclng of the ~lf~ reglon of ~ ~I eL~ndli revealed a gene wh~se translatlon product was slmllar to NIFA of K.
pneumonla~. The DNA sequence of 200bp upstream o~ DlfA revealed a partlal open rea~lng fr~me (ORF) whlch predlct~d ~ protcln wlth homology to the C-termlnal reg1~ns of the plfL and n~_~ gene products o~ K. pneumonlae. ~Bennett ~ al., 1986, Mol.
Mlc~oblol., ~, 315-321~.
It ~ould be d~slrable to be abla to lnduce ammonlum productlon ~n these nltrogen flxln~ bac-terla ~or a ~arlety o~
purposes. Attempts to de-regulate nltro0enase synthesls ln ~ Lslella pneumonlae by mutatlng elther th~ ~lfL or ntrC genes wQrQ unsuc~essful, ~he resultlng n1~ mutant was only weakly abl~ to escape ammon1um represslon, and the n~ mutant was nlfA~, whlch means th~t nltrogenass synth~s1s does not occur and the organlsm ls unable to flx nltrogen at ~11.
The problem ls that attempts to produce ammonla from nltro~en flxlng bacterla by de-regulatlon of nltrogenase productlon have been unsuccessful and althQugh attempts to produce ammonla by genetlcally manlpulatlng ammonlum as~lm11atlon enzymes have been succes~ful, th~ organlsm ltselP ls unable to ~row normally.
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E~RITISH TECH1~1nLClG`~ GROUP TEL: 44-71-403-7586 g.Sep.91 12:59 1~10.004 P.05 ~3~ ~ ~7;l Summary o~ the Inven~lon It has surprlslngly been foundl ~hat ln organlsms whereln nltrogen~se synthesls ls ~ontrolled by nl~ or nlfA-llke genes whlch are in turn re~ulated only by nl~ or ~EL-l1ke genes, 05 mutat~ons ln the nlfl or nlfL-llke genes regult ln mutant stralns o~ ~acterla whlch produce and excrete s~gnlflcant amounts of ammonlum. Surpr1slngly, these mutants grow as w~ s the parental stralns.
pe~s~1ptlon of thc pr.ef~LL~d embQdlment_ ~hls lnventlon ls appllcabls to any specles of nltrogen flxlng bacterla ~hlch contaln nlfL or nl~.~-llke yenes as part o~
thelr nltrogenase enzymQ system, sald nl~L or nlfL-llke genes solely controlllng the transcrlptlon of nlfA or nlfA-llke ~enes whlch ln turn regulate transcrlptlon of other nlf. genes necessary for nltrogenase stru~t~re and actlvlty. Fo~ convenlence herelnafter, n~f~ or nlfL-llke genes wlll be referred to slmply as nlfL and llkewlse nlfA or nlf~-llke gen0s wlll be ref~rr~d to as nlf~-Accordlng to a flrst aspect of the lnventlon there ls provlded a mutant of a nltrogen flxlng bacterlum as descrlbedabove, characterlsed ln ~hat lt contalns a mutant nlfL gene and a functlonal ~LE~ gene.
The preferred bacterla are t~ose whlch normally have stron~
plant assoclat~ons, elther by be1ng present ln the rhlzosphere or by belng attached to plant roots or llvlng lnsld~ plant roots or stems (so called systemlc andophyt~s)~ or all bacterla from the genus A7o~0b~~Qr wh~ther plant assoclated or not. Partlcularly preferred bacterla are from the genus Azotobacter, especlally A~otQbacter Ylnelandl1. The most preferred straln ls the subJect o~ a patent depos1t, dQposlted ln accordance wlth the prov1slons of the Budapest Treaty at the Na~lonal Collectlon o~ Industrlal and Marlne Bacterla Ltd., (NCIMB~, 23 St. Machen Drlve, Aberdeen, AB2 lRY, Scotland, Unlted Klngdom, o~ 30 August l9gl and glven the accesslon n~mber 40438, or ~ mutant or var~nt thereof havlng the deslred functlons of growth and productlon of ~mmonl~.
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BRITISH TECHl~l[lLDGY GROUP TEL: 44-71-403-7586 9.Sep.91 1~:5~ 1'10.004 P.O~
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Accordlng to a second ~spect of the lnventlon, there ls provlded a process ~or the 1solatlon of the nl.fL gene by, for example, standard heterologous hybrldlsatlon technlques, from the nltrogen flxlng bacterla descrlbed hlere1nbefore, the constructlon 05 of the mutatlons ln the n~ gene, and the relntroductlon of nl~L
mutatlons lnto th~ chromosome of the bacterla to r~place the wlld-type nlFL gene. It ls nec~ssary that mutat10ns lnactlvate the functlon of the ~lfL ~ene produst bu~ do not lnter~ere wlth expresslon of the downstream nlfA gene. Examples are presented whlch show that nl~L ~utants contlnue to produce nltrogenase and express nltrogenase genes (nlfHDK) in the presence of hlgh levels of ammon~um ~n the medlu~.
