INTRODUCTION

Biological nitrogen fixation, in which microorganisms reduce

dinitrogen ·to ammonia, is a very important process, since it is

the predominant natural source of nitrogen for agricultural crop

production. Due to diazotrophy's agronomic importance, one needs

basic knowledge of it at molecular and genetic level to solve the

present and future problems of nitrogen inputs effectively. One

of the major problems in the manipulation of nitrogen fixing genes

has been the inhibition of this process by oxygen in many of the

diazotrophs. However, Azotobacter vinelandii, a member of Azoto­

bacteriaceae family has a unique capacity to fix nitrogen in the

presence of oxygen. A. vinelandii which is widely distributed in

soil, is also known for its multiple copies of genome, 10-40 copies

per cell (Sadoff et ~ 1979). Recently it has also been proposed

that A. vinelandii possesses an "alternative pathway" (Bishop et

~ 1980) for reducing dinitrogen, which operates in the absence

of molybdenpm. Inspite of all these special features, very little

is known about its nif genetics, due to lack of genetic techniques.

However, lot of work has been carried out on the physiology of

nitrogen fixation and the structure and function of its nitrogenase

(Yates, 1974; Eady and Postgate, 1974; Mortenson et al., 1979;

Robson and Postgate, 1980; Eady, 1981). Development of recombinant

DNA techniques in recent years has made it possible to overcome

some of the technical barriers in the analysis of nif genetics in

A. vinelandii, It should now be possible to reveal the molecular

mechanisms involved in aerobic nitrogen fixation and the alternative

pathway.

- 2 -

The organization and expression of nitrogen fixation (nif)

genes has been thoroughly studied in Klebsiella pneumoniae, a

free living, microaerophilic diazotroph, due to its close relationship,

both physiolgically and genetically with Escherichia coli (Dixon,

1984; Cannon et ~ 1985). The cloned nif DNA fragments from

this organism has been used to identify and isolate nif genes from

other diazotrophic bacteria. The knowledge about nif genetics

in K. pneumoniae has been referred extensively in comparative

studies. Hence, some important features of nif genes in K. pneu­

moniae will be discussed.

Nif genetics of Klebsiella pneumoniae :

Nitrogenase enzyme complex which catalyzes the reduction

of dinitrogen to ammonia in diazotrphs, is composed of two proteins,

component I and component II. Component I (Mo-Fe protein or

dini trogenase) is a tetramer of two different polypeptides (c(l. p.J

and component II (Fe protein) is dimer of identical subunits. In

_!5_:_ pneumoniae component I has an average molecular weight (M W)

of 220,000 daltons and each subunit oC or p has a MW of 56,000

daltons. Whereas, the component II (Fe-S protein or dinitrogenase

reductase) has a MW of 66,000 and each subunit 34,000 daltons.

(Eady and Smith, 1979). Generally the reduced component II transfers

electrons to component I which is dependent upon MgA TP hydrolysis.

Component I in turn reduces the dinitrogen (Mortenson and Thornley,

I 979).

- 3 -

Initial genetic studies revealed that ni f genes were located

close to histidine (his) operon (Streicher ~ ~ 1971; Dixon

and postgate, 1971) on K. pneumoniae genome and that when

his operon and adjacent DNA were transferred by conjugation

into~ coli, the reciepient gained ability to fix nitrogen (Dixon

and Postgate, 1972). Using classical genetic techniques an

F' WN68) was constructed in~ coli (cannon ~ ~ 1976) carrying

Klebsiella his and ni f genes. Dixon ~ ~ (1976) constructed

a promiscuous his-ni f plasmid pRD1 (formerly called RP41)

derived by recombination between FN68 and the P-type drug

resistance (Kanamycin, tetracycline and carbenicilliit~ plasmid

RP4. Later when molecular cloning techniques were developed

pRD1 was used as source for cloning the K. pneumoniae genes.

