1. INTRODUCTION

INTRODUCTION

Nitrogen is an essential bioelement. Gaseous nitrogen is metabolically

unavailable to living organisms. Nitrogen has to be converted into ammonia for

consumption by plants and microorganisms through the process of biological

nitrogen fixation. Biological nitrogen fixation (BNF) is carried out by a number of

organisms called diazotrophs, belonging to the group of prokaryotes, including

obligate and facultative anaerobes, photosynthetic bacteria, some blue-greel). algae,

actinomycetes and some members of archea (Postgate, 1982).

BNF was first analyzed in the free-living, diazotrophic and facultative

anaerobic bacterium Klebsiella pneumoniae, a closely related organism to

Escherichia coil. This organism possesses a special set of genes called nitrogen

fixation genes or nif genes. Detailed genetic and biochemical analysis identified

about 20 nif genes arranged in 7 or 8 operons (Arnold et al., 1988). One of the nif

operons, nifLA, encodes for two proteins, NifA protein which is a positive

regulator of all other nif operons and NifL protein which antagonizes the action of

NifA in the presence of oxygen and fixed nitrogen (Ausubel, 1984).

Studies using the mutants and the inhibitors of the enzyme DNA gyrase,

indicate that the transcription of nifLA operon is influenced by gyrase-mediated

DNA supercoiling (Kranz and Haselkom. 1985; Dixon et al., 1988; Dimiri and

Das; 1988). DNA supercoiling influences the major cellular activities of DNA like

replication, recombination and transcription. DNA supercoiling has emerged as the

global regulator of many genes (Pruss and Drlica, 1989). The supercoiling of DNA

is catalyzed by the enzyme DNA gyrase discovered in 1976 (Gellert et al., 1976)

and is controlled homeostatically by other cellular topoisomerases. A brief account

of DNA supercoiling and its role on gene transcription has been given below.

1.1 DNA SUPERCOILING

Vinograd and his co-workers (1965) defined supercoiling as the twisting of

DNA upon itself or the spatial coiling of circular duplex DNA. Further studies

have elucidated that DNA supercoiling is an intrinsic property of circular DNA

extracted from natural sources and also of linear DNA molecules that lack ends

and are incapable of free rotation (Worcel and Burgi, 1972; Wang, 1980).

(a) Linking Number

DNA Supercoiling can be best explained in terms of linking number (Lk)

which can be defined as the number of times one helical strand goes around the

other in a double-stranded DNA ring in which both strands are continuous

(Vinograd et al., 1968; Cozzarelli et a/., 1990). Linking number is the sum of two

geometric properties of DNA, the twist (Tw) and the writhe (Wr);

Lk=Tw+Wr

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The twist is the number of times one strand rotates around the helix axis,

which corresponds to 10.5 bp per unit twist for a B-DNA in solution and the writhe

is a measure of global tertiary structure of the molecule or spatial contortion of

helix axis in space. Writhe characterizes the superhelicity or the supercoiling of the

DNA molecule.

Unwinding or overwinding of the duplex DNA can be accommodated by

decrease or increase in the twist value, but as the duplex DNA tends to maintain its

B-form, the values of Tw is always maintained in a narrow range. So the changes

in the linking number arising out of overwinding or underwinding can be

compensated by changes in the writhe, represented as

~LK=~Tw+~Wr

Since ~ Tw ~ 0,

~Lk~~Wr

The linking difference (~Lk) is the linking number difference between the

supercoiled (Lk) and the relaxed (Lko) forms of the same DNA molecule, given as:

~Lk=Lk-L~

Since Wr of relaxed form of DNA is equal to zero, Wr of supercoiled DNA

is equal to ~Lk. (Figure 1.1)

3

7

9

t:.Lk = -1

1

Wr= -1 Negatively supercoiled

8

2 6

t:.Lk = 0

1

Wr= 0 Relaxed

9

7

t:. Lk ~ +1

1

Wr= +1 Positively supercoi.led

4

Fig l.l. DNA Supercoiling parameters. The bar at the top of the figure represents a linear piece of DNA with 10 helical turns. By unwinding the linear molecule by one tum (-1) and ligating, the circular product on the left is generated: This molecule has a linking deficit of 1 i.e. llLk = -1. By overwinding the linear molecule by one helical tum and then ligating, the product on the right is generated. This has an enhanced linking value of 1, i.e. llLk = +I. By ligating the linear molecule without underwinding or overwinding, the relaxed molecule in the centre is generated (llLk = 0). The underwound molecule on the left can adopt a negatively supercoiled form, as shown at the bottom of the figure. Here, the change in the linking number has been converted to a change in writhe (Wr = -1 ). The overwound circular molecule on the right can adopt a positively supercoiled conformation in which the excess in linking number is converted to writhe (Wr = l ). The relaxed molecule in the centre remains in its thermodynamically most favourable form, with ~=~~ . .

(From, Dorman, 1994)

Another formulation of linking number takes into account of the fact that

the axis of DNA does not necessarily lie in a plane, for example, when it is

wrapped around a protein (White eta!., 1988). Here the linking number of surface­

wrapped DNA is expressed as:

Lk = SLk + <D

· Where SLk is the surface linking number, which takes account of the

effects of the surface configuration on twist and writhe, and <D is the winding

number, which is a function of the helical repeat relative to the surface on which

the axis lies. Both SLk and <D are integers, while Tw and Wr are not.

The DNA molecules having the same length but differing in linking

numbers are called topoisomers. The linking number can only be altered by strand

breakage, passage and reunion, a task carried out by cellular topoisomerases. The

linking number difference (~Lk) can be used to calculate the free energy

associated with a supercoiled DNA molecule as:

~G = [1100 RT/N] I ~Lk2

Where R is the gas constant and T is the temperature in Kelvin and N is the

number of base pairs in the DNA molecules.

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(b) Specific Linking Difference

The ratio of ~Lk /Lko gives cr, the superhelical density or specific linking

difference which is independent of DNA length and can be used to compare

superhelical densities of different DNA molecules. The DNA is said to be

positively supercoiled if cr value is positive, relaxed if cr value is zero and

negatively supercoiled if cr value is negative.

The superhelical density has been determined for DNA molecules of

various natural sources. In mesophiles either Bacteria or Eukarya, DNA IS

negatively supercoiled with cr values measured in vitro ranging from -0.05 to -

0.07, equivalent to one cycle of unwinding in every 17 tum (Wang, 1987). In

eukaryotes, the negative supercoiling is achieved by the wrapping of DNA around

. histone core and subsequent relaxation of intranucleosomal tension by

topoisomerases (Saavendra and Huberman, 1986). In bacteria negative

supercoiling is partially constrained in vivo by nucleoid architectural proteins like

histone-like proteins and as a consequence intracellular superhelical tension is

always reduced and often found with cr values in the range of -0.025 to -0.03

(Lukomski and Wells, 1994). Plasmid DNA isolated from the thermophilic

bacteria growing at 80°C was found to be negatively supercoiled with cr values -

0.059 (Charbonnier and Forterre, 1994).

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The degree of supercoiling has also been estimated in the third domain of

life, Archea. Halophilic archeal plasmids are highly negatively supercoiled

(Charbonnier and Forterre, 1994). In hyperthermophilic archea, growing at above

80°C, DNA is either relaxed or positively supercoiled (Lopez-Garcia and Forterre,

1997). The plasmids isolated from the hyperthermophilic archea Sulfolobus sp.

have been shown to have specific linking difference in the range of+ 0.006 to +

0.017 at 80°C.

(c) Shape & Size of Supercoiled DNA

The negatively supercoiled DNA of bacteria has been shown to exist in two

basic forms, solenoidal and plectonemic (Cozzarelli et al., 1990). The latter form is

the most energetically favoured form of negatively supercoiled DNA (Figure 1.2).

, Experimental data concerning the shape of supercoiled DNA in solution are not

free of ambiguity. The results of small-angle X-ray scattering are consistent with

the toroidal shape (solenoidal) but not with an interwound (plectonemic) shape

(Brandy et al., 1976). Dynamic light scattering and neutron diffraction studies

carried out in liquid crystalline solution favour interwound superhelical structure

(Torbet, 1989). The electron microscopic observations of a small negatively

supercoiled plasmid is in the plectonemically interwound form and about 40% of

plasmid DNA is estimated to be plectonemal in vivo (Boles et al., 1990).

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(a)

Fig 1.2 (a) Electron micrographs of negatively supercoiled, interwound DNA as prepared in pure form from E. coli bacteria. Each DNA plasmid or ring is 7000 base-pairs long, and has a mean Lk ,; -40. Courtesy of Christian Boles, Nickolas Cozzarelli and James White; and from Journal of Molecular Biology ( 1990) 213, 931-51. (b) Branched path of the interwound DNA shown in (a), in schematic form: Such branching has little effect on the parameters, Lk, Tw andWr.

Eventhough most electron microscopic works displayed images consistent

with plectonemic model, the changes in the DNA shape due to DNA absorption

and drying on the supporting film were not excluded. A drain et a!. ( 1990) observed

the interwound form of protein-free negatively supercoiled DNA by cryoelectron

microscopy of vitrified specimens. The authors showed that the shape of

supercoiled DNA molecules was strongly affected by Mg2+ ions; in 10 mM Tris

alone the diameter ofpUC18 molecules was about 12 nm, while it was reduced to

4 nm in 10 mM MgC12• Approximate values of partition of the linking difference

(~Lk) between ~Tw and ~wr was estimated to be 1:3 and 1:4. Recently,

Lyubchenko and Shylyakh~enko (1997) visualized supercoiled DNA with atomic

force microscopy (AFM) in situ. The DNA molecules are observed to be loosely

interwound supercoils with an irregular shape. Separation of superhelical loops by

close helix-helix contacts in the plectonemic DNA has also been visualized. The

overall geometry of DNA is also observed to be dependent dramatically on ionic

conditions.

Bacterial chromosomal DNA is circular but not a single topological

domain. Using hydrodynamic techniques and electron microscopic observations,

the groups of Pettijohn and Worcel showed that bacterial DNA is organized into

approximately 40 to 50 topologically independent domains, approximately 100 Kb

in size (Pettijohn and Pfenninger, 1980; Worcel and Burgi, 1972). This

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chromosome domain size has been shown to be modulated by type II

topoisomerases in vivo (Stacek and Higgins, 1998).

1.2 CONTROL OF DNA SUPERCOILING

(a) Topoisomerases and Supercoiling

The nature and the extent of negative supercoiling in bacteria is controlled

by the action of topoisomerases, enzymes that catalyze changes in the linking

number (Drlica, 1984; Wang, 1985,1996). Two major topoisomerases are· involved (

~f.

in the control of supercoiling in bacteria. One is DNA gyrase, a tetrameJc protein ~ ,,

constituted of two subunits of GyrA protein and two subunits of GyrB prdtein. The

second enzyme i~ topoisomerase I which is encoded by tapA gene and is capable of

relaxing negatively supercoiled DNA.

Early genetic analyses of gyrA, gyrE and tapA genes indicate that

supercoiling is controlled by their protein products. Deletion of tapA gene causes

cells to grow poorly and normal cell growth is restored by compensatory mutations

in gyrA or gyrB genes (Pruss eta!., 1982). Dinardo eta!., (1982) observed that

DNA isolated from bacterial cells, mutant for tapA gene, was found to have higher

than normal levels of supercoiling. On the other hand, inhibition of DNA gyrase

activity either by mutation _or by inhibitors leads to relaxation of DNA (Menzel and

Gellert, 1983). Menzel and Gellert (1983) observed that the synthesis of GyrA and

GyrB proteins increases when the DNA template is relaxed, while Tse-Dinh (1985)

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reported that topA gene expression increases when DNA template becomes more

supercoiled.