It wlll be apparent to those skllled ln the art that mutatlons may be carrled out ln a Yar~ety ~f w~ys. rhe mutatlons ~ay be carrled out by the addltlon of extra gen~tlc materlal lnto the n~fL gene ~so called "lnsertlon mutants"), deletlon of part of the nlfL g2na (so called "deletlon ~utants") or exchange o~
nuclelc acld sequences renderlng the proteln non-~unctlonal ~or those renderlng the proteln ~unctl~nal. Such exchan~es may be as large as the nlfL gene ltself, or an e~change of a slngle base ln the ~NA codlng ~or the gene ~so called "po~nt mutatlon") may bQ
sufflclent. Technlques for carrylng out the mutatlons wlll be apparant to those skllled ln the art.
In the bacterla of the ln~entlon, ths ~fL ~nd nlfA genes are normally f~und ln an operon, under the ~ontrol of one promoter, wlth nl~L transcr1p~on occurrlng prlor t~ nlfA transcrlptlon.
The ~unctlonallty of the nlf~ gene must be preserved 1n the mutant. That ls, the nlf~ gene must be retalned as ln the parent organl5m or, lf altered, the alterat~on must be non-lnter~erlng.
It ls lmportant that the mutatlon does not prevent n~
productlon, ~or example, by 1nsert10n of a polnt mutatlon 1nto nlf~ wh1ch lntroduces a term1natlon slgnal or by lntroductlon oF
a polar mutatlon. The mutatlon must therefore be one whlch does not ~nter~ere w~th mRNA productlon. The nlfA gene has lts own Shlne-Dalgarno sequence and hence the ~utatlon ln nlfL need not , , , ~ :
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~RITI~H TECHNnLQCY GRnup TEL: 44-71-403-7586 9.Sep.91 12:59 No.004 P.07 2~071 be 1n frame w~th the readlng frame of nlfA, s1ncQ transcrlptlon of ntfA wlll contlnue as normal, desplte mutatton of nlfL. It ls known that the n~f~l~ operon promoter ~5 ~uch that nl~A ls produced at levels wh~ch the bacter~a flnd physlologlcally 05 tolerable. Over-productlon of nlf~ by, for examplQ, mutatlons wh~ch result Sn the productlon of a strong promoter, ls detrlmenta~ to the cell. ~herefore mutatlons ln the nlfL ~ene must not result ln replacement o~ thQ natural gene promoter wlth a stronger promotar. The promoter of the nlfl~ operon must not be stron~er than the natural promoter found ln the wlld type bacterlum and ~referably ls the natural promoter.
When carrylng out the lnventlon ln the deposlted straln of eas~obacter ylnelandll lt ~s therefore preferable, ln order to achleve normal expresslon of ~lf~ ln the nl~L ~tant, to retaln a 200 base palr r~glon betwee~ the BglII ~n~ ~aI sltes upstream of the nlfL codlng reglon. Thls reglon carrles the promotQr of the ~l~LA operon ln the A. vlnelandll straln.
According to a th~rd aspect of the lnventlon there ls al50 proYlded a mutated nlfL gene whlch may be lnserted lnto wlld type ~0 bacterla tn order to render them nlfL-. Pr~erably the mutant gene ~5 from ~z ~ C~ ~ and ls lnserted lnto other ~ oto~a~ter specles. More preferably, the gene ls from the deposlted straln Of 2ZQ~ er vln~landll.
Accordlng to a fourth aspect of the 1nventlon there ls provlded a method of contro111ng the ~evel o~ ammonlum productlon by bacterla descrlb~d here1nbe~ore. It ls ~nown th~t tho lncrease ln pH of the cultur~ medlum whlch occur~ c~ncomltantly wlth ammonlum excretlon, llmlts the amount of ammonlum excreted because the hlgh pH lnactlvates nltrog~nase. Control of p~l, elther by lncreased buffer capaclty of the medlum or by removal o~ the ammonlum as lt ls excretecl, can be utl11zed to glve hlgher levels of ammonium productlon, or lower levels as requ1red, for exa~ple ln the use o~ ~lfL mutants of ~. vlnelandll, or other nltrogen ~lxlng organlsms descrlbed herelnb~fore to produce ammonlum whllst cells are tmmoblllzed on calctum alglnate BRITI~H TECHNOLQGY GROUP TEL: 4~l-71-403-7586 9.Sep.91 12:59 l~lo.004 P,Og 2 ~ 7 1 part1cles or beads.