A series plasmids carrying overlapping restriction DNA fragments

were constructed (Cannon ~ ~ 1977, 1979; Puhler and Klipp,

1981) which were used in the mapping of ni f and in the study

of various nif gene products. Genetic mapping of K. pneumoniae

ni f DNA so far revealed that atleast 17 genes are present

which are contiguous and clustered in a 23 Kb region close

to the his operon. The 17 ni f genes arranged (Fig.1) in the

order his ••• ni f QBALFMUSUXNEYKDHJ •••• ShiA and are organised

in 7 or 8 operons (Dixon, 1984). Most ·of the nif genes are

transcribed in the direction of his operon except nif F and

nif J which are transcribed in the opposite orientation. Table

lists the molecular weights and functions of various gene

products (Cannon et ~ 1985). ni f H codes for the

component II, ni f D and ni f K code for o( and J3 subunits

respectively of component I. These three genes and another gene

his n 1f

DG Q 8 A L F M V S UXN E y K D H J genetic map \ r I I II I I I , I I I R R R H RX X R R: H R X restriction ~I · pCRAIO

map

I I R R

pGR Ill pMC2 r-t R R

pGRII2 H X

R R pWK25

R R I

pGRII3 II· R R·

pSA30

pCRA37 R R

, pGRII9

R pCMI

R R R

------R R H H

K, obase po1rs

" 5 10 15 20 25 30 35 '-'

Fig. I. Genetic and physical map of K. pneumoniae his nif region

(Ausubel ~ ~. 1982).

Gene

Q

B

A

L

F

M

v s u X

N

E

y

K

D

H

J

TABLE I

nif gene products and their function in K. pneumoniae

(From Cannon et al., 1985)

Mol. wt. of product (xl03 daltons)

Unknown

49

57

45

19

28

42

45

25

18

50

40

24

60

56

35

120

Function

Mo uptake

FeMoCo Synthesis

Transcription activation

Transcription repression

Flavodoxin subunit

Kp2 processing

FeMoCo synthesis

Kpl processing

Kpl processing

Unknown

FeMoCo synthesis

FeMoCo synthesis

Unknown

Kpl ~ - subunit

Kpl ~- subunit

Kp2 subunit

Pyruva te-Fla vodoxin-oxido-reduc-

tase subunit.

- 4 -

nif Y (whose function is not known) are transcribed as a single

operon from a promoter located upstream of nif H. nif M is involved

in the processing of component II and the mutations in nif M produce

inactive component II. The nif B, nif V, nif S, nif U, nif N and

nif E are involved in the synthesis of Fe-Mo cofactor of component

I nif Q may be involved in the Mo uptake. Function of nif X is

not clearly known. nif F and nif J encode components of a specific

electron transport pathway to nitrogenase. The nif F product is

a flavodoxin whereas nif J product derives reducing power from

anaerobic metabolism as a pyruvate: flavodoxin oxidoreductase.

nif A gene product activates the transcription of all other nif

genes and nif L is involved in the repression of nitrogenase synthesis

in the presence of oxygen and ammonia. nif A and nif L are trans-

cribed as a single operon from a promoter located upstream of

nif L.

Unique feature of the nif genes IS the unusual primary struc­

ture of their promoters (Ow et al., 1983; Beynon et al., 1983). -- -- -- --

The nif promoters have an atypical consensus sequences 5'

-CTGGCACN; TTGCA-3' between positions -27 and -11 rather

than the characteristic sequence at -35 and -10 found in most

of the bacterial promoters (Rosenberg and Court, 1979; Hawley

and McClure, 1983).

Regulation of nif genes :

Nitrogenase synthesis was known to be repressed by fixed nitrogen

- 5 -

(Tubb and Postgate, 1973) and by the presence of Oxygen (Eady

et al., 1978). However, the exact mechanism of this control was

elucidated only after the development of lac fusion techniques.

where the (d -galactosidase gene (without its promoter) is cloned

downstream of the regulatory elements of the gene of interest

Expression of these regulatory elements is monitored by assaying

p -galactosidase activity. Most of the results discussed below have

been obtained using this powerful technique (Casadaban et ~

1980).

The nif genes are regulated at two levels. The first level

involves a centralized nitrogen regulation system (ntr system)

mediated by the products of ntr A (~F), ntr B ~L) and ntrC

(~G). The second level is specific control of nif operon mediated

by the products of nifL and nifA .