Thus two aspects of topoisomerases tend to reduce variation in supercoiling

under stable growth conditions (Fig. 1.3). One is the response of the enzymes to

the topological state of DNA. Gyrase is more active on a relaxed DNA substrate

and topoisomerase I on a more negatively supercoiled one. Another is the

homeostatic effect of supercoiling on expression of gyrA, g,vrB and topA genes.

Lowering negative supercoiling raises gyrase expression and lowers topoisomerase

I expression. Raising supercoiling raises the levels of topoisomerase I expression

(Menzel and Gellert, 1983; Tse-Dinh, 1985; Tse-Dinh et al., 1988) Recent studies

by Zechiedrich et al. (1997) indicates that topoisomerase IV can also remove

negative supercoils generated by gyrase in vivo and they have suggested that topo

IV also plays a role in setting the supercoiling levels in bacteria. The extent of

DNA relaxation by topo IV has shown to be much higher than that of topo I in

VIVO.

(b) Cellular Energetics and DNA Supercoiling

Gyrase catalyses the introduction of negative supercoils in the presence of

· ATP and removes negative supercoils in the absence of ATP (Gellert eta!., 1977).

It was shown that the. ratio of ATP and ADP concentrations [ATP/ADP] strongly ,.

influences gyrase activity (Westerhoff et al., 1988). Higher values of [ATP/ADP]

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ratio generate greater negative superhelic:al density. Thus ADP appears to interfere

with the supercoiling reaction of gyrase with DNA while allowing a competing

relaxing reaction to occur.·

Several physiological correlations between supercoiling and [ATP/ADP]

ratio have emerged form the study of environmental changes by the group of Karl

Drlica (Hseih et al., 1991a; 199lb). These studies indicate that intracellular DNA

supercoiling might depend· on the ratio of ATP to ADP. One correlation was

observed shortly after lowering oxygen tension: a parallel decline in [ATP/ADP]

ratio and supercoiling occurred. Later both [ATP/ADP] ratio and supercoiling

increased in a non-parallel manner, eventually reaching a situation in which both

were higher than under aerobic conditions. Another correlation was seen when

cells were suddenly exposed to high concentrations of sodium chloride. Both

[ATP/ADP] ratio and supercoiling increased rapidly. After few minutes, both

gradually declined to a steady-state level that was higher than at low salt

concentration. Thus [ATP/ADP] ratio may be another factor involved in the

control of supercoiling (Fig. 1.3) Under most steady state conditions [ATP/ADP]

ratio is probably maintained within a narrow range (Chapman and Atkinson,

1977), keeping the supercoiling within a narrow range. No evidence has been

reported whether topoisomerase I also plays a role in the influence of adenine

nucleotide concentration on DNA supercoiling in eubacteria.

10 .

SUPERCOILED

DNA ~ol

gyrase {activated) •

. ATP

RELAXED DNA

Fig 1.3. Gyrase, [ATP]:[ADP], and topoisomerase I affect DNA supercoiling. ATP and Gyrase interact to form an activated complex that then introduces negative supercoils into topologically constrained DNA. ATP hydrolysis (not shown) follows the introduction of supercoils. In the absence of A TP, gyrase can also remove negative supercoils. The level of supercoiling is determined by [ATP]:[ADP], as if ADP interferes withthe interaction of ATP and gyrase. Topoisomerase I (topo I) relaxes highly negatively s~percoiled DNA. When the [ATP]:[ADP] is so high that gyrase introdu~es an unacceptably high level of supercoils, topoisomerase I could actto lower supercoiling. (From, Drlica, 1990).

(c) Transcription Induced Supercoiling

Liu and Wang (1988) reported that pBR322 DNA, when isolated from tapA

mutant, was observed to be more negatively supercoiled than that of a closely

related plasmid unable to transcribe tet gene. Liu and Wang subsequently proposed

that tracking of transcription complex along the DNA generates positive supercoils

ahead of the complex and negative supercoils behind, called the twin-domain

model (Fig. 1.4). Topoisomerase I normally removes the negative supercoils and

gyrase the positive one. Thus negative supercoils will accumulate in a tapA mutant . .

and positive supercoils will accumulate when gyrase is inhibited. It has been also

reported that induction of very high levels of transcription from a plasmid results

in abnormally high levels of negative supercoils (Figueroa eta/., 1988)

Transcriptional effects on supercoiling are more pronounced for some genes

than for others. One factor is the anchorage of the transcribed gene so that rotation

of the transcription complex around the DNA is retarded. These effects are

particularly evident with genes encoding membrane proteins, when nascent

polypeptide chains may be simultaneously bound to membrane and ribosomes. In

pBR322, the tet gene encodes a membrane protein, and deletion of the

transnational signals from tet lowers abnormally high levels of negative

supercoiling found in tapA mutant (Lodge et a/., 1989).

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Non-rotatable supports

Non-rotating complex moving left to right between strands .of the DNA duplex

Non-rotatable . supports

· Fig ·1.4. Differential supercoiling of a DNA template during transcription. A length of · duplex DNA is shown connected to two non-rotable supports. If a body, sw;;h as a transcription complex, moves through the duplex, the template becomes partioned into a domain of underwound (i.e. negatively supercoiled) DNA behind the complex and a . domain of relaxed or even positively supercoiled DNA (as shown here) ahead of the complex. The only ways to resolve this situation are:(i) for the DNA to rotate; which it cannot; (ii) for the transcription complex to rotate, which it can only do with difficulty due to the torsional drag of the transcribing proteins, the attached mRNA and ·the translational machinery with its nascent polypeptides; and (iii) for DNA topoisomerase I to relax the domain of negative supercoils and DNA gyrase to relax the. domain of : positive slmercoils. These topoisomerase-catalysed events probably · represent the principal way in which this situation is resolved in vivo (From Dorman;l994).

(d) Extracellular Environment and Supercoiling

Various environmental factors influence the degree of supercoiling that has

profound influence on expression of certain genes. The changes brought out in the

gene expression profile lead to efficient adaptation to the environment (Fig. 1.5)

The first evidence for this came from the report of Y amomoto and Droffner

in 1985 that some mutations that prevent anaerobic growth, map at or near the

gyrase genes. Subsequently, it was shown that plasmids extracted from

anaerobically grown cells exhibited higher negative superhelicity than those of

aerobically grown cells. Drlica et al. (1990) found that nucleoides isolated from

cells growing exponentially under anaerobic conditions require 25 to 35% higher

dye (ethidium bromide) concentrations for titration of supercoils than do

nucleoides isolated from cells growing aerobically, indicating that the superhelical

density is 25 to 35% higher. The increased supercoiling under anaerobic condition

correlates with the increased presence of gyrase on the chromosome. They have

also shown that under anaerobic conditions, oxonolic acid plus sodium dodecyl

sulfate produces DNA fragments that are smaller that those obtained with cells

grown aerobically, suggesting the increased presence of gyrase on the

chromosome. Plasmid DNA supercoiling also becomes more negative when

growth medium osmolarity is increased (Higgins et al., 1988).

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\ I

I t

Environmental stress

Fig. 1.5. Environmental stress can alter DNA supercoiling: Changes in environmental parameters (such as growth medium osmolarity) can result in a change in the level of supercoiling in bacterial DNA.

Temperature is another environmental factor that influences the levels of

DNA supercoiling. In vitro, temperature variations alter the DNA helical pitch and

thus alter the level of supercoiling. Goldstein and Drlica ( 1984) have reported that

exposure of E. coli cells grown at 17°C to 37°C caused plasmid DNA supercoiling

to increase. These results were interpreted as follows: temperature upshift

decreases intra-cellular DNA supercoiling leading to relaxation of chromosomal

superhelicity and the level of gyrase is temporarily favoured until supercoiling

regains its preshift level. Another study showed _that upshift of temperature from

30 to 42,45,47, or 50°C led to an increase in the level of supercoiling of a reporter

plasmid. Inhibition of this increase by novobiocin, a gyrase inhibitor, and the

protein synthesis inhibitor, chloramphenicol, indicates that gyrase and some other

proteins are involved. The intracellular [ATP/ADP] ratio also increased rapidly

after temperature upshift from 30 to 47°C and then reached a level above that

observed at 30°C, suggesting that gyrase and some other proteins synthesized

during heat shock are responsible for changes observed in supercoiling (Camacho­

Carranza eta/., 1995)

When the effect of cold shock on DNA supercoiling was examined, it was

seen that DNA supercoiling increases transiently after cold shock This effect was

not observed with mutants deficient in the expression of HU protein and cells

pretreated with nalidixic acid, suggesting the involvement of HU protein and DNA

gyrase (Mizushima et al., 1997). Jones et al. (1992) Using 2-D acrylamide gel

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electrophoresis found that cold shock increases the synthesis of GyrA protein,

probably mediated by the binding of cold shock positive regulator, CS7.4 to the

promoter.

Growth temperature also influences DNA supercoiling levels. Mutations

that allow growth at 48°C in E. coli and S. typhimurium have been mapped to gyrA

gene (Droffner and Yamamoto, 1991 ). A spontaneously occurring nalidixic acid­

resistant gyrA mutant of E. coli was found to grow at 48°C and DNA supercoiling

was 12% less negative than in the parental strain (Droffner and Yamamoto, 1992).

Friedman et al. (1995) suggested that helical twist of DNA decreases with

increasing temperature, there may be a level of twist below which cells may not

grow. This observation also help to explain why topoisomerase I mutants have

decreased survival at high temperature;

(e) Histone-like Proteins and DNA Supercoiling

Histone-like proteins are small, basic, abundant and relatively nonspecific

DNA binding proteins that play a vital role in the bacterial chromosome

organization (Drlica and Rouviere-Y aniv, 1987). The most abundant architectural

proteins associated with the nucleoid are HU and H-NS proteins. Bliska and

Cozzarelli ( 1987) showed that one half of supercoiling in bacteria is constrained by

proteins while whole of it is constrained by histones in eukaryotes. The HU protein

is a 9.5 kDaprotein whose aminoacid sequence is conserved among many bacteria

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(Brait et al., 1984). Native HU is a _heterodimer of two highly homologous

components, HU-1 and HU-2, encoded by hupA and hupB genes respectively

(Kano et al., 1986)). It has been suggested that HU binds preferentially to

supercoiled DNA rather than to relaxed DNA (Holck and Kleppe, 1985). HU

protein also has been shown to introduce negative supercoils into a relaxed circular

DNA molecule in the presence of topoisomerase I and also to condense DNA into

nucleosome-like structures (Broyles and Pettijohn, 1986). However, mutations in

genes for HU, hupA and hupB, resulted in only a 10% lowering ofsupercoiling of

pBR322 plasmid DNA (Hillyard et al., 1991). A recent study by Bensaid et al.

( 1996) showed that mutations · in HU increases the relaxing activity of

. topoisomerase I, while increase in the intracellular concentrations of HU decreases

topoisomerase I activity. These results demonstrate that ·HU contributes to the

maintenance of DNA superhelical density in living bacteria both by condensing

DNA and by modulating topoisomerase I activity.

H-NS is another nucleoid associated protein of molecular weight 16.5 kDa

(Drlica and Rouviere-Yaniv, 1987). Although it binds relatively nonspecifically to

DNA, it exhibits a preference for curved sequences. H-NS can influence

supercoiling (Yamada et al., 1991). Tupper et al (1994) showed that hns mutations

affect the topology of chromosomal DNA and H-NS can constrain supercoiling in

vitro. Using trimethylpsoralen to measure DNA supercoiling, Mojica and Higgins

15

( 1997) have shown that plasmid and chromosomal DNA supercoiling increases in

a hns mutant.