The lnYantlon 1s partlcularly use~ul ~or those nltrogsn flxlng bacterla whlch are normally abundant ln the rhlzosphere or found assoclated wlth speclflc plants ~lthor by beln~ bound tOr 05 g1ycoprotelns such as lectln on plant roots or actually llvlng lnslde plant roots and ste~s ~systemlc endophyt~s).
Accordlng to a flfth aspect cf the lnventlon there ls provlded a method o~ prov1dlng plants ~lth ~ sourcs o~ ftxed nltrogen by lntroductlon o~ mutant nttrogen flxln~-bacterla as descrlbed herelnbefore lnto nltrogen-flxlng assoclatlon wlth plant ~Issue ~lncludlng parts of plants seed whole plants etc.). Nltrogen-flxlng assoclatlon wlth plants can manlfest ltsel~ ln several ways. Once lntroduced lnto n~trogen-flxlng assoclatlon ~lth plants the bacterla may bs pr~sent ln the rhl~ospher~ or be assoclated wlth p~ant~ as d~scrlbQd aboYe.
Th~ mutant n~trogen flxlng bacterla may be lntroduced to the plant tlss~e ln a varlety of ways. They c~n be applled to the plants themselves before or after plan-tlng the 5011 before or after plantlng has occurred or to sQeds. Convenlently the bacterla are applled as an aerosol or ln a l~quld or solld form.
The mutant bacterla may be ~f a totally dlf~erent ~amlly genus or specles to the wlld t~YpQ bacterla normally ~ound ln nltrogQn-flxlng assoclat~on w~th plants.
The mutatlons ln the nl1 gene may be lntroduced by mutatlng the wlld type bacterlum ltself by methods herelnb~for~ descrlbed or may be tntroduced by gens replacement. The bacterla may be mutants of the wlld type bact0rla whlch are normall~ found assoclat~d wlth plants. Gene replacement 1nvolves the repla~ement of a gene from one orsanlsm wlth in thls case a mutant nl~L gene from another organtsm. Pre~erablY~ the two organl sms are of the same fam11y or genus mor~ pre~srab1y of the same specles or straln.
The lnventlon also provldes a way o~ lntroduclng a source o~
flxed nltro~en to those plants to whlch there may be no nltrogen flxlng bacterlum notmally assoclated. Preferably the mutant , ~ : ~ :` :
BRITJ~H TEcHNnLnG~ GRnup TEL: 44-71-403-7586 ~.Sep.91 12:59 No.004 P.09 7 ~ ~
bacterla provldln~ the sourc~ o~ ammonl~ to these plants ls ~Q~o~a~hL vlnelandll, whlch ls able to blnd to lectlns present on plant roots~ Such Azotobac~er. Yi~Q1~ndll capable o~ lectln bindlng ls constructed accordlng to the teachlng descrl~ed ~y 05 Blshop et ~1., 1977 Sclence, l9~, 938-939 and Dlaz ~
Nature, 1989, ~, 579-5~l. These papers deserlbe the constructlon of ~. ylnelandll stralns that hlnd to lectlns produced by two specl0s of legumlnous plants, 7rlfollum repens and PlsumLsatlva. The ~enes lmportant for provldln~ the ablllty of le~tln blndln~ wlll be transferred ~rom ~hlzoh9um tr~QLl~m and ~ um~nosarum to ~. Yl,~,elandll nlfl mutant stralns. The gene for lectln productlon from Plsum s~,tlya wlll be transform~d lnto plant cells of a varlety of specles as descrlbed ln Blshop et al., and Dlaz Q~ . above. The p~oductlon of ~. satlva lectln receptors ~n ~. ~I,nelandli wlll allow lt to blnd to roots or other tlssues of plants carrylng genes that determlne lectln blndlng ~rom Rhlzoblum legu,m,l,nosarum. The blndln~ o~ a . y~lnel,and~l nlfL mu~ants to plant roots or other tlssues may allow the provlslon of flxed nltrogen ~rom bacte~la dlr~ctly to plants.
Ç9~s.rlpt.iQn o~ the drawln~s Elgure 1 shvws a restrlctlon map of the Az~obac~
vlne~andli nlEL~ operon, and constructlon of plasmlds.
E~gu~ _2 shows the levels of ammonla productlon by mutant and wlld type Azotoba~ter ~,ne,landll.
Fmbodlments of ~he lnventlon wlll be descrlbed by way of Example only.