The nitrogen assimilation is controlled by ntr system in all

the enteric bacteria (Magasanik, 1982). The ntr genes of K. pneu-

moniae have also been cloned and found to have organization similar

to that of ..£:.._ coli (Espin et ~ 1982; de Bruiin and Ausubel, 1983;

Merrick and Stwart, 1985). The ntr B and ntrC genes are linked

to R!..!:!A (structural gene for glutamine synthetase) and form a

single operon, which is transcribed from ntr B to ntrC either from

their own promoter or by readthrough transcription from the stronger

~A promoter. ntr B o.na ntr C gene products of K. pneumoniae

have a MW of 31,000 daltons and 54,000 daltons respectively (Espin

- 6 -

et al., 1982). ntr A gene is not linked to ~A ntr BC and its gene

product has a MW of 75,000 daltons (de Brujijn and Ausubel, 1983).

The expression of ntr A is independent of nitrogen status of the

cell (Merrick and Stewart, 1985).

Fig. 2 shows the current model of how ntr genes control

the transcription of nif genes (Cannon ~ al., 1985). At low levels

(4- mM) expression of ~A/ntr BC is greatly enhanced

from the ntr promoter preceeding ~A, due to autogenous activation

by ntrC gene product in concert with ntr A. ntrC protein activates

the transcription from nifA promoter and other operons like hut

(histidine utilization), ~(proline utilization) aut (arginine utilization).

The nifA gene product in turn activates the transcription from

all other ni f operons and again ntr A is necessary for this activation,

while nifL product is maintained in an inactive form (Buchanan­

Wollaston et ~ 1981). In the presence of high levels of NH4(>

20 mM), ntrC in conjunction with ntrB product represse s the trans­

cription of nifLA promoter (Drummond et al, 1983). This regulation

is not affected by oxygen.

The second level of regulation, which occurs at intermediate

levels of NHl) 4- mM) and at low levels of dissolved oxygen(>o.l

uM) involves nifL product, where repressor form of nifL is predo­

minant and most likely inactivates the nifA protein.

The role of nifA was clarified by constructing plasmids which

Act1ve---~

1nact1ve

nlfl ndA c:=J

t nl r C c===J

t Inactive

ntrB ginA r==J

N

_. ----- P3-----P2-PI

!\ Low N

All other nd operons

nfr and nJf promoters

t j Mod 1 11 e d R N A-p-o -1 y-fTl-e -r o-s-e~j

t S1gmo subunll

t n IrA

Fig. 2 Regulation of nif genes (Cannon ~ ~. 198 5).

- 7 -

express this gene constitutively (Buchanan ~ ~ 1981 ), when

these plasm ids were present, ni f expression was not subjected

to repression by NH4 or 02

and it was also independent of

mutations in ntr genes. It was also found that less amount

of nifA was enough for the activation. Thus showing, ntr genes

activated ni f expression which is inhibited in repressing condi­

tions (+NH4

or o2

) bythe interaction of nifA with nifL. These

experiments also demonstrated that lack of nitrogenase activity

in vivo at 37°C was due to the inactivation of nifA.

The nifLA operon is also subjected to autoregulation

by ni fA gene product. In concert with ntrA gene product,ni fA

protein can substitute for the ntrC gene product and activate

the operons of other nitrogen metabolism genes, .9!!:!_A, hut,

~ and cut (Ow and Ausubel, 1983; Drummond ~ ~ 1983).

However, ntrC, ntrA cannot activate nifH promoterofKlebsiella.

This led to the proposal that ntrA could act as an alternate

sigma factor for RNA polymerase (Beynon ~ ~ 1983; deBruijn

and Ausbel, 1983). Since the nif promoters have an atypical

structure, modification of RNA polymerase might be necessary

to allow transcription at these promoters. Merrick and Gibbins

(1985) sequenced the ntrA gene and identified some sequences

which are similar to those found in known site specific DNA

binding domains present in five sigma factors from I:_ coli

and B. subtils and may be involved in the recognition of -35

and -10 promoter sequences (-27 and -11 incaseofnifpromoters).