1.3 DNA SUPERCOILING AND GENE EXPRESSION

Vinograd et a!. ( 1968) pointed out that one of the most important properties

of supercoiled DNA is that supercoiling is associated with an unfavourable free

energy and any process that reduces the degree of supercoiling occurs more readily

in a negatively supercoiled DNA than in a linear or nicked circular DNA. Specific

examples of processes that are favoured in a negatively supercoiled DNA are the

denaturation of a helical segment, the binding of intercalative dye, and 'possibly

the binding of RNA polymerase at the initiation site for transcription and the

formation of transient hybrid between the growing RNA chain and the template

DNA strand'.

An understanding of relationship between supercoiling and transcription

has· been developed primarily by in vitro studies. Hayashi and Hayashi in 1971

noticed that the initial rate. of RNA synthesis is about three times higher for a

supercoiled bacteriophage template than for a relaxed one. Richardson_ (1974,

197 5) provided evidences that superhelical tension facilitates initiation of

transcription. First, chain initiation, measured by incorporation of y-32P labeled

ATP and GTP, is greater with a supercoiled template. Second, maximum number

of RNA polymerase molecules that can form heparin-resistant complexes with

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DNA (i.e., RNA polymerase-promoter open-complexes capable of initiating RNA

synthesis) is greater with supercoiled DNA. In vitro studies also indicate that

supercoiling does not affect all genes equally. In the case of bacterophage lambda

DNA, increasing superhelicity raises the amount of late genes RNA synthesis and

lowers the relative amount of early genes transcription (Botchan et a/., 1973 ).

Transcription of tyrT gene in vitro has been reported to be 100 times stronger on a

supercoiled template as compared to a linear template at physiological salt

concentration (Lamond, 1985). Bramhs et a/. (1985) reported that transcription of

various pBR322 genes like Ampr, Tcr and rep gene is enhanced by increasing

negative superhelicity upto the natural level. Lei Sun and Fuchs (1994) have

recently reported that the nrd operon of E. coli is sensitive to supercoiling and

transcription increases with increasing superhelicity.

Boroweic and Gralla (1985) determined the rate of open complex formation

. of lac P promoter in vitro in response to increasing levels of negative supercoiling.

The presence of low to moderate levels of supercoiling gave a large stimulation in

the rate relative to that measured on a relaxed DNA template lacking superhelical

turns. Further increases in supercoiling past these levels caused a sharp inhibition

of rate from the maximum, suggesting a limit beyond which supercoiling can not

favour transcription. Boroweic and Gralla ( 1987) further examined the

supercoiling response in four closely related lac promoter variants each differing

by a single base pair mutation in one of the consensus, -10, -35 or spacer elements.

17

They showed that promoter mutation differently affects the response of the rate of

open complex formation to negative supercoiling and thus the promoter as a whole,

rather than individual element is shown· to be more important for mediating the

effects of DNA supercoiling.

First in vivo study to show that the transcription rates are higher in a

negatively supercoiled template carne from the studies of the mutants of the phage,

<j>X174. The phage protein A converts supercoiled replicative form of DNA into

nicked-circular DNA and in the protein A mutants, replicative form remains

supercoiled. Puga and Tessman (1973) found that the phage mutant for protein A

has template efficiency four to five times higher than cells infected with wild type

phage.

The clearest evidences that the regulatory potential of DNA supercoiling is

used in vivo come from the genetic studies of the leucine operon in Salmonella

typhimurium and Escherichia coli. These studies used topoisornerase mutants or

inhibitors that could alter the steady-state levels of DNA supercoiling and thus

gene expression. A point mutation of A to G transition, described as leu-500

mutation, in the -1 0 region of leu operon rendered the cells auxotrophic for leucine

(Dubanau and Margolin, 1972). The mutation in topoisornerase I gene, tapA, has

been found to suppress leu-500 mutation (Trucksis et al., 1981 ). Richardson et al.

(1988) observed that the tapA-dependent suppression of leu-500 mutation occurred

18

only when the promoter is located in its original position in the chromosome and

not when subcloned into a plasmid, supporting the notion that changes in local

rather than global supercoiling is involved in the activation of the leu-500

promoter. One interpretation of leu-500 suppression by tapA mutation is that

overall negative supercoiling is increased due to the absence of the functional

topoisomerase I, which would energetically favour open complex formation.

In 1992 Chen et a!. presented that leu-500 mutation can also be suppressed

m extrachromosomal plasmid DNA in a tapA background, if a divergently

transcribing · tetA gene is placed maximum 250 bp apart. These results were

explained based on the fact that transcription can both respond to and promote

changes in supercoiling. The excessive negative supercoiling generated by tetA

gene transcription is responsible for the suppression of leu-500 mutation, thus also

proving that the movement of RNA polymerase along the template DNA generates

negative supercoils behind and positive supercoils ahead. Another elegant study by

Wu et al. in 1995 indicates that the divergent transcription of ilv!H promoter,

located L9kp uprestream of the leu-500 promoter in the chromosomal DNA, is

responsible for the suppression of leu-500 mutations. Through deletion analysis of

the intervening 1.9 Kb sequences, they also showed that the transcription-mediated

local supercoiling changes are relayed to a distant promoter by intervening

sequence element(s). Fang and Wu in 1998 reported that activation of leu-500

mutation can also occur in a tapA+ strain due to the ilv!H transcriptional activity

19

initiated promoter relay, while the ab~ence of topoisomerase I enhances the

proinoter relay-mediated leu-500 activation by two-fold.

Another type of study linked gyrase to transcription. Altering the steady

state levels of supercoiling by mutations in gyr genes or by inhibitors of gyrase,

differently affects expression of many genes. Addition of any of the gyrase

inhibitors to intact growing cells was found to inhibit the expression of many

genes. These include catabolite sensitive operons, lactose and galactose operons

(Sanzey, 1979) and cysB gene (Bielinska and Hulanicka, 1986). This gyrase­

specific inhibition is not observed in strains resistant to the particular drug.

Expression of certain other genes, on the other hand, has oeen reported to

be-enhanced by inhibiting DNA gyrase, such as lacUV5, !del (Sanzey, 1979), heat

shock genes (Neidhardt et al., 1984), SOS repair system (Little, 1982) and the gyr

genes themselves (Menzel and Gellert, 1987a). In S. typhimurium, by making

random operon fusions, percentage of coumermycin induced promoter has been

found to be 70% while decreased expression was noticed only for 16% and rest

13% did not show any change (Jovanovich and Lebowitz, 1987). Topoisomerase I

mutants also have pleiotrophic cellular effects including increase in alkaline

phosphatase activity (Dubau et al., 1973). and increased sensitivity to UV light

damage (Overbye and Margolin, 1981).

20

Various environmental factors may also influence the degree of

supercoiling and thereby affect gene expression, leading to efficient adaptation to

the environment. Balke a:nd Gralla (1987) have shown that external nutritional

environment can force a cell to reorganize their global metabolism and these

changes are accompanied by changes at the DNA supercoiling level. The severe

nutrient downshift reduces the level of DNA supercoiling. The promoters of

'stringently controlled' operons of ribosomal RNA and transfer RNA (e.g. rrnBI

and tyrT) are sensitive to negative supercoiling and are inhibited when bacteria

experience aminoacid starvation (Condon eta!., 1995). In 1987, Rudd and Menzel.

l (1987) found that hisR operon promoter of S. typhimurium required negative

~

is particularly refractory to melting, apparently due to the high G-C content of the

'discriminator sequence' between -10 sequences and transcription ·start site. Tqe

tortional energy of negatively supercoiled DNA helps in opening the promoter.

The sequence changes that lower the barrier to melting lessen the supercoi1ing,

requirement for initiation. The same changes relieve transcriptional response to the

stringent, control suggesting that 'resistance to melting' is the basis for

susceptibility to stringent regulation.

21

Higgins et a/. in 1988 reported that increasing extracellular osmolarity of

the growth medium increases DNA supercoiling level resulting in the induction of

proU locus in S. typhiniurium and E .. coli. Another osmotically inducible gene

osmE has been shown to be growth-phase dependent (Conter eta/., 1997).

In S. typhiinurium, the iron siderophore uptake system, is very sensitive to

supercoiling changes associated with aerobic or anaerobic shifts, which appear to

account fully for aerobic-anaerobic regulation of tonE transcription (Dorman et

a/., 1988). Kranz and Haselkom in 1985 has shown that nitrogenase activity of

Klebsiellapneumoniae under anaerobic conditions is completely abolished by the

gyrase inhibitor, coumermycin. They failed to detect polypeptides of nitrogenase

complex in wes~em blot of coumermycin-treated cell extracts. Another study by

Dimiri and Das (1988) showed that the plasmid pRDI, containing the entire nif

gene cluster, which when transferred into E. coli, conferred the ability to fix

nitrogen, could not do so in a E. coli host that is mutant for DNA gyrase. Further

studies by Dixon et a/. ( 198 8) concluded that gyrase influences the expression of

only the regulatory operon of nif genes, namely nifLA operon. The nifLA promoter

is cr54-dependent promoter and activated by the positive regulator NtrC under

nitrogen limiting conditions. It has been suggested that higher negative

supercoiling under anaerobic conditions may facilitate DNA looping, promoting

the effective interaction between NtrC and cr54 -RNA polymerase.

22

Environmental regulation of virulence genes of pathogenic bacteria, which

experience changes in oxygen tension, osmolarity, temperature, pH, etc., during

infection, is shown to be mediated through DNA supercoiling (Dorman, 1991).

Supercoiling-induced helix destabilization can drive the formation of alternate

DNA structures of appropriate sequences, like cruciform extrusion by inverted

repeats (Palecek, 1991 ). Occurrence of such structures near or in the promoter .

region has been shown to influence gene expression (Mukerji and Mahadevan,

1997). The topology of DNA can also modulate the influence of DNA bending at

the promoter region and subsequently gene expression (Perez-martinet al., 1994).

The mechanisms by which DNA supercoiling influences gene expression

can be summarized in the following ways: ( 1) supercoiling could promote the

melting of duplex DNA to form an open complex of RNA polymerase and the

promoter; (2) ·it could participate in communication between distantly located

regulatory elements by increasing effective DNA site concentration; (3} it could

provide the energy required to bend a short segment of DNA or for the formation

of an alternate DNA structure; ( 4) it could assist the winding of duplex DNA

around a protein complex; and (5) it could enhance the binding of transcription

initiation complex around the transcription start site.

23

1.4 DNA TOPOISOMERASES

Topoisomerases form a sub-class of nicking-closing enzymes that catalyze

changes in linking number and are vital in solving topological problems arising out

of entangling of duplex DNA(Luttinger, 1995; Wang, 1996). Fourtopoisomerases

have been discovered in bacteria and are grouped into type I and type II

topoisomerases. Type I topoisomerases, topoisomerase I and topoisomerase III,

catalyze the change in the linking number in steps of one by transiently introducing

a single strand nick in the DNA, passing the opposite strand through the nick and

resealing the nick. Type II· topoisomerases, DNA gyrase and topoisomerase IV,

introduce a transient double strand break in the DNA, catalyze the translocation of

another intact double strand DNA through the break and ligate the break. In this

process they either alter the linking number in steps of two or help form catenanes.

The properties of bacterial DNA topoisomerases are shown in Table 1.1 and those

of DNA gyrase have been described later in details.

1.5 DNA GYRASE

DNA gyrase was discovered by Gellert et a{ (1976). While studying in

vitro site-specific recombination of A. DNA with purified int protein, Gellert et al.

found that an ATP-dependent host factor, capable of converting relaxed form of

DNA into supercoiled DNA form, is necessary for site-specific recombination.