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~RITI~H TECHNOLnGY GROUP TEL: 44-7l-403-7586 9.Sep.91 12:59 No.004 P.10 2~ 0~
EXAMPLE l . Growth oF A. ~lnelan~ll ~ tralns of ~. Ylnelandll were grown aeroblcally at 30~C ln ~urk`s sucrose medlum as descrlbed prevlously by To~kdarlan ~ 1., 1986, Embo J~, 5, 399-407. Llcluld 25ml cultures, contalned 05 ln 125ml ~lasks, were lncubated on a rotary shaker (lBOrpm~, Competence medlum ~CM) was Burk's sucrose m~dlum prQpared wlthout the addltlon of Fe and Mo salts. L~ medlum was used for growlng E. ~Ql~ Antlb10tlcs for selectlon of reslstan~e genes on plasmlds or ln genomlc transformants wsre added at concent~atlons prevlously reported ~Santero Q~ ~1-, 1988, Mol. Mlcroblol., ~, 303-314).
~MPLE 2 : Isolatlon o.f.an L ~ og~ G~IC~ Ir~.gmen~
carrylng the re~lon 1151~mL~f_DlE~
About 300bp of th~ 3'-end of A gene ~ncodlng a protetn wlth partlal sequence homology to the C-termlnl of the nlfL and n~r~
gene products o~ ~. 4ne~mQnl~Q had been cloned ln the plasmld pDB150 and sequenced by Bsnnett ~ al., ls~8, Mol. Mlcroblol., ~, 315-321. In order to lsolate and clone the entlre upstream gene, plaques rrom a lambda llbrary of ~. vlnelandll genomlc DNA were ~0 screened fvr hybrldlzatlon to a DNA probe labelled w1th 32P-dcTp~ Th~s probe was a 1.2Kb ~ nI ~ra~ment from pDB150 wh1ch cont21ns the 3'-end of the nlflln~R-llke ~ne and the 5'-~nd of nlfA (ses Flg. 1) ~Bennett Q~ al., 1988, Mol.
Mlcroblol., ~, 315-321). An ~pproxlmately 12.5~b EcoR~ ~ragment was ldentlfled ln the lns~rt DNA prepared ~rom tha progeny of one hybrldlzlng plaque; )t was subcloned lnto pBR~25 to glve pAB21.
~Thls 1~.5Kb fragment corresponded ln slze tv a ~enomlc fragment prevlo~sly reported to hybrld ke to a nl~B probe; a nlfA::Tn5 mutant, MV3, conta1ned a new nlfA-hybrldlzlng fragment of about l9Kb ln slze ~Santero ~t ~1., 1~88 Mol. M~croblol, 2, 303-814).
A restr1ctlon map o~ the lnsert tn pAB21 showed that one end oF
the 12.5KB ~QRl fragment had restrlctlon sltes correspondlng to those report~d by Bennett ~ al. (see a~ove) ~or pVBlSO. Part o~
thls fragment and the n1~/n.lfB re~lon, and subclones der1ved for thls work, are sho~n ln Flg. 1.
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Methods ~or blottlng and screenlng the lambd~ llbrary and ~or clonlng DNA fragments were standard procedures d~scrlbed ln Sambrook ~ al., 1989, Molecular Clonlng, Cold Sprlng Harbor Laboratory Press. Cold Sprln~ Harbor, and by lnstru~tlons 05 provlded by supp11ers of restrlctlon en~ymes.
E~M~LE 3 : Constructlon of a D1~9-l~cZ fuslon ae.nq The transposon Tn5-B21 carryln~ and ~ gene encodln~
tetracycllnQ reslst~nce ~Tcr) was lntroduced lnto E. coll carrylng pAB~, (a 5maI-~hI ~ragment of pA~5 ln pACYC177), (see ~19. 1) by lnfectlon wlth ~ ::Tn5-B21, ~Slmonet Q~ al. 19~9, Gene, ~Q, 161-169). Plasm1d derlvatl~s c~rrylng Tn5-B21 lnsertlons were lsolated and characterlzed as descrlbed prevlously. (Thomas ~ ~1-, Appl. Envl~on. Mlcroblol., ~, 3499-~S04~.
XAMPLE 4_ ; .Transf~r~ o~lL~ D~l eLll Cornpetent cells were prepared by a slmpllf1eatlon of a ~ethod descrlbed prevlously (Page et ~1., 197B, Can. J. Mlcroblol., 24 1~90-1594): ~hstead of growlng c~l~s 1n l~quld eompatence medlum (CM) prlor to transformatl~n, cells were resuspended dlrectly from the second round o~ growth on CM agar lnto lml of llquld CM
~6mM MgS04 to a dens1ty of ab~ut 108 cells ml~l. Approxlmately 50~1 of resuspended cells wera spotted onto a CM agar plate ~nd 0.1-l~g of plasm1d ~NA was mlxed ~1th the c~lls. After ~ncubatlon at 30 for two days, approxlmately S x 107 cells wsra ~5 transferred to ~electlY~ medlum contalnlng ~pproprlate antlbl~tlcs. Cells not mlxed wlth DNA were plated as controls.