- 8 -

It has also been shown that nucleotides -72 to -184 in K. pneu­

moniae ni fH promoter is necessary for the activation by ni fA

(Buck et ~ 1985). Deletion of these sequences relieves the

inhibitory effect on nif gene expression by multiple copies

of nifH, nifU and nifB promoters and it can also be activated

weakly by ntrC (Buck ~ ~ 1986). This upstream sequence

seems to be a specialisation among certain ni f promoters,

because no such effect was found in case of ni fl. promoter,

thus permitting the efficient activation byregulatory proteins

functionally homologous to ni fA.

Azotobacter vinelandii

Nitrogenase complex of A. vinelandii is also composed

of two proteins, component I and component II (Bulen and

Leconte, 1966). Component has an average MW of 245,000

daltons, containing 2 atoms of Mo per molecule. The two sub­

units and have an average MW of 61 ,ODD daltons each (Swisher

~ ~ 1977; Shah and Brill, 1977). Molybednum was found asso­

ciated with component I also as a co factor, FeMoCo (Nagatani

et ~ 1974) which contained Fe, S and Mo in the ratio 8:

6: 1 (Shah and Brill, 1977). Component, II (MW 60,000) is a

dimer formed of two identical subunits of 31,200 daltons each

containing 289 aminoacids (Hausinger and Howard, 1980).

Nitrogenase complex of A. vinelandii J..!:!. vivo or J..!:!. vitro crude

extracts is more resistant to oxygen (Bullen ~ ~ 1964) than

that of other diazotrophs. Different mechanisms have been

- 9 -

proposed to explain the oxygen tolerance of A. vinelandii

nitrogenase. The more widely accepted one has been respiratory

protection supported by conformational protection (Postgate

et §.1 1981 ). During aerobic growth, nitrogenase is protected

by high rate of respiration, which scavenges the excess oxygen.

When 0 2 concentration is strong enough to overcome the rate

of respiration, nitrogenase forms a complex with a protective

protein and in this conformation nitrogenase is inactive but

remains undamaged. When the oxygen concentration is lowered,

the complex dissociates the nitrogenase becomes active.

Shethana ~ ~ (1966) isolated an FeS protein also called 'Shethna

protein' (Fe-S proteinii) of MW 24,000 daltons, which in its

oxidized form binds to nitrogenase and might act as the protec­

tive agent during the conformational protection (Scherings,

1977).

Another important physiological factor which i fluences

the nitrogen fixation is fixed ammonia. In A. vinelandii ammonia

or any easily assimilated nitrogen source represses the synthesis

of nitrogenase enzyme (Shah et §.1 1972). However, the extent

of inhibition of nitrogenase activity was found to be variable,

anywhere between 30-100% inhibition, depending on growth

conditions (Eady, 1981 ). Klugkist and Haaker (1984) explained

these variable results. Inhibition of nitrogenase activity by

NH4

CI is dependent upon : [a] concentration of dissolved

02

in the medium: under extreme concentrations of oxygen

(very low or very high) nitrogenase activity is low and inhibition

by NH4

CJ is very strong. Whereas, at optimal concentrations

- 10 -

of oxygen, nitrogenase activity was maximum and inhibition

by NH4

CJ is small. [b] pH of the medium: inhibition by NH4

C I

was very strong at low pH values. [c] stage of growth: at

the end of growth, nitrogenase activity is more strongly inhibited

than at an early phase of growth.

Genome Organization :

Genetic studies of A. vinelandii was hampered due to

lack of auxotrophic mutants. Though nif mutants could be

isolated at high frequency, it was difficult to isolate auxotrophic

mutants. Very few auxotrophic mutants were isolated,adenine

(Mishra and Wyss, 1969), purine and pyrimidine auxotrophs(Page

and Sadoff, 1976) and mutants lacing sulfate reductase. The

difficulty in the isolation of mutants was attributed to genetic

redundancy in the form of an unusually high chromosome copy

number, 10-40 copies per cell (Sadoff, 1979).

