Download The structure and mechanism of DNA gyrase from divergent

Biochemical Society Transactions ( 1 996) 24
The structure and mechanism of DNA gyrase from
divergent bacterial species
DEREK S. GILCHRIST and ANDREW D. BATES.
Department of Biochemistry, University of Liverpool,
P.O. Box 147, Liverpool, L69 3BX, UK.
Type I1 topoisomerases are a class of ubiquitous enzymes
that alter the level of DNA supercoiling and play a major
role in replication, transcription and recombination [ 11.
DNA gyrase, a bacterial type I1 topoisomerase, is unique
among type I1 enzymes in its ability to introduce negative
supercoils into DNA. The active form of the enzyme is
an A2B2 tetramer encoded by the gyrA and gyrB genes.
The enzyme alters the level of supercoiling through the
concerted passage of a segment of DNA through a
double-stranded DNA gap made by the A subunits. This
process utilises the free energy from the hydrolysis of ATP
by the B subunits.
The sequences of several type I1 topoisomerases from
phage, bacteria and eukaryotes have been determined,
revealing considerable homology; however, the subunit
arrangement varies considerably. Among bacteria the
only significant difference lies in the presence of a 160
amino acid region in the C-terminal domain of B
subunits from Gram-negative species. No function has
been ascribed to this region though some evidence
suggests that it may be of importance in the initiation of
replication [2,3].
To date the main focus of our investigation has been the
importance of the 160 amino acid insert region in DNA
gyrases from Gram-negative bacteria, together with a
more general characterisation of any differences between
gyrases from Gram-negative and -positive species.
We have examined the ability of gyrB genes from both
Gram-negative (Pseudomonas putida) and Gram-positive
(Bacillus subtilis) species to complement an E. coli gyrBtS
mutant (N4177). The results showed that the P. putida
g y r B gene was able to complement E . coli gyrBts at a nonpermissive temperature (42"C), while B. subtilis gyrB
alone could not, and appeared toxic in N4177 at 3TC, a
normally permissive temperature. B. subtilis gyrA plus
g y r B were able to complement at 3TC and 42°C (Table 1).
These results confirm previous in vitro data which
showed that heteromeric DNA gyrases composed of E.
coli GyrA and B. subtilis GyrB were not able to support
initiation of DNA replication from an E. coli origin of
replication, despite possessing supercoiling activity [4]. It
is possible that this is due to the absence of the 160 amino
acid region from the B subunits of Gram-positive species.
The role of DNA gyrase during initiation of replication
Table 1. Complementaion of an E . coli gyrBts by gyrB
genes from Gram-negative and -positive species.Growth of E. coli gyrBts (+/-)
Plasmid
Geno tvDe
P. putida g y r B
B. subtilis g y r B
B. subtilis gyrA,g y r B
E. coli g y r B
negative control
30°C
37°C
42°C
+
+
+
+
+
+
+
+
+
+
+
+
42 1 S
may be more specific than the provision of a negatively
supercoiled DNA substrate for the replication machinery;
gyrase may be structurally important to the initiation
complex. Mutations in gyrB genes have been shown to
specifically affect initiation [5].
We have constructed clones based on a T7 expression
system in BL21(DE3) which overproduce B. subtilis GyrB
and GyrA proteins, together with P. putida GyrB and an
engineered version of P. putida GyrB in which the 160
amino acid Gram-negative insert region has been
specifically deleted (GyrAB). Both P. putida genes were
cloned as PCR products into the T7 translational vector
pT7-7, while B. subtilis gyrA and gyrB genes were
subcloned directly into the transcriptional vector pT7-5.
In all cases target proteins of the correct approximate
molecular weights were produced; however only B.
subtilis GyrA accumulated in a soluble form, all other
target proteins formed insoluble inclusion bodies.
Both P. putida GyrB and GyrAB have been overproduced, refolded and purified to near homogeneity.
Heteromeric DNA gyrase composed of P. putida GyrB/E.
coli GyrA was shown to be capable of introducing
negative supercoils into a relaxed pBR322 substrate in the
presence of ATP. The activity of P. putida GyrB was of the
same order as that of E. coli GyrB. Similarly P. putida
GyrAB was shown capable of negative supercoiling when
present with E.coli GyrA, however only to a superhelical
density significantly lower than that achieved by either P.
putida or E. coli GyrB. Whether this represents a
significant difference between Gram-negative and
-positive gyrases in the initiation of replication is under
investigation.
The ability of the B. subtilis gyrB gene to complement an
E. coli gyrB*s mutant when present along with B. subtilis
gyrA does not itself rule out a requirement for the 160
amino acid insert region in the replication complex.
Potentially heat inactivated E. coli GyrB could fulfil
structural requirements while being unable to introduce
supercoils.
Acknowledgments: We thank the BBSRC for financial
support.
References.
l.Bates, A.D. and Maxwell, A. (1993) DNA Topology In
Focus. IRL Press, Oxford.
2. Orr, E., Fairweather, N.F., Holland, I.B. and Pritchard,
R.H. (1979) Mol. Gen. Genet. 177,103.
3. Orr, E. and Staudenbauer, W.L. (1981) Mol. Gen. Genet.
181,52.
4. Orr, E. and Staudenbauer, W.L. (1982) J. Bacteriol. 151
(l),
524.
5. Filutowicz, M. (1980) Mol. Gen. Genet. 117, 301.