Transformants arose at 104 - 105 (~g DNA)-l.
EXAMPLE S :. Isolatlon of tlle KlXX C~ ..t~
The reglon upstream of nlfA ln ~ n~l3ngl~ was ldentlfled and clon~d as descrlbed ln Example 1. The KIXX cassette, conta~nlng the KMr ~ene ~ h) from TnS, was 1solated as a 1.5Kb ~m~I fragment ~From the plasmld pUC4-KIXX ~Pharmacla ~td., UK).
~hls fra~ment was ll~ated wlth pAB26, whl~h had been dlgested wlth ~I ànd ~m~I; the S~lI overhang was blunt-ended uslng Klenow polymerase and dfloxynuclaotldas ~Fl~. 1), ThQ r~sultlng : .
. ~: . , , ~ITT~ ECHNQLOGY GROIIP TEL: 44-71-403-7586 ~.Sep.~1 12:59 No.004 P.12 2~5~)71 plasmlds pABzs ~nd pAB30, wlth th~ KIXX cassette 1nserted In opposlte orlentatlons, ~re transformed as dQscrlbed ln Example 4 lnto two stralns o- ~. Y.inelandll~ wlld type UW136 and the nlf~-lacZ fuslon straln M~101. Kmr transformants were screened 05 ~or reslstance to carbenlclllln ~Cbr); CBs derlYatlves were assumed to have arlsen from a double erossover recomblnatlon event ln whlch the wlld-type n1~LJ~L~ ke gene was replaced by th~ KIXX-contalnlng DNA. Kmr CB5 tr~nsformants o~ UW136 werQ
obtalned w~th pAB29 but not wlth pAB30. The pAB29-der1ved straln, MV376, was Nlf~. Southern blots o~ DNA from MV376, MV378, and ~lV380, d~gested w~th ~lI, were hybrldlz~d to a 32P-labelled probe from thQ mutat~d reglon (the 0.3Kb ~AlI-S~hI
fragment from pAB31; see Flg. 1). Southern hybrldl2atlon experlments showed that ln all three mutants, the 3.5K~ wlld-type 5~1I fragment was absent and replaced by a 6.2Kb hybrldl~lng fragment.
Although the KIXX lnsertlon ln both or1entatlons ln the ~lf~
gene of ~. vlnela~dll resulted ln a Nlf+ phenotype, l~sertlons o~
Tn5 or the n (transcrlptlon termlnator) cassette ln A. vlnelao~11 nlfL resu1ted ln A Nlf- phenotype. Therefore, the nlfLA genes are probably cotranscrlbed ln ~. vlnelan.~ as ln ~. ~n?~m~nl3Q.
Although the KIXX ~h cartrldye ~nserted upstre~m to oppos~
transc~lptlon can lnhlblt axpresslon of downstream g~nQs ln ~- Ylnelandll the nlfA sene ln the nlfL (KlXX) mutants MV376 and MV378 ls obvlously express~d. Thls could ar1se from unexp~cted promoter ac-tlvlty ln the cassette or from promoter-llke sequences generated by the KIXX lnsert10n ln th~ nlf~ r~glon.
Insert~on of KIXX ln the orlentatlon where expresslon of ~4h was ln the same dlrectlon as nlf~ resulted ln somewhat hlgher constltutlve levels of ~-galactosldase (MV380) than when lnserted ln the opposlte dlrectlon ~MV37~). However, lt was not posslble to construct a Nlf~ ~lfL2-KIXX derlYatlvQ ln the wlld-type ~qulvalent to MV380, whlch, as lt carrles a ntg~-lacZ fusion, 15 Nlf-. There~or~ excesslve expresslon ~f n.lfA dr1ven by th~ very strong ~4h promoter ls probably lethal ln a Nlf+ ~. v~nelandll ~ ' , ~ .'' .
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_ 12 -background, posslbly dua to dlverslon o~ too much ATP towards nltrogenase synthesls ~nd actlvlty or to NH4~ toxlclty.