Gellert et al. purified the enzyme and named it as DNA gyrase. The discovery of

24

:Table Lt. Properties of DNA Topoisomerases. (From, Sinden, 1994)

Siz.t Enzyme Type Gene (kDa) ~L Co factors Activities

Prokaryotic top A 97 increase L; Mg2+ Relaxes ne~atively sudercoiled DNA; (will form E. coli Topoisomerase I ilL= 1 knots an catenate DNA in nicked molecules);,

DNA transiently bound by a 5' phosphotyrosine bond

E. coli DNA gyrase II gyrA 105 decrease L· ilL = - 2 ATP, Mg2+ ATP-dependent negative supercoiling (to cr < gyrB 95 increase L; ilL = 2 -0.1 ); ATP-independent relaxation

of negatively supercoiled DNA; relaxes positively supercoiled DNA; (will form knotted and catenated DNA); responsible for supercoiling the chromosome; DNA transiently bound by a 5' phosphotyrosi~e bond

E. coli Topo III topB 73.2 increase L; ~L = + 1 Mg2+ Decatenase activity, also relaxes negatively supercoiled DNA; active in chromosome decatenation following replication in vitro

E. coli Topo IV II parC 75 increase L; ilL = + 2 ATP, Mg2 + Similar to DNA Yyrase; has a DNA relaxing parE 70 . activity but wi I not negatively supercoil DNA;

involved in chromosome decatenation following replication

DNA gyrase was soon followed by works related to two classes of DNA synthesis

inhibitors, the quinolone class of synthetic drugs like nalidixic acid, oxonolic acid

and ciprofloxacin, and the coumarin class of antibiotics (e.g., novobiocin,

coumermycin and chlorobiocin) (Goss eta!.; 1964, Ryan et al:, 1976). The genetic

loci conferring resistance to nalidixic acid, nal, has been mapped to gyrA gene

encoding for GyrA subunit of DNA gyrase, while the genetic loci conferring

resistance to coumermycin, cou, has been found as gyrE gene, encoding GyrB

subunit of DNA gyrase (Gellert, et al., 1977, Sugino et al., 1978). The gyrA and

gyrE genes have been cloned, sequenced and expressed (Adachi et al., 1987,

Swanberg and Wang, 1987, Hallet et al., 1990) and were found to encode proteins

of 874 (97 kDa GyrA protein) and 804 (90 kDa GyrB protein) aminoacids

respectively. The gyrA and gyrE genes have been mapped at 43 minutes and 83

minutes respectively on a standard K-12 E. coli chromosome map (Bachman and

Low, 1980).

Gyrase is found only in bacteria and plays an indispensable role in

chromosome architecture and function. It is ·also the target of one of the most

successful anti-bacterial agents, quinolones. The following description summarizes

whatever is known on DNA gyrase.

25

(1.5.1) Reactions of Gyrase

DNA gyrase performs a number of topological interconversions of DNA

molecules. It was first known for its supercoiling activity. Gyrase is also capable of

relaxing negatively supercoiled DNA in the absence of ATP, as well as catenating

and decatenating two duplex DNA circles and resolving a topologically knotted

single DNA duplex. In the presence of ATP or the nonhydrolyzable analog 5 '­

adenylyl-P-y-imidodiphosphate (ADPNP), gyrase can also relax positively

supercoiled DNA. in a reaction considered analogous . to the introduction of

negative supercoils.

(a) Supercoiling and relaxation

The DNA supercoiling reaction requires in addition to ATP, a divalent

cation, such as Mg2+,. and is stimulated in the presence of spermidine (Gellert et a!.,

1976). Incubation of gyrase with a single purified DNA topoisomer has indicated

that the enzyme alters the linking number in steps of two (Brown and Cozzarelfi,

1979). This has been interpreted mechanistically in terms of the translocation of

DNA segment through a double-stranded DNA break. There is a limit to the degree

of negative supercoiling that can be introduced into DNA molecule. When the

plasmid pBR322 is isolated from E. coli cells, it is found to have a specific linking

difference of approximately- 0.06, corresponding to a linking number deficit of 25

(Bauer, 1978). The maximum specific linking difference achievable by gyrase is -

26

. 0.11, which corresponds to the linking number deficit of about 46 to 4 7 for

pBR322 DNA (Westerhoff et al., 1988). This limit of specific linking difference

has been found to be the same for small (150 to 400 bp) and large (4 Kb) DNA

circles (Bates and Maxwell, 1989). The ATP-dependent negative supercoiling of

gyrase is completely inhibited by ADPNP (Sugino et al., 1978). ADPNP alone can

support limited negative supercoiling by gyrase in such a way that binding of

nucleotide to gyrase is sufficient to. allow a single cycle of supercoiling. But the

hydrolysis of ATP is required to return the enzyme to its starting state for another

round of supercoiling. The negative supercoiling reaction performed by gyrase is

inhibited by both quinolones and coumarins.

In the absence of ATP, gyrase can relax negatively supercoiled DNA

(Sugino et al., 1977, Higgins et al., 1978). The relaxation activity of gyrase is

much less efficient than the supercoiling reaction, with about 20 to 30 times as

much enzyme required for a comparable rate. Gyrase relaxation is inhibited by

quinolone drugs but not by coumarins. It has been speculated that DNA relaxation

is simply the reverse of supercoiling reaction, and that ATP hydrolysis is required

to derive DNA strand passage in one direction only.

(b) Catenation, Decatenation and Unknotting

Gyrase can catalyze the formation and resolution of DNA catenanes and

can unknot knotted DNA. These reactions require ATP and are inhibited by both

27

coumarins and quinolones. Catenation and decatenation reactions are stimulated by

spermidine (Kreuzer and Cozzarelli, 1980).

· (1.5.2) Subunits of DNA gyrase

DNA gyrase has been found to be composed of two subunits each of GyrA

and GyrB proteins. This A2B2 composition has been supported by biochemical

studies. Mixing different ratios of the gyrase proteins with DNA, followed by

separation of the DNA-protein complex from free protein by gel filtration, showed

that the gyrase-DNA complex contains equivalent amount of the A and B subunits

(Sugino et al., 1980). Sedimentation analysis of gyrase complexed with DNA

fragments suggested a molecular mass of 470 kDa corresponding to A2B2.complex

bound to about 140 bp of DNA (Klevan and Wang, 1980). Cross-linking of M

luteus gyrase, using dimethyl subermidate, yielded a number of protein complexes

of molecular masses 420 kDa, 330 kDa and 230 kDa (Klevan and Wang, 1980).

These spe9ies have been tentatively assigned A2B2, A2B and A2 complexes

respectively. Cross-linking of the purified A protein also yielded the 230 kDa

species, again suggesting A2 dimers, while purified B protein gave no cross-linked

products using diethyl subermidate. ATPase experiments using the GyrB protein

show a nonlinear dependence of enzyme activity on protein concentration,

consistent with oligomerization of this subunit (Maxwell and Gellert, 1984). The

molecular mass of E.. coli gyrase has been calculated from small-angle neutron

28

scattering by measuring the scattering intensity at zero angle (Krueger eta!., 1990).

The value obtained, 353 kDa is again strongly suggestive of an A2B2 composition

for the active enzyme complex.

(a) GyrA Subunit

Treatment of GyrA protein with either trypsin or chymotrypsin results in

the generation of two large fragments with approximate molecular masses of 64

kDa and 33 kDa, which are relatively stable to further digestion (Reece and·

Maxwell, 1989) (Fig. 1.6). The 64 kDa tryptic fragment comprising residues 7 to

571 of the intact protein, was found, in the presence GyrB subunit to perform low

rate of DNA supercoiling which can be inhibited by quinolones. The 64 kDa

protein contains the active tyrosine residues involved in the covalent attachment of

the protein to the DNA and also contained all the sites in the intact GyrA protein

that confer resistance to quinolones when mutated. The 33 kDa fragment was not

able to support any of the reactions of gyrase, but the supercoiling reaction

catalyzed by the 64 kDa fragment was found to proceed more efficiently in the

presence of the 33 kDa fragment. It is understood that theN-terminal portion of A

protein is principally involved in the breakage and reunion steps of the gyrase

reaction, while C-terminal portion of the protein seems to impart stability to the

complex. It has been shown that the N-terminal 6 aminoacids of A protein are

completely dispensable for enzymatic activity. The deletion of N-terminal 67

29

(a)

83 106 122 214 462

,; ' ......

Proteolytic cleavage

~ 571 875.

amino . - \:- ,_ .·-...·.h

carboxyl

(b)

DNA breakage-reunion, interaction with quinolones

;

Structural domain

~' 136 220

Proteolytic cleavage

~ 394 426447

~:

DNA wrapping

. 751 804

~-. . amino 47 kDa.:. carboxyl

' ' :fill( ,., : : : Interaction with : : coumarins : :~ I

Interaction with ATP Interaction with GvrA and DNA

Fig. 1.6. Domain structure of Escherichia coli DNA gyrase. The (a} GyrA (97 kDa) and (b) GyrB (90 kDa) proteins are represented as linear blocks with proposed domain

· boundaries indicated. Some of the key amino acids referred to in the text are also shown. Amino acid 122 in GyrA i.s the active-site tyrosine. Amino acids whose mutation leads to drug resistance are numbe~ed in italics. (From, Maxwell, 1997).

aminoacids resulted in the inactivation of GyrA protein. The smallest fragment of

the protein that is able to perform quinolone-directed cleavage of DNA was found

to be GyrA of 7 to 523 aminoacid residues of the intact GyrA protein (Reece and

Maxwell, 1989).

Reconstituted DNA gyrase with 59 kDa GyrA fragment, devoid of 33 kDa

C-terminal fragment behaved much like a conventional type II topoisomerase,

which is able to perform catenation but unable to perform supercoiling. The unique

property of gyrase, the supercoiling activity, i·s attributable to the positive

wrapping of DNA by 33 kDa C-terminal fragment (Kampranis and Maxwell,

1996).

The crystal structure of 59 kDa breakage-reunion domain of GyrA protein

has been recently solved at 2.8A0 resolution by Morias Cabral et a!. (1997).

Comparison of the structure ofthis 59 kDa domain with that of a 92 kDa fragment

of yeast topoisomerase II reveals very different quaternary organization, and the

authors propose that the two structures represent two principal conformations that

participate in the enzymatic pathway. The gyrase structure reveals a new dimer

contact with a grooved concave surface for binding the G segment (DNA breakage

segment) and a cluster of conserved charged residues surrounding the active-site

tyrosine. Mutations that co~fer resistance to the quinolone antibacterial agents

cluster at the new dimer interface.

30

(b) GyrB subunit

GyrB protein consists of two domains, 43 kDa N-terminal and 47 kDa C­

terminal domains (Fig. 1.6). In the presence ofGyrA, the 43 kDa domain can not

supercoil DNA but is able to perform all the ATP-dependent reactions of gyrase

and also binds to coumarins (Reece and Maxwell, 199la; Ali eta!., 1993; 1996).

· This protein dimerizes in the presence of ATP (Wigley et a_!., 1991) and this

process facilitates B proteins to capture the trans segment of DNA and to direct it

through the double strand break made by the enzyme on the gate segment. ATP

hydrolysis allows the enzyme-DNA complex to return to its original supercoiling

conformation (Bates et al., 1989) Mutation of Glu42 to Ala in GyrB subunit

abolished ATP hydrolysis but not nucleotide binding (Jackson and Maxwell,

1993). When gy~ase complex containing one mutant GyrB Ala42 and one wild type

GyrB subunit were formed, the resulting heterogeneous tetramers were able to

perform supercoiling at a lower rate, with wild type GyrB ·subunit independently

performing ATP-hydrolysis (Kampranis and Maxwell, 1998). ATP hydrolysis by

GyrB subunit alone has been found to be very low, which is stimulated when GyrA

subunit and double- strand DNA are present (Maxwell and Gellert, 1984). This

stimulation is dependent on the length of DNA; DNA molecules below 70bp in

lenth of DNA can only stimulate the ATPase at high concentration. The kinetics of

ATP hydrolysis by gyrase has been studied (Maxwell et al., 1986). The B protein

31

alone was found to have a Km for A TP of 1. 7 mM and turnover number of about

1/s. The presence of GyrA protein and DNA lowers the Km to o-.5 mM.