Sadoff et al

A. vine Iandi i ce lis

(1971) found that exponentially growing

14 contain as much as 15x1 0 g DNA per

cell which is 40 times more than present in an~ coli cell,where-

asin cyst form, the amount of DNA was 10 times more than

that of an ~ coli cell. Studies with purified chromosomal DNA

(Sadoff ~ ~ 1979) showed that average G+C content was

65%. The denaturation kinetics follow second order kinetics in the

first 60 min. Cot 1/2value is similar to that of E.coli DNA. Even

the average sedimentation coefficient (1600 svedberg units) was

similar to that of E.coli chromosome(1300 svedberg units). These

data suggested that A. vinelandii genome might exist in 40 copies per

cell in mid-exponential phase where as it excists in 10 copies per

- 11 -

cell in cyst form (Sadoff et ~ 1979). Later Terzaghi (1980) found

that the rate of survival of UV irradiated A. vinelandii cells was

similar to that of ..£:._ coli cells, suggesting that all the copies may

not be biologically functional.

Genetics of nif in Azotobacter vinelandii

The niC mutants isolated in earlier stages of work (Wyss

and Wyss, 1950; Green et ~ 1953) could not be characterized

due to Jack of purified nitrogenase proteins and efficient transfor­

mation system. Later several stable nif mutants were obtained

after mutagenesis with nitrosoguanidine and enrichment with peni­

cillin (Shah et ~ 1973). These mutants have been well characterized

by titrating the extracts of niC strains with purified components

of nitrogenase, determining the amount of antigenic cross reactive

material for each component in the extract, locating the nitrogenase

components on polyacrylamide gels by an iron staining (Brill et

~ 1974) tecpnique and by electron paramagnetic resonance spectros­

copy of whole cells, which indicates the presence or absence of

a signal (g = 3.65) unique to component I. Table 2 shows the charac­

teristics of each mutant strain (Shah et ~ 1973; Brill et ~ 1974).

Mutants UW 1 and UW2 do not synthesize component I and component

II, therefore, mutation may be regulatory in nature. Mutants UW3

and UWlO make inactive component I, whereas in strains UW6

and UW38 component I was not detected. UW91 makes inactive

component II. In UW45 component I is inactive but it can be activa­

ted by the addition of FeMoCo to the crude extracts (Nagatani

Strain

Wild type

UWl

UW2

UW3

UW6

UWlO

UW38

UW45

UW91

TABLE 2

nif- mutants of A. vinelandii (Bishop et ~ 1977)

Activity

I+II+

I II

I II

I II

(II+

(II+

(II++

I+II+

1+1(

CRM

I+II+

(II-,

I II

I+I(

(II+

I+II+

(II+

I+II+

I+II+

Derepreseed activity

+

- 12 -

et ~ 1974) Later it was found that UW38, which lacks component

I activity makes more than 5-8 fold component II than the wild

type (Shah ~ ~ 1974). nif+ revertants of this strain synthesized

equivalent amounts of both the components, suggesting that the

releaxed control of component II and the nif- phenotype were caused

by a single mutation. Using strain UW2 some mutants derepressed

for NH 4 were obtained, which make 1 o5 fold more nitrogenase

in the presence of NH4 than the wild type (Gordon and Brill, 1972).

Using a transformation procedure described by Page and

Sadoff (1976), some of the nif mutations were mapped (Bishp and

Brill, 1977; Bishop et ~ 1977) and figure 3 shows the genetic

map obtained. Mutations nif-1 and nif-2 are tightly linked and

may be located in the same gene. Mutation nif-45 was closer to

nif-1 and nif-2 than to any other mutations. Mutations nif-6 and

nif-38 appeared to be tightly linked to each other but they can

be separated by recombination. !\.4wte.tigr:~s .!:!.!!_ 91 ~ .!:!.!!_ l 0 ~

~ lecaliz.eEI +H ~ ~ rec9FR9ir:~e.tier:~. Mutations nif-91 and nif-10

may be localized in the same region. From these results they con­

cluded that nif genes do not fall into one cluster as in Klebsiella

pneumoniae and may be spread over a large region of A. vinelandii

genome. It may be pointed out that crude chromosomal DNA was

used for transformation in these experiments and the linkage observ­

ed between two mutations could as well be due to the large size

of the transforming DNA.

Phenotype 1-11---;----+----------~+---~----~--+--

Mutation nif-1 nif-45 nif-6 nif-10 nd-91 nif-3 n if -2 nif-38

Fig. 3. Genetic map of niC mutants.