EXAMPLE 6 : .En~yme assays Nltrogenase and ~-galactosldase actl~ltles were mea5ured as 05 descrlbed prevlously. (Sambr~ok qt ~1.. 198~, "Molecular Clonlng". Cold Sprlng Harbor Laboratory Press, Cold Sprlny Harbor, ~nd ~almslsy ~ 9l, Appl. Envlron. Mlçroblol., 57, 522-524).
The results of ~hese ~ssays are shown 1n Table l below:- :
IA~L~
Nltrogenase and ~-galactosldase act~vltles ~n wlld-type and nlf~ ~utants of ~. Ylnelandll Straln Genotype ~N +NH4 UWl3S wlld-type 4~ 0 ~V376 nlf~l :KIXX ~8 4~
MVl01 nlf~ acz l$~4lb 432 MV378 ~lf~ ~ ll9G7 377Z
n1~ KIXX
MV380 ~lf~ ç~ lS384 6474 ~lf~2:KI~
lO -N N free med1um, a Nltrogenase actlvl~y ln UWl36 and MV376 me~sured as acetylene reductl~n ~nmol ethylene produced ~ln~l ~mg proteln)~~).
b ~-galactosldase act~vlty ln MVlOl, MV378, ~V380: measured as Mlller unlts (26~.
Each value ls the mean from 2 - 3 lndependent determlnatlons;
varlatlon was < lOX ~rom ~he mean.
~RITI~H ~ECHNOLOBY ~Rnup TEL: 44-71-403-7586 ~.Sep.91 12:5g No.004 ~.14 2 ~ 7 1 The data ln Tabl~ 1 show that MV376 was Nlf~ and ~xpressed wlld-type acetylene reductlon actlYlty both ln N-free medlum and ln medlum wltl~ ammon~um at 15mM ~ concentratlon that repressed nltrogenase ~ctlvlty ln the ~lld-type straln UW136. Sl~llarly 05 both MV378 and MV3~0 expr~ssed ~-galactosldas~ actlvlty ln the absence or presenc~ of ammonlum whl~r~as actlv1ty ln thc nlfL~
straln M~101 was repressed by lSmM ammon3um.
Other actl~tles r~pressed by ammonlum ln ~. vlng]andll are those of nltrate reductase and nltrlte reductase. (LuquQ et al.
l9B7 Arch. Mlcroblol. 148 231-235) the ~xpresslon o~ whlch requlres both the n~r~ and rpoN gene products (Luque ~ al.
Supra Santero ~ al. 1986 3. Bacterlol. 1~6 541-544 and Toukdarlan et ~. 1986 EMBO J. ~ 39g-407) The KIXX
lnsertlon mutat~on had no eff~ct on normal re~ulatlon of n1trate reductase; thls actlv7ty was prssent ln N03~-~rown but not NH4~ N03~)-grown cultures of both ~lld-type UW136 and mutant MV376 Sdata not shown~.
Th~se rssults demonstrate that ~he gene upstr~am o~ n1fA ln ~. vlnel~dl~ medlates ammonlum represslon o~ nlf gene expresslon but not o~ at least one other gene under ammonlum control. Thls phenotype 1s slmllar to that of PlfL mutants o~ ~. pneumoni~
where nlfl ls locatQd l~medlately upstream o~ nlfA. translatlon of the entlra ~. ~lnelandll upstream gene sequentQ tapproxlmately 1.4kb) sho~s a prote1n wlth greater homology to ~. ~n~umorl~
nlfL than to NT~B (31X vs. Z4% ldentlty).
EXAMPLE 7 :. Ammonlum estl~a~lQn Culture supernatants of ~. ~lnelan~ll wlld-type straln UW136 and mutant straln MV376 grown on nltrogen werQ tested for thQ
present o~ ammonlum.
Samples of cultur~s WQr~ taken at dl~ferent tlmes and centrlfu3ed. 0.5ml of supernatant or flltrate was tested for the presence of ammonlum by the lndophenol me~l-od ~Bergersen 1980 Methods for evaluat1ng blologlcal nltrogen flxa~lon John Wlley & Sons Ltd. London). ~hls conslsted of the addltlon ln order of a) O.Sml phenol-~odlum nltroprussld~ solut10n (phenol 509 1~
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~RITI~ I TECHMOLOG~' GROUP TEL: 4'1-71-40~-7586 9.Sep.91 12:59 No.OO~l P.15 2 ~ 3 3 0 7 ~
~ sodlum n1troprusslde, 0.25g 1-1); b)O.5ml of sodlum hypochlorlte solut~on ~O.lm); and c~ 2ml of d~st~lled water. The mlxture was lncubated for 30 mln at room temperature. Absorbance at 625nm was measured and ammonlum conc~ntratlon estlmated from a 05 standard curve obtalned wlth ammonlum solu~lons at varlous concentratlons assayed uslny the same reagent solutlon.