The 43 kDa N-terminal domain of GyrB protein has been crystallized in the

presence of non-hydrolysable ATP analogue, ADPNP, and the structure has been

solved at 2.5A0 resolution (Wigley et a!., 1991). The space-filling model of this

structure has been shown in Fig. 1.7. This protein monomer consists of two

domains; an N-terminal domain (residues 2 to 220) that contains the bound

ADPNP and a C-terminal domain (221 to 392). The protein isproposed to be a

dimer with most of the monomer-monomer contacts occurring between the N­

terminal domains. This includes anN-terminal arm that protrudes from the surface

of the monomer and wraps around theN-terminal domain of the other subunit. The

C-terminal domain forms the sides of a hole through the protein dimer

approximately 20A 0 wide. It is possible that this hole forms a part of a gateway

through which DNA is passed during the supercoiling reaction.

(1.5.3) DNA Binding by DNA gyrase

Liu and Wang (1978) initially studied the characteristics of gyrase-DNA

complex using M luteus enzyme. They found that incubation of gyrase with

nicked circular DNA species and the following ligation of the nick by DNA ligase,

resulted in the positive supercoiling of the DNA species suggesting that DNA is

wrapped around the enzyme with a unique handedness equal to nearly one

32

Fig. L7. Space-filling model of theN-terminal fragment of the gyrase B protein. The two monomers are picked out in different shades. of gray. The N-terminal domains are in the upper part of the figure and the C-terminal domains are in the lower part, forming the sides ofthe hole between the two monomers. (From~ Reece and Maxwell, 1991).

complete positive superhelical tum. Furth~r evidences of the wrapping of DNA by

gyrase came from the analysis by footprinting techniques (Morrison and

Cozzarelli, 1981 ). Gyrase was found to protect approximately 100 to 155 bp of

DNA from nuclease attack, with a central region, of some 40 to 50 bp, being most

strongly protected. The DNA flanking this region is less well protected, and there

is evidence for sites of enhanced sensitivity to DNAse I spaced 10 to 11 bp apart;

these sensitive sites are staggered by two bases on the complementary DNA

strands, indicating the wrapping of DNA by gyrase. Surprisingly, no or little

protection of DNA to methylation by dimethyl sulfate is afforded by gyrase,

suggesting that the entire length of DNA in the gyrase-DNA complex is accessible

to solvent (Kirkegaard and Wang, 1981 ).

Gyrase-DNA complexes can also be detected by the retention m

nitrocellulose filters. The complex, consisting of equal amounts of the gyrase A

and B proteins, appears to be very stable, with a half -life at 25°C of 60 to 70 hours

and equilibrium dissociation constant of 0.1 to 0.5 nM (Higgins and Cozzarelli,

1982). It was also noted that gyrase binds more strongly to relaxed or linear DNA

than do supercoiled DNA by a factor of 10. A preference in the order of positively

supercoiled > relaxed > negatively supercoiled DNA is expected from the right­

handed wrapping of a DNA segment around the enzyme (Higgins and Cozzarelli,

1982).

33

The strength of binding of gyrase to a particular linear DNA is dependent

on the length of the fragment used. Maxwell and Gellert (1984) noted that gyrase

readily forms complexes with a 117 bp or larger, but will only form a complex

with a 55 bp fragment at high DNA concentrations. The binding curve for gyrase

and the 55 bp fragment was consistent with at least two DNA molecules binding to

a single gyrase molecule in a cooperative manner suggesting the possibility of

gyrase containing two or more DNA-binding sites.

Kirchhausen et a!. (1985) based on the interpretations of electron

microscopic observations of gyrase-DNA complex, suggested that the complex is

heart-shaped and that the DNA is wrapped around the protein such that the centre

of DNA is located between the heart's upper lobes. Small-scale angle neutron

. .

scattering of gyrase-DNA complexes also predicted a model for the complex that is

similar to the electron microscopic model and suggested that channels or cavities

exist within the complex (Krueger eta!., 1990).

The figure 1.8A shows gyrase-DNA model based on electron microscopy

and scattering data. In the gyrase-DNA complex about 120 bp of DNA is wrapped

around the protein. The DNA entry and exist points are located close together, and