- 13 -

However, in the past few years some progress has been made

in understanding the organization and expression of A. vinelandii

nif genes. This was largely due to the availability of a good trans-

formation system in A. vinelandii and also the use of molecular

cloning methods.

Transformation system on plates of BNF agar, described

by Page and Sadoff (1976) was modified to allow competence indue-

tion in liquid medium, where competence was induced by growing

A. vinelandii in iron limited BNF medium (Page and vonTigerstrom,

1978). The cells wee readily transformed by crude lysate or purified

DNA (Page and vonTigerstrom, 1979) achieving transformation

-3 -2 frequencies of 10 to 10 . Competence development was found

optimal at pH 7.2 to 7.4- and required restricted aeration of the

culture. It was also found that nitrogen fixing cell were less compe-

tent than those grown with fixed nitrogen because of the increased

demands for reducing power by respiration and nitrogenase (Page,

1982). This transformation procedure was further refined so that

plasmid DNA can be used resulting in transformation at a frequency

4- -2 of 3 x 10- to 5 x 10 depending on the plasm ids used (Glick

et ~ 1985).

Most of the information available to date on A. vinelandii

was obtained using the cloned K. pneumoniae nif DNA fragments.

Initially, Ruvkun and Ausubel (1980) showed that nif structural

genes (nifHDK) of K. pneumoniae hybridized to chromosomal DNA

- 14 -

of many nitrogen fixing bacteria, including A. vine1andii. K. pneu­

moniae nifHDK fragment hybridized to 5 fragments of 12.5, 8.1,

3.5, 2.2 and 0.8 Kb in size, with EcoRI digested A. vinelandii (strain

UW 1 0) chromosomal DNA. This indicated that nif genes in A.

vinelandii might be reiterated or they are dispersed on the chromo­

some. However, Krol et al. (1982) analyzed nif transcrips by hybridi­

zing RNA blots of A. vinelandii with K. pneumoniae nifHDK probe

and demonstrated that nif structural genes in A. vinelandii are

transcribed as a single operon, suggesting that at1east nif structural

genes are clustered. Medhora et al (1983) reported the construction

of a genomic library of A. vinelandii in a cosmid, pHC79, from

which four recombinant cosmids, pMP1, pMP2, pMP3 and pMP4

having sequence homology with K. pneumoniae nifHDK DNA were

isolated. When Bglll digests of these cosmids were probed with

Klebsiella nifHDK fragment, a 22.5 Kb fragment in pMP 1, a 4.3

Kb fragment in pMP2 and pMP3 and a 6 Kb fragment in pMP4

showed hybridization. It was also argued that nif sequence in pMP 1

may exist on a different Bglll restriction fragment of A. vinelandii

chromosome as compared with nif sequences that have been cloned

in pMP2 or pMP3. Therefore, this organism contains more than

one copy of nif sequences.

When pMP1 ~nd pMP2 were used for the marker rescue test

by transformation of nif- mutant UW6 ((II+), pMP2 transformed

UW6 to ampr, nif+ phenotype. Whereas, pMP1 failed to transform

it to nif+ phenotype, though ampr transformants were obtained

- 15 -

(Medhora, 1984 ). Bishop et al. (1985) reported the construction

of a gene library of A. vinelandii in the EcoRI site of pBR325

and isolation of recombinant plasmids pLBl and pLB3, which hybri­

dize with nifHDK probe of K. pneumoniae. pLBl and pLB3 contained

2.6 and 1.4 Kb EcoRI fragments respectively. Marker rescue tests

by genetic transformation indicated that pLB1 contained wild type

allele for nif-6 and nif-38 mutations carried by nif strains UW6

and UW38 and pLB3 contained wild type allele for nif-1 0 (UW 10

strain) mutation, indicating that these fragments contained DNA

sequences for atleast part of the nifK (2.6 Kb fragment) and nifD

(1.4 Kb fragment) genes. The authors have also demonstrated that

these two fragments are contiguous on A. vinelandii genome.