The rssults are drawn ln Flgure 2, whereln ~ denotes U~136 and O denotes MY376.
In contrast to UW136, M~376 excreted ammonium rather suddenly towards the end of ~ponentla1 growth. F~nal amounts measured ln MV376 statlonary phasQ cultures wer~ 5 - 10 mM. Excretlon was not llmlted by carbon sourcQ avallablllty because half of the supplled sucrose remalned at lts cessatlon.
~AMPLE 8 : Constructlon of nlfL deletlon mut~t of ~- vln~
The plasmld pAB27 carrylng tha nlfL reglon was dlgsstQd wlth lII and llgated to a 3.8 Kb ~mHI fragment from pJ017 whlch carrlQs the gene cartrldge, sac ~Hynes ~ al., Gene, 198g 1~, 111-120. The ~3~ gene confers a sensltlvlty to sucrose when transferred to certaln gl~am negatlve bacterla, l.e. organlsms carrylng the sac 0ens cannot grow on sucroso-conta~n1ng medla.
The resul~lng plasmld, pLA5:Sac-Km, c~rrles the sac cartrldg~
whlch replaces a 585 base palr ~g~II fragment, dsletlng the S -end of the ~ene and 20 basQ palrs upstream from thQ
translatlon start ~odon. In order to transfer the nlpl:
mutatlon from pLA5:Sac-Km lnto the chromosome, the plasmld ~as transformed lnto ~. Ylnelandl1 U~135 ~nd kanamycln-reslstant colonles were selected. These were found to be unabl~ to ~row on sucrose-contalnlng med~um but could grow normally on ~lucose as carbon sourc8. One colonles was purlfled several tlmes an~ had stable s~crose-sensltlve, kanamycln-reslstant phenotype Thls mutant was MV399. MV399 had a Nlf mlnus phenotype because the ~c cartrldge lnsert prevented expresslon of the downstr~am nlf~
gene whlch ls essentlal for expresslon of n~f genes.
In order to replace the nl~.L~ mutatlon wlth a nlfL
deletlon mutatlon unlnterrupted by ~ DNA, pAB27 was d!~ested .
~RITT~H T~:CHI~IOLOGY GROUP TEL: 4~1-71-403-75~6 9.Sep.91 1~:59 No.004 P.16 ~51~71 wlth ~lI1 and ligated to ttself. Plasmld pLA4 was lsolated and found to contaln tha 585 base palr ~lII deletlon wlthtn the nlfL
gene. MY399 was transformed wlth pLA4 and colonles growln~ on sucrose-contalnlng am~onlum-free medlum were selected, These Nlf 05 plus colonles were also kanamycln-sensltlvo lndlcatlng that the Km cartrldge had been rep1aced. One such colony was purlfled and tested for ammonlum excretlon, Thls straln, MV440, was found to excrete up to lOmM ammonlum lnto the growth medlum and the pH
of cultures lncreased to about 8.5 ~hen no further ammonlum was excreted. Thus a deletlon o~ the ~'-end o~ the nlfL gene, whlch does not affect expresslon of the downstream nlfA gene, leads to a mutant stratn of ~. Y1neland~ wh~ch excretes large amounts of ammontum s~mllar to the phenotype o~ the nl~L:KIXX mutant MV376 descrlbed prevlously.
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Claims (22)
1. A mutant nitrogen fixing bacterium wherein nitrogenase activity is controlled by a nifA or nifA-like gene which is in turn regulated solely by a nifL or nifL-like gene characterised by the modification that it has a mutation in the nifL or nifL-like gene, but a functional nifA or nifA-like gene.
2. A bacterium according to Claim 1 which is of the genus Azotobacter.
3. A bacterium according to Claim 2 which is of the species Azotobacter vinelandil.
4. A bacterium according to Claim 3 which is the strain deposited at the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 31 August 1991 under the Accession number 40438 or a mutant or variant thereof having the same function of overporduction of ammonia.
5. A process for the production of a mutant bacterium according to Claim 1, 2, 3 or 4 comprising the speps of isolating the nifL
gene of a parent nitrogen-fixing bacterium wherein nitrogenase activity is controlled by a nifA or nifA-like gene which is in turn regulated solely by a nifL or nifL-like gene, mutating the nifL gene and replacing the nifL gene of a parent nitrogen-fixing bacterium by the mutated nifL or nifL-like gene, while preserving the functionality of the nifA or nifA-like gene.
gene of a parent nitrogen-fixing bacterium wherein nitrogenase activity is controlled by a nifA or nifA-like gene which is in turn regulated solely by a nifL or nifL-like gene, mutating the nifL gene and replacing the nifL gene of a parent nitrogen-fixing bacterium by the mutated nifL or nifL-like gene, while preserving the functionality of the nifA or nifA-like gene.