the DNA tails are thought to be at an angle of 120°. Assuming the smooth

wrapping of DNA around gyrase, then the diameter of the resulting circle, at the

outside edge of the DNA, will be about 150 A0• The size of gyrase particle has

34

175 A

1

52 A

175 A

DNA cleavage site

I

A

175 A

~~

~~

~~

~~

B

~~~ ~~

~~

Fig. 1.8. Proposed structure of the DNA gyrase-DNA complex. (A) The DNA is shown . as a shaded loop· wrapped around the A and B subunits. The A proteins are in the upper part of the model and the B proteins in the lower. -N and -C indicate the amino- and carboxy-terminal domains of the proteins. The black dots represent the sites of covalent attachment between the enzyme and DNA. (B) A transverse· section of the model indicating the DNA around the protein complex. (From, Reece and Maxwell, 1991).

been estimated to be 175 A0 by 52 A0 in dimention. The DNA is likely to be

embedded into the protein structure, which will extend beyond the wrapped DNA.

Gyrase has been shown to cleave DNA in both strands, with a four base pair

stagger between single-stranded breaks (Sugino et al., 1977). The cleavage site is

shown in figure on one side of the DNA only. Since DNA is helical with about

10.5 bp per tum in the B-form, a difference of four bases will mean that

nucleotides in different strands will be approximately in the same side of the helix,

and separated by 15A0• Therefore, cleavage site could be at the mouth of the

channel as shown in the figure 1.8A.

Figure 1.8B shows a side in section of the proposed gyrase-DNA complex.

The DNA is wrapped at an angle around the oblate gyrase particle. In this

configuration, the DNA should be accessible to nuclease attack, yet the binding of

DNA to gyrase would not appreciably alter the size and shape of the particle in

agreement with the neutron-scattering data (Krueger et al., 1990).

(1.5.4) DNA Cleavage by DNA Gyrase

The binding of gyrase to DNA results in the formation of an organized

DNA-protein complex. In the presence of quinolone drugs ( oxolinic acid,

ciprofloxacin etc.) and following subsequent addition of a protein denaturant like

SDS, DNA gyrase induces double-stranded DNA cleavage with a four base pair

35

stagger at the cut-site (Sugino et al., 1977). Linear, relaxed, and supercoiled DNA

are all shown to be substrated for cleavage by gyrase. Following cleavage, the

protein is covalently attached with the DNA. Morrison and Cozzarelli (1979)

reported that the 5' end of the cleaved DNA are blocked to labeling by T4

polynucleotide kinase, while 3' hydroxyl ends of the cutsite remain free and able to

prime a DNA polymerase reaction. Horowtiz and Wang (1987) found the precise

point of attachment between the protein and the 5, end of cleavage site by series of

proteolytic digestions and sequencing of the labeled peptides as tyrosine residue

122 of the A subunit of gyrase.

Efficient DNA cleavage by gyrase can also occur in the absence of

quinolone drugs if Ca2~ is substituted for Mg2+ in reaction mixtures. Cleavage

induced by Ca2+ occurs at the same loci as that induced by oxolinic acid but with

different relative efficiencies (Reece and Maxwell, 1989). Lockshon and Morris

. (198;i) based on the analysis of oxolinic acid/gyrase-induced DNA cleavage site in

vivo has suggested the following consensus sequence:

+ S'RNNNRNNRTGRYCT YNYNGNY 3'

(G) G (T)

36

T and Gat the 13th position are equally preferred and the G and T in brackets are

preferred seeondarily. O'conner and Malamy (1985) reported a preferred cleavage

site at nucleotide position 990 for the pBR322 DNA as shown below:

S'GGCTGGATtGCCTTCCCCAT 3'

They also mapped a total of 74 quinolone-induced cleavage sites in vivo

using the same plasmid.

In a hydroxy radical footprinting study in which the major cleavage site in

pBR322 was examined, gyrase, in the absence of ATP,protected about 120 bp

DNA, 50 bp on one side of the gyrase-dependent cleavage site and 70 bp on the

other (Orphanides and Maxwell, 1994). When a non-hydrolyzable analog of ATP,

ADPNP, was used, a conformational change was observed in which additional

DNA was wrapped around gyrase. Cove et al., in 1997 reported that DNA gyrase

could cleave short DNA fragments as small as 20 bp in the presence of quinolone

drugs.

The mechanism of DNA reunion after cleavage by gyrase is not known. There is

no direct experimental evidence available to suggest how this reaction occurs.

Gellert et al. (1977) have shown that when the gyrase-DNA complexes are briefly

heated to 80°C before the addition of SDS in a quinolone directed cleavage

reaction, the supercoiled substrate remains intact. This result suggests that either

37

that cleavage occurs only after SDS addi~ion, or, more probably, that the broken

DNA ends are efficiently resealed prior to protein -DNA dissociation.

(1.5.5) Mechanism of DNA supercoiling by gyrase

A possible scheme for the supercoiling reaction of closed-circular DNA by

gyrase based on various experimental evidences is shown in Figure 1.9. Gyrase is

represented as an avoid structure, which is able to bind to DNA, 120 bp is wrapped

around the protein with a positive superhelical sense. This necessitates the

formation of a negative writhe elsewhere in the molecule to maintain the

in variance of topological linking. Gyrase then cleaves the wrapped DNA segment

in both strands. The 5 '-ends of each of the break sites are covalently attached to the

A subunits of gyrase via phosphotyrosine residues (Horowitz and Wang, 1987).

The 3 '-ends of the broken DNA must be held by non-covalent forces, which may

include direct interactions between the enzyme and the free 3 '-hydroxyl groups,

forces between the wrapped DNA segment and the protein, and the integnty of the

DNA double helix itself. Such interactions stabilize the break site and will not

allow the DNA to untwist to relieve the strain of the negative writhe.

DNA strand passage then occurs through the break-site, and also

presumably through at least part of the protein structure. Reece and Maxwell

( 1991) propose that intersubunit channels within the protein structure facilitate this

process and suggest that the translocated DNA segment is close to or possibly part

38

DNA gyrase

\.

Positive writhe Negative writhe Relaxed closed­circular DNA

(Alk'"' 0)

Negativeiy supercoiled DNA

(Alk- -2) .

DNA .._J ' 1/

1/ 1/

1/ .(l Processive

if supercoiling

·' A'

gyrase

1 ··' Q

Break resealed

Linking number reduced by 2

' Double-stranded· break in DNA

DNA strand passage through DNA gap and protein

2ATP

! 2ADP

+ 2 Pi

Fig. 1.9 The mechanism of DNA supercoiling by DNA supercoiling by DNA gyrase (From, Reece and Maxwell, 1991).

of, the wrapped DNA. Evidence for the existence of a DNA channel between the

gyrase B subunits has recently been provided by X-ray crystallographic analysis of

theN-terminal protein of the B protein (Wigley et al., 1991).

The passing of a DNA duplex through the double stranded break causes the

linking number of that DNA to be reduced by 2. After the translocation of the

DNA through cleavage gate, the break in the DNA is resealed and the gyrase

dissociate from the DNA. It is also assumed that the break may not or may be

resealed and gyrase remain attached with the DNA to perform other reaction

cycles. In the reaction two ATP molecules are hydrolyzed~

The relaxation of positive supercoils by gyrase is likely to occur by the

same mechanism as that of negative supercoiling, the only difference being in the

starting state of the DNA and the fact that reaction is energetically favourbale.

Catenation and decatenation can be viewed as the intermolecular counterparts of

the supercoiling reaction, the major difference being that the DNA ~egment to be

translocated comes from another DNA molecule. Unknotting can proceed via the

same type of mechanism that is essentially the reverse of the supercoiling reaction,

where DNA passes through the enzyme and the break site in the opposite direction.

(1.5.6) Inhibitors of DNA gyrase

The indispensable role of gyrase in the bacterial cell and the apparent lack

of gyrase activity in eukaryotes make DNA gyrase an ideal drug target. A large

39

number of gyrase specific antibacterial agents have been reported (Maxwell, 1997;

1999). Most of them can be classified into two groups, the quinolones and the

coumarins (Fig. 1.1 0).

(a) The quinolones

Quinolone drugs are entirely synthetic and are excellent antibacterial agents

(Maxwell, 1992; Maxwell, 1997). The first quinolones to be synthesized were

nalidixic acid and oxolinic acid, but these compounds were largely superseded by

fluoroquinolones such as ciprofloxacin as antibacterial agents (Lesher et al., 1962).

Following the discovery of gyrase, it was shown that addition of quinolone drugs

to an in vitro reaction containing gyrase, relaxed circular DNA and ATP leads to

the inhibition of supercoiling (Gellert et al., 1976a). Quinolones also were shown

to inhibit relaxation, catenation and decatenation activities of DNA gyrase

(Kreuzer and Cozzarelli, 1980). The quinolones were found to bind strongly to the

complex formed by gyrase and DNA, and the ATP appears to assist this ·

interaction. Treatment of quinolone-gyrase-DNA complex with SDS produced

DNA cleavage with the gyrase A protein covalently attached to the newly formed

5' termini (Sugino et al., 1977). Mutation of tyrosine at 122 of GyrA subunit to

serine or phenylalanine abolished supercoiling activity and quinolone-induced

DNA cleavage but the mutant enzyme could still bind the drugs equally as that of

wildtype (Horowitz and Wang, 1987).

40

Coumarins

Novobiocin

Quinolones

ci= H3C I; Nl WO COOH

0

<0 N . . I C.Hs

Nalidixic acid

I C.Hs.

Oxolinic acid

H:JCf-\0 . HO 0

CH:J CH, CH:J ·~ CHp~· O'O:AO O C'-' O 0¢"' 01\0'~0CH3

Pi! I -..: ~·'3 · I CH:J .....: .....: 7 ::-... ::-...

OOH ~ I ~ . ob- OH H , H OH

I 111-t H

.....: Cti:J Coumermycin A1

0

FroCOOH I" I rN ~ N

HNJ A .. Ciprofloxacin

Clerocidin

Fig. 1.10. Compounds acting on DNA gyrase. Examples of antibacterial agents thought to have gyrase as their target. (From, Maxwell, 1997).

Using limited proteolysis of gyrase, DNA and quinolone complexes,

Kampranis and Maxwell (1998a) recently found that quinolones stabilize a

conformational change that involves the C-terminal 4 7 kDa domain of B protein in

the gyrase-DNA complex. This quinolone stabilized conformational change has

been proposed to be responsible for the inhibition of gyrase functions. At the

molecular level, this conformation is interpreted as a state of enzyme where DNA·

gate is trapped in the closed form irrespective of the DNA cleavage-relaxation

state. When the complex is in the quinolone-trapped state, it can hydrolyze ATP

with a characteristic rate. Kampranis and Maxwell (1998b) using sensitive ATPase

assays also found that the binding of the drugs to the gyrase-DNA complex is a

relatively fast reaction while DNA cleavage is a subsequent slow step and DNA

.cleavage is the result rather than a pre-requisite of quinolone binding.

Many mutations that confer resistance to quinolones were often mapped to

gyrA gene. Most of these mutations fall within a small area of the gyrase A protein,

between aminoacids 67 to 106, which has been termed the 'quinolone resistance

determining region' (QRDR) (Yoshida eta!., 1986). The mutation of Ser 83 to Trp

results in high levels of drug resistance both in vivo and in vitro (Willmott and

Maxwell, 1993). Some quinolone-resistance mutations have also been mapped to

gyrB gene, not exhibiting high level resistance to drugs (Nakamura et al., 1990).

One interpretation of these findings is that gyrase A protein is the primary target of

the quinolones and that mutations in gyrB have secondary effects on quinolone

41

interaction mediated via protein-protein contacts. Recently Zhao et al. (1997)

reported that the potent quinolone, ciprofloxacin containing methoxyl group at C-8

position was particularly lethal to cells producing no mutants. Quinolone-

. resistance mutations of the gyr genes are recessive to sensitive· alleles (Hane and

Wood, 1969). Cells containing nalidixic acid-resistant (NaiR) and nalidixic acid­

sensitive (Nals) alleles ofgyrA gene are phenotypically drug sensitive, hinting that

mode of action of the drugs in vivo is not simply the inhibition ofsupercoiling by

gyrase.

Exact mechanism by which gyrase-DNA-quinolone interaction leads to cell

death is not clear. One hypothesis was that the complex forms a DNA lession that

acts as a poison and initiates the events that lead to cell death. Kreuzer and

Cozzarelli (1979) showed that growth of some bacteriophages is very sensitive to

nalidixic acid but not to the elimination of gyrase supercoiling activity; they

proposed that the gyrase-nalidixic acid complex on DNA form a barrier to the ..

passage ofpolymerases. Willmott et al. (1994) observed that the gyrase-quinolone

complex with DNA lead to the blocking of transcription by E. coli and T7 RNA

polymerases in vitro. Another study by Chen et al. (1996) proposes that the

liberation of DNA ends from quinolone-gyrase-DNA complexes account for the

-bactericidal action of the quinolones. Chen et al. showed that when cells were