Recently, Brigle ~ ~ (1985) reported the isolation of 6

Kb Smal fragment, which carries nifHDK genes. This fragment

was sequenced and the sequence of nif H, D and K genes was

compared with that of other nitrogen fixing bacteria. Sequence

analysis revealed that, the region preceeding nifH contains sequences

of striking homology with the K. pneumoniae nif H promoter (Beynon

et ~ 1983). Since this homology was contained within the consensus

nif promoter region, it might contain nifH promoter of~ vinelandii.

nifH and nifD are separated by 129 bp and 101 bp separate nifD

and nifK. There was no consensus nif promoter sequence in these

intercistronic regions indicating that all the three genes are trans­

cribed from the nifH promoter. nifH and nifD sequences end with

tandem stop condons, which along with secondary structures found

- 16 -

in intercistronic region might play regulatory role in differential

expression of the individual gene products.

The search for the nif regulatory genes in A. vinelandii

was carried out by inducing various cloned regulatory elements

of nif genes of K. pneumoniae which were fused to ~·-galactosidase

gene.

When nifA gene of Klebsiella cloned into broad host range

plasmid pKT230, was introduced into A. vinelandii nif mutant

UW 1 ((I(), it could activate the nif genes of A. vinelandii, indicating

the presence of an activator gene, analogous to K. pneumoniae

nifA (Kennedy and Robson, 1983). Drummond and Kennedy (1985)

introduced plasmids carrying nif genes of Klebsiella fused to lacZ

gene into A. vinelandii strains UW (wild type), UW 1 and UW2 (both

(II-). Results of this experiment showed that ntrC and nifL genes

of Klebsie:la had no effect on nif expression in A. vinelandii. Whereas

nifA carried on a low copy number plasmid corrected these mu­

tations, Klebsiella nifL-lacZ and nifF-lacZ fusion:; expressed very

strongly and were not repressed by ammonium. However, nifH­

lacZ fusion failed to express even in the wild type strain (UW)

under any conditions, except when Klebsiella nifA was also present,

that too weakly. Based on these findings, it was suggested that

nif regulatory mechanism in Azotobacter includes an ntrC like

activator, but the nif specific regulation is less like that of ..!5.:_

pneumoniae. The differences were attributed to the different

- 17 -

physiological conditions under which they fix nitrogen which may

impose certain constraints on the regulatory systems involved.

Alternative pathway for nitrogen fixation :

Almost all the nitrogen fixing bacteria require Mo for the

nitrogenase activity, which was rationalized by the isolation of

an essential FeMoCo (Shah and Brill, 1977). This protein, is proposed

to contain at least part of the active site of component I and

may be involved in N2 binding (Hawkes et al., 1984). Molybdenum

is also involved m the regulation of nitrogenase synthesis (Eady

et ~ 1982). However, several evidences show that in A. vinelandii

there may be another enzyme complex which reduces nitrogen

in the absence of molybdenum. Initial evidence came when some

Nif+ pseudorevertants of nif- strains (UW6, UW 1 0) of A. vinelandii,

which could fix nitrogen in the presence of 1 mM tungsten (W) wu·c. isol<a.~ (Bishop et ¥1 1980). Characterization of these pseudorevertants

revealed that two new ammonia repressible proteins were synthesized

and the conventional nitrogenase proteins were not made. When

wild type A. vinelandii of nif parental strain of above mentioned

pseudorevertants were grown in broth with no added Mo, they

also showed the presence of the two new proteins (Bishop et ~

1982). Based on these evidences, Bishop et al (1980, 1982) proposed

that there is an "alternative pathway" and it is Mo independent.

Another group isolated a resistant mutant (WD2) of wild type A.

vinelandii (UW), which can grow in Burk's nitrogen free medium

- 18 -

containing 10 mM W, whereas, N2 independent growth of wild

type is inhibited by 0.01 mM W (Riddle et ~ 1982). WD2 strain

exhibited 22% of whole cell acetylene reduction activity of the

wild type in broth containing Mo and had lowered affinity for

acetylene. Two dimensional gel electrophoresis and ESR analysis

of cell extracts showed that instead of native nitrogenase proteins,

some new ammonia repressible proteins were formed. Premakumar

et ~ (1984) provided further evidence by demonstrating the presence

of an alternative reductase (component II) in the extracts of nif

mutants U W 1, UW3 and UW9 grown under Mo deficient conditions.