6. A process according to Claim 5 wherein a nifL gene from one bacterium, (recipient) is replaced by a mutant nifL gene from another (donor) bacterium.
7. A process according to Claim 6 wherein the donor and recipient bacteria are of the same genus of family.
8. A process according to Claim 7 wherein the donor and recipient bacteria are of the same species.
9. A process according to Claim 7 wherein the donor and recipient bacteria are Azotobacter.
10. A process according to Claim 8 wherein the donor and recipient bacteria are Azotobacter vinelandil.
11. A process according to Claim 8 wherein the donor and recipient bacteria are the Azotobacter vinelandii strain deposited at National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 30th August 1991 under the Accsssion number 4043a or a mutant or variant thereof having the same functions of growth and production of ammonia.
12. A method of providing plants with a source of fixed nitrogen which comprises introducing a mutant nitrogen fixing bacterium according to Claim 1, 2, 3 or 4 or produced by a method claimed in any one of Claims 5 to 11 into nitrogen fixing association with plant tissue.
13. A method according to Claim 12 wherein the mutant bacteria are introduced to the soil before or after planting.
14. A method according to Claim 12 wherein the mutant bacteria are introduced to the plant before or after planting.
15. A method according to Claim 12 wherein the mutant bacteria are introduced to the seed before or after planting.
16. A method according to Claim 12 wherein the bacteria are introduced as an aerosol or liquid.
17. A method according to Claim 12 wherein the bacteria are capable of binding to lectins produced by leguminous plants.
18. A method according to Claim 12 wherein the genes providing ability to bind to lactin are introduced into Azotobacteria vinelandil.
19. A method according to Clalm 18 whereln the genes are obtalned from Rhizobium trifolium and Rhizobium leguminosaum.
20. Plants wherein the source of fixed nitrogen is supplied by a method according to any of Claims 12-19.
21. A mutant nifL gene for insertion into a recipient bacterium by gene replacement.
22. A gene according to Claim 21 characterised in that it originates from the strain of A vinelandil deposited at the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, UK on 30th August 1991 under the accession number 40438 or a mutant or variant thereof having the same functions of growth and production of ammonia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2051071 CA2051071A1 (en) | 1991-09-10 | 1991-09-10 | Ammonia production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2051071 CA2051071A1 (en) | 1991-09-10 | 1991-09-10 | Ammonia production |
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| Publication Number | Publication Date |
|---|---|
| CA2051071A1 true CA2051071A1 (en) | 1993-03-11 |
Family
ID=4148339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2051071 Abandoned CA2051071A1 (en) | 1991-09-10 | 1991-09-10 | Ammonia production |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10384983B2 (en) | 2015-07-13 | 2019-08-20 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US10662432B2 (en) | 2011-06-16 | 2020-05-26 | The Regents Of The University Of California | Synthetic gene clusters |
| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11678668B2 (en) | 2018-06-27 | 2023-06-20 | Pivot Bio, Inc. | Agricultural compositions comprising remodeled nitrogen fixing microbes |
| US11946162B2 (en) | 2012-11-01 | 2024-04-02 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
-
1991
- 1991-09-10 CA CA 2051071 patent/CA2051071A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10662432B2 (en) | 2011-06-16 | 2020-05-26 | The Regents Of The University Of California | Synthetic gene clusters |
| US11946162B2 (en) | 2012-11-01 | 2024-04-02 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
| US10384983B2 (en) | 2015-07-13 | 2019-08-20 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US10556839B2 (en) | 2015-07-13 | 2020-02-11 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US10919814B2 (en) | 2015-07-13 | 2021-02-16 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US10934226B2 (en) | 2015-07-13 | 2021-03-02 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11739032B2 (en) | 2015-07-13 | 2023-08-29 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11479516B2 (en) | 2015-10-05 | 2022-10-25 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored NIF clusters |
| US11565979B2 (en) | 2017-01-12 | 2023-01-31 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| US11678668B2 (en) | 2018-06-27 | 2023-06-20 | Pivot Bio, Inc. | Agricultural compositions comprising remodeled nitrogen fixing microbes |
| US11678667B2 (en) | 2018-06-27 | 2023-06-20 | Pivot Bio, Inc. | Agricultural compositions comprising remodeled nitrogen fixing microbes |
| US11963530B2 (en) | 2018-06-27 | 2024-04-23 | Pivot Bio, Inc. | Agricultural compositions comprising remodeled nitrogen fixing microbes |
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