treated with chloramphenicol to inhibit protein synthesis, oxolinic acid failed to

kill the cells, thus predicting a protein factor involved in releasing DNA ends from

42

the quinolone-gyrase-DNA complexes. ~he potent quinolone, ciprofloxacin does

not need the assistance of these protein factors for the release of DNA breaks.

(b) The Coumarins

The Coumarin class of antibiotics like coumermycin and novobiocin are

Streptomyces products and are inhibitors of the gyrase supercoiling and ATPase

activities, by apparently competing with ATP for binding with the enzyme, even

though they lack structural resemblance with ATP (Maxwell, 1992). The inhibition

constant (ki) values for coumarins have been estimated in the 1 0"7- 1 o-9 range.

Enzymatic analysis of the 43 kDa amino-terminal domain of GyrB shows

that this protein possesses a coumarin-sensitive ATPase activity, which localized

the coumarin drug-binding site to this part of the protein (Ali et al., 1993). Kinetic

analysis of ATPase reaction of the 43 kDa protein by Gormley eta!. (1996) reveals

that the drug is a non-competitive inhibitor. They have shown that novobiocin

binds to 43 kDa monomer, whereas QOumermycin, which resembles a dimer of

novobiocin, stabilizes a dimer form of the 43 kDa fragment. The 23 kDa amino

terminal of this 43 kDa protein was able to bind to coumarins almost as tightly as

intact GyrB, but not to ATP (Gilbert and Maxwell, 1994).

Analysis of coumarin-resistant bacterial strains from severaa species has

identified point mutations that· map to the 24 kDa fragment (Contreras and

Maxwell, 1992). The most prevalent of these mutations are an qrginine residue

43

(Argl36) and GyrB protein bearing this mutation at Arg 136 shows low

supercoiling and ATPase activities and are relatively resistant to the drugs in vitro.

Recently Lewis et a!. ( 1996) has showed the crystal structure of 24 kDa fragment

complexed with novobiocin. The structure indicates that Argl36 residue hydrogen

bonds to the coumarin part of novobiocin. In addition the protein forms a network

of hydrogen bonds with the novobiose sugar moiety and several hydrophobic

contact with the drug. Recently Kampranis et a!. (1999) studied coumarin-gyrase

complexes using limited proteolysis and found that binding of coumarins induced a

characteristic conformational change in gyrase.

{c) Other Compounds

Although majority of compounds that target DNA gyrase are members of

the quinolone and coumarin classes, there are growing number of inhibitors that

fall outside this group.

Microcin B17 (MccB17) is a glycine rich peptide of molecular weight 32

kDa produced by enterobacteria carrying pMccB 1 7 or related plasmids. This

peptide is subject to extensive post-transnational modification, generating a novel

structure (Yorgey et a!., 1994). MccB 17 is active against many enterobacteria and

has been shown to be an inhibitor of DNA replication, leading to rapid arrest of

DNA synthesis, induction of SOS response and DNA degradation. Vizan et a!.

(1991) independently isolated MccB17-resistance mutants of E. coli and found that

44

they both contain a single point mutation that converts Trp751 to Arg in GyrB

protein. They reported that MccB 17 is able to induce DNA cleavage, in fashion

similar to quinolones.

·The protein CcdB is an 11-kDa protein produced by F plasmid as a part of

two component system for plasmid copy maintenance. CcdB acts as a toxin and

CcdA as the antidote (Jensen and Gerdes, 1995). In the absence of CcdA,

expression of the CcdB protein causes cell filamentation, induction of SOS

pathway and ultimately cell death. Bemand and co-workers (1992) found that

mutations conferring resistance to CcdB map to GyrA and produce aminoacid

substitutions Arg 462 to Cys. Bernard et al. ( 1993) reported that if CcdB is

expressed in vivo in the absence of CcdA, treatment of cells with SDS reveals,

DNA cleavage-analogous to that seen with quinolone drugs. Recently Critchlow et

al. (1997) reported that cleavage reaction by CcdB requires ATP hydrolysis when

the substrate is linear DNA, but is independent of hydrolysis when the DNA is

negatively supercoiled. They also reported that cleavage reaction requires intact

GyrA protein and a minimum length of 160 bp DNA. The poisoning of gyrase­

DNA complex by CcdB protein has been recently studied by Bhassi et al. (1999).

The crystal structure of CcdB has also been solved recently by Loris et al. (1999).

Study of MccB 17 and CcdB proteins may yield novel pepide-based inhibitors:

45

Cinodine is an antibiotic of glycocinnamoyl spermidine class produced by

Nocardia species and this has been shown to inhibit DNA supercoiling by M

luteus DNA gyrase in vitro (Osbume et al., 1990). Clerocidin is a terpenoid

antibiotic isolated from Fusidium visidae, which has been shown to inhibit gyrase­

catalyzed DNA supercoiling and to cause DNA cleavage in a manner similar to

quinolones (Osbume, 1995).

(1.5.7) Roles of DNA gyrase

DNA gyrase nearly influences all maJor cellular activities of DNA

involving strand separation and DNA-protein interaction like replication,

recombination and transcription. The role of DNA gyrase in transcription has been

discussed in detail previously as a part of regulation of gene expression by DNA

supercoiling previously. Temperature sensitive mutants of gyrA and gyrE genes in

E. coli block DNA replication at non-permissive temperature and gyrase inhibitors

are also inhibitors of replication (Orr and Staudenbauor, 1981). The gyrA

mutations lead to rapid arrest of chain elongation. Lather et a!. ( 1984) have shown

a preferred gyrase-binding site at E. coli origin of replication ( oriC). The nucleoids

isolated from cells carrying gyrE temperature-sensitive mutants are found to be

doublet when isolated at _the non-permissive temperature suggesting gyrase

involvement in the decatenation of daughter chromosomes (Mizuuchi and

Mizuuchi, 1978).

46

DNA gyrase is a host factor required for phage A site-specific integration

into the E. coli chromosome (Gellert et al., 1976). Gyrase can be dispensed within

this reaction if the A DNA is already negatively supercoiled. DNA gyrase is also

thought to participate in other types of recombination. Generalized recombination

in gyrB mutants is reduced compared with that of wildtype cells (Von Wright and

Bridges, 1981 ). In bacteriophage Mu, a centrally located sequence is proposed to

be a strong DNA gynise binding site that is required for replicative transposition

(Pato et al., 1990). DNA gyrase mediated illgetimate recombination has been

shown to be suppressed by HU protein (Shanado et al, 1998) The gryA mutations

that confer hyper-recombination phenotypes have been characterized recently by

Ashizawa et a!. ( 1999)

DNA gyrase maintains the chromosome architecture of the cell. It has been

found that DNA gyrase binds with DNA sequences containing REP (repetitive

extra-genic palindromic) sequences with 10-fold higher affinity as compared to

non-REP DNA (Yang and Ames, 1992). REP sequences are distributed throughout

the chromosome (100 to 200 per chromosome). Reports of in vivo treatment of E.

coli cells with oxonolic acid have suggested that the chromosome has 50-100

major cleavage sites (toposites). It has been proposed that REP-gyrase interaction

is required for maintaining the supercoiled domains of the chromosome (Drlica et

al., 1992). Gyrase along with topoisomerase IV has been shown to modulate

supercoiled domains of bacterial chromosome (Stacek and Higgins, 1998).

47

(1.5.8) The gyr genes

Cloning, sequencmg and expression of gyr genes from various bacterial

sources have been carried out (Reece and Maxwell, 1991) These include

Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, Neisseiria

gonorrhea, Micoplasma pneumoniae, Haloferax sp, Bacillus subtilis, Streptomyces

sphaeriades and S. aureus. Without cloning, the gyrase enzyme has also been

purified from Micrococcus luteus and Citrobacter freundii (Table 1.2). From such

studies, it can be seen that the gyrase subunits fall into size ranges, with GyrA being

about 90 to 100 kDa and GyrB being about 70 to 90 kDa. The proteins from gram­

positive or-negative species correlate poorly into separate size classes. This leaves

unexplained differences in the anti-gyrase drug susceptibilities of these bacterial

groups. In many cases both the gyrA and gyrB genes have been found to be

contiguous within genome. For example, the Bacillus subtilis gyrase genes are both

located near to the origin of replication and are separated by only 214 base pairs.

One exception to this is E. coli where the gyrase genes are widely separated. The

gyrA gene of Mycobacterium leprae has been found to harbour a 1260-bp in-frame

insertion encoding an intrin, a homing endonuclease (Hafida et al. 1996)

(1.5.9) Regulation of gyr genes

Regulation of gene expressiOn is a pivotal process in the adaptability of

bacterial cells to their vicissitudes. Gene expression is regulated by several

48

Table 1.2 Characteristics of the DNA Gyrase Genes from Different Organisms. (from, Reece and Maxwell, 1991 ).

Length Distance Amino Size of of gene between acids in protein

Organism Gram- Gene (bp) genes (bp) protein (kDa)

Escherichia coli gyrA 2625 -1.5 1()6 875 97 gyrB 2412 804 90

Citrobacter freundii gyrA 107 gyrB 96

Pseudomonas gyrA -400" aeruginosa gyrB

P. putida gyrA 806 90 gyrB 2418

Klebsiella gyrA 2628 8i6 97 pneumoniae gyrB

Borrelia burgdorferi gyrA 2430 14 810 90 gyrB 1917. 639 71

Mycoplasma gyrA 5' only<' 1 bp overlap pneumoniae gyr8 1953 650 72

Haloferax gyrA 5' only<' 640 gyrB 1920 71

Neisseria gyrA gono1rhoea gyrB 2313 771 86e

Bacillus subtilis + gyrA 2463 214 821 92 gyrB 1914 638 71

Micrococicus luteus + gyrA 115 gyrB 97

Staphylococcus + gyrA 2667 39 889 100 au reus gyrB 3' only"

Streptomyces + gyrA >3000 sphaeroides gyrB 7g.

. - • a G~am-negative organism; +. a Gram-posik,•e organism . b. Molecular weight of intact gyrase complex. c Data of W. M. Huang, personal communication. d Only partial.sequence information is available. . Resistance gene cloned . I Data of L. M. Fisher, personal communication.

mechanisms in the bacterial cells. DNA supercoiling represents global

transcriptional regulatory mechanism (Pruss and Drlica, 1989). Environmentally

induced changes in DNA topology have been suggested to play an important role

to cell adaptability to stress conditions. Since DNA gyrase regulates the degree of

supercoiling in bacteria, one might expect that the genes for DNA gyrase are also

subjected to regulation.

The intracellular levels of DNA gyrase are indeed determined by the

transcriptional regulation of gyr genes by supercoiling itself. In E. coli, total

deletions of topA gene for topoisomerase I have been found to be accompanied by

mutation in gyrA or gyrB genes, leading to the production of a partly defective

GyrA or GyrB protein to net decrease in supercoiling level (Pruss et al., 1982).

These genetic s~dies show that when topo I activity is removed, there is a strong

selective pressure for compensating alternations in DNA gyrase activity. Such a

relative pressure is being viewed as 'homeostatic' selection for 'normal'

superhelical density. Based on these observations, it has been proposed that there is

a homeostatic mechanism for controlling the expression of DNA gyrase and

topoisomerase I (Menzel and Gellert, 1983).

Menzel and Gellert in 1983 studied the rate of synthesis of DNA gyrase by

immunoprecipitation of gyrase by gyrase-specific antibody. They found that

inhibition of DNA gyrase activity by sub-lethal concentrations of coumermycin or

49

novobiocin or by heat inactivation of a mutationally altered temperature-sensitive

gyrase, leads to 10 to 20 fold increased synthesis of gyrase. They also showed

using a S-30 cell free protein synthesis system that a relaxed DNA is a good

. template for gyrase synthesis, while a supercoiled DNA is a poor template. In vivo,

2 to 3 fold increase in the expression of gyrA-galK and gyrB-galK fusions were

observed upon in the inhibition of DNA gyrase by coumermycin (Menzel and

Gellert, 198za). These results suggest that regulation of gyr genes occurs at"the

level of transcription and only a small piece of 5' sequences approximately 110 bp

upstream of the translational initiation codon, is sufficient to cause induction by

coumermycin, also called as relaxation stimulated transcription (RST). A deletion

analysis of gyrA and gyrE promoters fused to ga!K gene revealed that the deletion

upto the -35 sequence did not affect either promoter strength or RST (Menzel and

Gellert, 1987b). A 20 bp sequence including -10 sequences,

CTGTGTTAT AA TTGCGACC, has been found to be responsible for RST

(Straney et al., 1994). The mutational changes in the -10 sequences affected both

promoter strength and response to RST. The -10 mutants with similar basal levels

of expression exhibited differential induction in gene expression to DNA

relaxation (Straney et al., 1994). A protein factor(s) in E. coli, whose inhibitory

properties are minimal on a relaxed DNA template, has also been proposed to play

a role in gyrA gene activation (Carty and Menzel, 1990). The characterization of

such a factor(s) has not been reported so far.

50

