Note: Descriptions are shown in the official language in which they were submitted.
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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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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
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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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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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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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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
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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
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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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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
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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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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.
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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
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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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