They also showed that "alternative reductase" is repressed by Mo

and W, whereas Vanadium (V) stimulated the synthesis.

These studies, coupled with the hybridisation of multiple

fragments with K. pneumoniae nifHDK fragment (Ruvkun and

Ausubel, 1980; Medhora et ~ , 1983) lend support to the existence

of an "alternative pathway" for nitrogen fixation in A. vinelandii.

Transposon Tn5 mutagenesis :

Transposons, the mobile genetic elements, have been very

useful in the mapping of genes in bacteria. Among them, transposon

Tn5 has been used extensively for mutagenesis. Tn5 is 5700 bp

in size, with two inverted repeats of 1535 bp. The central 2700

bp region confers Kanamycin or neomycin and streptomycin resis­

tance (Fig.4). The inverted repeats contain genes necessary for

Tn 5

r r r Km I Nm Sm )

IS50 L IS50 R

I I )I I

I I 1 X p H B p Sm PS X Bm B H p X

II) 0 II) II) 00 <0 M"~t <0 0 0 0 II) 0 00 00 Ol .... N .... 11)00 N ...... <0 00 Ol Ol 'It <0 .... II) ...... II) <0<0 00 0 N II) 0 N .... .... .... N NN N M 'It 'It II) II)

Fig. 4. Restriction map of Tn5.

Bg- Bglll; H-Hindlll; Bm- BamHI; R- EcoRI; P- Pstl;

Sm- Smal.

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transposition (Berg and Berg, 1983). Since the transposon Tn5 does

not have any insertional specificity and the resultant mutants

are very stable and exert strong polarity effects on the downstream

genes in a polycistronic operon, it was the most preferred transposon

(Lupski and deBruijon, 1983).

In general two types of techniques have been used to create

Tn5 mutagenesis :

1. Generalized mutagenesis, where genomic DNA is mutagenized

randomly by introducing a suicide plasmid or phage carrying Tn5.

Selection for Kanamycin or neomycin results in the detection of

transposition of the Tn5 from the suicide vector into the gencrre

of the host and concamitant loss of the vector. In.£:.. coli, defective

)\-phage ( )\ 467) was used as vector. Among suicide plasmids

pSUP2021, which contains ColEI replicon, mob region from RP4

plasmid and tn5 (Simon et ~ 1983) was very useful for mutagenizing

non-enteric bacteria, where colEI replicon was not stable.

2. Site directed mutagenesis : Tn5 was used initially to cons­

truct a physical map of the K. pneumoniae nif cluster (Riedel

et ~ 1979). Later Ruvkum and Ausubel (1981) developed a method

for site directed mutagenesis of Rhizobium genome, which is also

called marker exchange. In this technique cloned DNA fragment

in a plasmid was mutagenized with Tn5 and the mutated plasmid

was introduced into the wild type bacterium. Another plasmid

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which was incompatible with this plasmid was introduced and selec­

tion was made for the incoming plasmid and Tn5. This resulted

in the transfer of Tn5 insertion from the plasmid to genome by

homologous recombination and simultaneous loss of the vector.

Position of Tn5 on chromosome is confirmed by hybridizing blots

of restriction enzyme digested genomic DNA with 32P-Iabelled

ins~. Using this technique, they mapped the symbiotic genes of

Rhizobium melioloti (Ruvkun et ~ 1982). Later, this method of

site directed mutagenesis was used extensively to map the nif

genes in many nitrogen fixing bacteria.

Aim of the present work is to subclone the DNA fragments

of cosmids isolated from A. vinelandii gene library and have se­

quences homologous to K. pneumoniae nifHDKY DNA (Medhora

et ~ 1983) in mobilizable broad host range plasmids (Ditta et

~ 1980, 1985), to identify the nif genes present on these fragements

by complementation of various nif mutants of A. vinelandii and

to map the nif genes using transposon Tn5 mutagenesis (Ditta,

1985). The aim is also to find whether nif genes in A. vinelandii

are present in multiple copies, if so, whether they are functional.

These studies might provide some insight into the organization

and expression of nif genes in A. vinelandii.