Based on above studies, some models have been reported to explain RST

(Straney et al., 1994). The movement of RNA polymerase away from the promoter

region· following open complex formation and transcription initiation is a possible

step at which DNA relaxation could act to increase the productive transcription.

Transcription initiations at such a promoter would lead to abortive full length

transcripts, which trap the promoter by R-loop formation. Such R-loop in the

promoter with abortive transcripts could be stabilized by negative supercoiling,

while relaxation of DNA supercoiling would tend to destabilize such R-loop

formation (Masse and Drolet, 1999).

A recent study by Gomez et al. shows that gyr A gene of E. coli is under the

control of CRP or cyclic AMP receptor protein, the global regulator in response to

glucose depletion (Gomez-Gomez et al., 1996). Increased (4-fold) expression of

gyrA-lacZ fusion observed in a crp + (CRP gene) background at the exponential

phase was not observed in a crp background, indicating that CRP positively

regulates gyrA transcription. The possible regulatory role of the bent region,

located between the -35 and -10 regions, of the gyrA promoter of Streptococcus

pneumoniae has been reported (Balas et al., 1998).

(1.5.10) The gyrA gene of Klebsiella pneumoniae

The findings that DNA gyrase activity influences the expression of the

regulatory operon of the nif genes, the nifLA operon, in Klebsiella pneumoniae, led

51

to the cloning and sequencing of gyrA gene encoding for GyrA subunit of gyrase

from that organism (Dimiri and Das, 1988, 1990). Bases of about 3.5 Kb DNA

have been sequenced to locate the gyrA gene and an open reading frame of 2628

nucleotides encoding for a 97 kDa protein has been identified. The translational

initiation codon, the potential SD sequence, the -10 consensus elements and the -

35 consensus elements were all located. A potential GC-rich transcription

terminator sequence has also been identified (Fig. 1.11)

Sequence companson at the nucleotide level between E. coli and K.

pneilmoniae gyrA genes, shows a homology of about 90% in the 5' coding region,

84% in the middle region and 79% in the 3' coding region. The composition of

aminoacids, derived from the nucleotide sequence exhibits close similarity with

that of E. coli, with an overall homology of 90%. Homology is more than 95%

with E. coli at the NHTterminal region and less homology towards COOH­

terminus, particularly in the regions 865 to 876, 658 to 677 and 408-417. When

residues, which when mutated, caused resistance towards nalidixic acid (serine 83

and glutamic acid 1 06) in E. coli were compared, glutamic acid 106 was found to

be conserved in K. pneumoniae, but serine 83 was found to be changed to

threonine.

On analyzing the promoter sequence, it was found that the gyrA gene of K.

pneumoniae differs from the promoter region of E. coli in some respects. K.

52

-287 G~~TTCGCCTTCT~~~TCCC~CC~GCGCG~GGCG~CGGCTTC~~~TTT~GCGATTTC -231

-170

-·11 0

-50

10

70

130

190

250

310

370

430

490

550

610

670

730

790

850

910

970

1030

1090

1150

1210

1270

1330

~TT~TG~TCAACATTGTGGGCCACCGACGGTTTTTCGGCATTCATGGGCGCTTTTACTCC

TGTTC~GTTC~GACCCGCGAGTATATCAGGCCTCAGGGATGAATAAACCGTCTATCCCTG

TCGCCTTCCGCCGCGCGAAAATCATTGGGTATAGGTTT~~~~~!~TCACl~~~99!GTGT

1 T~TAATTTGCGACCTTTGAATCCGGGATACAGTAGAGGGATAGCGGTTAG ATGAGCGAC J----- : f - - --- - H S D

CTTGCGAGAGAAATTACACCGGTCAACATTGAGGAAGAGCTGAAGAGCTCGTATCTGGAT L A R E I T P V N I E E E L K S S Y L D TACGCGATGTCGGTCATTGTTGGCCGTGCGCTGCCGGATGTCCGAGATGGCCTGAAGCCG y A H S V I V G R ~ L P 0 V R D G L K P GTACACCGTCGCGTACTATACGCCATGAACGTATTGGGCAATGACTGGAACAAAGCCTAT V H R R V L Y A H N V L G N D W N K A Y AAAAAATCTGCCCGTGTCGTTGGTGACGTAATCGGTAAATACCACCCTCATGGTGATACT K K S A R V V G D V I G K Y H P H G D T GCCGTGTATGACACCATTGTACGTATGGCGCAGCCATTCTCCCTGCGTTACATGCTGGTA A V Y D T I V R ~ A Q · P F S L R Y M · L V GATGGCCAGGGTAACTTCGGTTCTGTCGACGGCGACTCCGCCGCAGCGATGCGTTATACG 0 G Q G N F G S V D G 0 S A A A M R Y T GAAATCCGTATGTCGAAGATCGCCCATGAACTGATGGCCGACCTGGAAAAAGAGACGGTC E I R H S K I A H E L H A 0 L E K E T V GATTTCGTCGATAACTATGACGGCACGGAAAAGATCCCTGACGTTATGCCGACCAAAATC 0 F V 0 N Y 0 G T E K I P 0 V H P T K l CCGAACCTGTTAGTCAACGGTTCGTTCGGTATCGCGGTAGGTATGGCGACCAACATTCCG P ~ L L V N G S F G I A V G H .. A T N I P CCGCACAACCTGACCGA~GTGATCAACGGTCGTCTGGCCTACGTTGAAGACGAAGAGATC P H N L T E - V. I N G R L A Y V E 0 E E I ~GC~TTG~AGGGCTGATGGAACATATTCCGGGCCCGG~CTTCCCGACCGCCGCGATCATC SIEGLHEHIPGPDFPTAAII A~CGGTCGCCGCGGCATTGA~GAGGCCTATCGTACCGGTCGCGGTAAAGTGTACATTTGC N G R R G ! E E A Y R T G R G K V Y I C GCCCGCGCGGAAGTGGAAGCTGACGCGAAAACCGGTCGCGAAACCATCATCGTGCATGAA A R A · E V E A 0 A K T G R E ~T I I V H E ATTCCGTATCAGGTGAACAAAGCGCGCCTGATTGAGAAAATCGCTGAGCTGGTCAAAGAC I P Y Q V N K A R L I E K I A' E L V K D AAACGCGTCGAAGGCATCAGCGCGCTGCGTGACGAGTCTGATAAAGACGGCATGCGTATC K R V E G I S A L R 0 E S 0 K 0 G H R I GTGATTGAAGTGAAGCGCGATGCGGTGGGTAGGGTTGTGCTCAACAACCTCTACTCGCAG V I E V . K R 0 A V G R V V L N N L Y S Q ACTCAGCTGCAGGTCTCCTTCGGTATCAACATGGTTGCCCTGCACCATGGTCAGCCGAAG T Q L Q V S F G I N H V A L H H G Q P K ATCATGAATCTGAAAGAAATTATTGCCGCCTTCGTGCGCCACCGCCGCGAAGTGGTGACC I H· N L K E I I A A F V R H R R E V V T CGCCGTACGATTTTAGCACTGCGTAAAGCCCGTGATCGGGCGGACATCCTTGAAGCGCTG R R T I L A L R K A R 0 R A D I L E A L TCGATTGCCCTGGCCAACATCGATCCGATTATTGAGCTGATTCGCCGCGCGCCGACGCCG S I A L A N I 0 P I I E L I R R A P T P GCGGAAGCGAAAGCTGGCTTAATCGCCC~TTCATGGGATCTGGGCAACGTTTCCGCGATG A E A K A G L I A R S W 0 L G N V S A H CTGGAAGCGGGCGATGACGCCGCGCGTCCGGAATGGCTGGAACCTGAATTCGGCGTGCGC L E A G D 0 A A R P E W L E P E F G V R GACGGCCAGTACTACCTGACCGAACAGCAGGCACAGGCGATTCTGGATCTGCGTTTGCAG 0 G Q Y Y L T E Q Q A Q A I L 0 L R L Q

-171

-Ill

-51

9

69

129

189

249

309

369

429

489

549

609

669

789

849

909

969

1029

1089

1149

120'::1

1269

1329

1389

Fig 1.11 A portion of DNA sequence of gyrA gene of K. pneumoniae. The underlined sequences are the SD sequence immediately upstream of the base numbered I . of the initiator ATG and· the TAT A sequence further upstream of the TAT A box. The 7 bp inverted repeat is shown by horizontal arrows upstream of the TAT A box. The vertical arrow indicates the site where transcription starts in E. coli gyrA gene. Sequences are numbered relative to A of the translation initiator ATG codon. (From, Dimiri, 1992).

pneumoniae gene does have the 20 bp sequence necessary for the relaxation

stimulated transcription (CTGTGTTATAATTTGCGACC). But the presence of

additional 39 bases, at position -85 to -123 upstream, which are totally absent in

the upstream region of E. coli gyrA gene, has been identified in the K. pneumoniae

gyrA gene (Fig. 1.12). Another unique difference is the occurrence in the K.

pneumoniae gyr A gene of a 7 bp GC rich inverted repeat just upstream of TAT A

sequence at the position -55 to -72 (with respect to the translational initiation

codon). This sequence can adopt a cruciform structure with a 7 bp GC rich stem

and a loop of 4 bases (Fig. 12). A part of -35 consensus sequence comes in the left

hand arm of the cruciform and so the cruciform is included in the 18 bp distance

between the -10 and -35 sequences. This cruciformresembles a rho-independent

transcription terminator and has a GC rich stem followed by an AT rich sequence.

Sequences between -69 and -86 also can adopt cruciform structure with a 7 bp

(having one mismatch) stem and a small loop of 2 bases (Fig. 1.13).

The presence of the 39 bp additional sequences and the 7 bp inverted repeat

in the 5' control region of gyrA of K. pneumoniae and not in that of gyrA of E. coli

raises many questions. The extrusion of cruciform by the inverted repeats in the

region between -10 and -35 sequences could interfere with the promoter

recognition and the initiation of transcription. The role of such sequences is being

recognized (Horwitz and Loeb, 1988). One distinctive character of K. pneumoniae

is its ability to fix nitrogen. The enzyme nitrogenase is sensitive· to oxygen and

53

-287 GAATTCGCCTTCTAAATCCCACCAGCGCGAGGCGACGGCTTCAAATT -241 I I I t I I I I I I t I I I I I I I I I I I I I I I I I I I I It I I I I I I I I I I I I I I t I I I I I Itt t I If I I I I I I I I I I I I I I I I I I I I I I If

-248 GAACTCACCTTCCAGATCCCACCAGCGGGAGGCGACGGCTTCAAATT -202

-240 TAGCGATTTCATTATGATCAACATTGTGGGCCACCGACGGTTTTTCGGCA -181 I I I I I I I I I I I I f I I I t I I I I I I I I I I I It I I I I I I I I I I I I I I I I I f I I I I I I I I I I f I I I I I I I It I I I I I I

-201 TAGCGATCTCTTCGTGGTCTACGTTATGGTTTACCGGCGATTTTTCGGCA -152

-190 TTCATGGGCGCTTTTACTCCTGTTCAGTTCAGACCCGCGAGTATATCAGG -141 ·1 It I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

I It It t I I I I I I It It I I I I I I I I I I I I I I If I I

-151 TTCATTGGCACTTCTACTCCGTAATTGGCAAGACAAACGAGTATATCAGG. -102

-140 CCTCAGGGATGAATAAACCGTCTATCCCTGTCGCCTTCCGCCGCGCGAAA -81 : : : ::::::::

-101 CATTGGATGTGUTAAA ............ -..................... -85

-90 ATCATTGGGTATAGGTTTACCCGTATCACTACGGGTQTGTTATAATTTGC -41 I I I I I I I I I I I I I I I I It I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I t I I I I I I 1 It I It I I I I I

-84 ...... GCGTATAGGTTTACCTCAAACTGCGCGGCTGTGTTATAATTTGC -41

-40 GACCTTTGAATCCGGGATACAGTAGAGGGATAGCGGTTAG -1 I I I I I I I I It I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I If I I I 1·1 I I I I I I I I I It I I I It I I I I I I I 1 I I I Itt I I I

-40 GACCTTTGAATCCGGGATACAGTAGAGGGAlAGCGGTTAG -1

Fig 1.12 DNA base sequence comparison in promoter region of K pneumoniae gyrA gene (upper line) and E. coli gyrA gene (lower line). The double vertical dots between the bases indicate homology. The region between residues -85 to -123 is extra in the K. pneumoniae gyrA gene (From, Dimiri,l990).

C A T C

A - T T - A G - C C - G C - G C - G

<a>A - T <b> -103 CCGCCGCGCGAAAATCATTGGGTATAGGTTT GTGTTATAATTTGCGACCTTTGAATCCGG - 26

I I I I I I I I I I 1 I I ,I I I I I It I I I I I I I I I I I I" I I It I I I I I I I I I I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I I I It I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

-103 GGCGGCGCGCTTTTAGTAACCCATATCCAAA CACA~TATTAAACGCTGGAAACTTAGGCC- 26

G A

G - T

T - A G - C G - C G - C C - G A - T T - A

A G G T

T T A - T T - A <a> G - C G - C G - C <b>

CCGCCGCGCGnAAATCATT GTATCACTACGGGTGTGTTATAATTTGCGACCTTTGAATCCGG - 26 I I I I I I I I I I I I I I I I I I I I I I I If I I I I I I I I. J 1 t I I

I I I I I I I I I I I I I I I It I I I I I I I I I I I I I I I I I I I I I I I I I I It I I I I If I I I It I It I I I I I I I t"t I I I I I I I I I I I I I I It I I

-103 GGCGGCGCGCTTTTAGTAA CATAGTGATGCCCACACAATATTAAACGCTGGAAACTTAGGCC - 26

2

C - G C - G C - G A - T T - A

A A T - A c c

Fig 1.13 Potential cruciform (stem and loop) structures in promoter region of gyrA gene. 1. Cruciform in between -:10 and -35 Sequences. 2. Another alternative cruciform which· includes -35 sequences. a-the region of -35 sequence; b-the region of -10 sequence (From, . Dimiri, 1992).

oxygen is known to downregulate the level of DNA gyrase (Yamamoto and

Droffner, 1985; Drlica eta!., 1992) and on the other hand the expression of the nif

genes in K. pneumoniae is dependent on DNA gyrase (Kranz and Haselkorn,

1985). These considerations lead us to· postulate a special regulatory mechanism

for gyrA of K. pneumoniae. Understanding this mechanism could be of immense

value for harnessing the nitrogen fixation in bacteria. Hence the study relating to K.

pneumoniae gyr A promoter structure and function has been taken up to understand

the novel regulatory mechanism, if any, of gyrA gene.

(1.6) Strategy of the present work

Majority of genes is regulated by cis-acting sequences present m the

promoter region .or trans-acting proteins interacting with the cis-acting sequences,

thereby enhancing or reducing the level of tran~criptional activity. Hence the

structural and functional analysis of K. pneumoniae gyrA gene was of immediate

concern to us.

We first decided to find the mRNA start-site for the gyrA gene by the

primer extension analysis of its transcripts. Knowledge of the start-site will enable

us to locate the putative -10 and -35 sequences and also the relative positions of

the 7 bp inverted repeat and the 39 bp extra-sequences.

To know whether, the 7 bp inverted repeat and the 39 bp extra-sequences

(hitherto also collectively called as 'special sequences') are involved in the

54

regulation of gyrA gene transcription, we decided to carry out mutational analysis

of these sequences and measured the strength of the wild type and mutated

promoters by constructing and studying a series of gyr A -lacZ transcriptional

fusions in the host K. pneumoniae and its close relative E. coli.

The special sequences can also act as binding sites for DNA-binding

proteins that could play a regulatory role. To detect and analyze such proteins, we

partially purified DNA-binding proteins from K. pneumoniae and E. coli. We

studied the interaction of these proteins with the 'special sequences' by

electrophoretic mobility shift assay. The location of the binding site was also

unraveled at the nucleotide l€vel by DN ase I footprinting analysis. Identification of

the nature and size of these proteins was carried out using southwestern blot

techniques.

55