Genetic transfer in bioleaching microorganisms

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Dec 11, 2012 (4 years and 7 months ago)

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16/03/2013

Genetic transfer in bioleaching microorganisms

Violaine Bonnefoy

Laboratoire de Chimie Bactérienne, Institut de Biologie Moléculaire et de Microbiologie,
C.N.R.S., 13402, Marseille Cedex 20, France


Genetic transfer techniques allow the
introduction of ge
netic material into cells.
The three classical approaches are
:




transduction

-

the transfer of genetic
information via a bacteriophage
(virus) particle (Figure 1)



conjugation

-

the transfer of
conjugative or mobilizable plasmids
from one bacterium to anoth
er by
cell
-
to
-
cell contact (Figure 2)



electrotransformation, by exposing
the cells in the presence of free DNA
to a pulsed electric field which
destabilizes transiently the bacterial
membrane and permits the entry of
the DNA into the cell (Figure 3).


Gene
tic transfer from one
microbe

to
another

can be used to express heterologous genes in
the recipient bacteria or to bring back genes
that have been modified in more accurate
hosts by genetic engineering. The later
approach allows the construction of mutants
,
precisely defined at the molecular level,
which can help elucidating the physiology of
these
microbe
s or improve some of their
specific metabolic properties.


Genetic transfer in bioleaching
microbe
s is a
real challenge because their life conditions
are
extreme and quite different from the
"classical" bacterium
Escherichia coli
.
Genetic engineering is usually performed in
this eubacterium which is consequently often
used as donor cells. While the former are
strict or moderate acidophiles and obligatory
or

facultative chemoautolithotrophs, the later
is neutrophilic and heterotrophic. The main
problem is therefore to find the conditions in
which both donor and recipient cells have
enough energy to survive. Furthermore,
bioleaching
microbe
s grow slowly with v
ery
low cell yields making them difficult to
culture
.



Nevertheless, genetic transfer was made
possible in some bioleaching
microbe
s
(Table 1). This has allowed the expression

of
the phosphofructokinase gene (
ptkA
) of
E.
coli

in
At. thiooxidans

(12). More

interestingly the transposon Tn5 was shown
to be able to transpose into the chromosome
of
At. ferrooxidans
, opening the way to
random transposon insertion mutagenesis
(9). Finally, conjugation by marker exchange
mutagenesis has allowed the construction of

the
recA

mutant in
At. ferrooxidans

(7), the
only "constructed" mutant described so far

in
bioeaching microbes.


Bacterium

Genetic transfer
technique

Reference

Acidiphilium

sp.

electropermeabilization

2; 3


conjugation

1; 2; 10; 11

Acidithiobacillus
t
hiooxidans

conjugation

4; 12

Acidithiobacillus
ferrooxidans

electropermeabilization

5

Acidithiobacillus
ferrooxidans

conjugation

6; 7; 8; 9


References:

1.

Bruhn, D. F. and F. F. Roberto (1993).
Maintenance and expression of enteric arsenic
resistance g
enes in Acidiphilium.
Biohydrometallurgical Technologies
. A. E. Torma,
Wey, J. E. and Lakshmanan VI. Warrendale, PA, The
Minerals, Metals and Materials Society. II: 745
-
754.

2.

Glenn, A. W., F. F. Roberto, and T. E. Ward
(1992). Transformation of Acidiphil
ium by
electroporation and conjugation.
Can J Microbiol

38:
387
-
93.

3.

Inagaki, K., J. Tomono, N. Kishimoto, T.
Tano and H. Tanaka (1993). Transformation of the
acidophilic heterotroph Acidiphilium facilis by
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electroporation.
Biosci Biotechnol Biochem

57:
1770
-
1.

4.

Jin, S. M., W. M. Yan, and Z. N. Wang
(1992). Transfer of IncP plasmids to extremely
acidophilic Thiobacillus thiooxidans.
Appl. Environ.
Microbiol.

58: 429
-
430.

5.

Kusano, T., K. Sugawara, C. Inoue, T.
Takeshima, M. Numata and T. Shiratori (199
2).
Electrotransformation of Thiobacillus ferrooxidans
with plasmids containing a mer determinant.
J
Bacteriol

174: 6617
-
6623.

6.

Liu, Z., F. Borne, J. Ratouchniak and V.
Bonnefoy (2001). Genetic transfer of IncP, IncQ and
IncW plasmids to four Thiobacillu
s ferrooxidans
strain by conjugation.
Hydrometallurgy

59: 339
-
345.

7.

Liu, Z., N. Guiliani, C. Appia
-
Ayme, F.
Borne, J. Ratouchniak and V. Bonnefoy (2000).
Construction and characterization of a recA mutant of
Thiobacillus ferrooxidans by marker exchange
m
utagenesis.
J Bacteriol

182: 2269
-
2276.

8.

Peng, J., Yan, W. and Bao, X. (1994).
Expression of heterologous arsenic resistance genes
in the obligately autotrophic biomining bacterium
Thiobacillus ferrooxidans.
Appl. Environ. Microbiol.

60: 2653
-
2656.

9.

Pe
ng, J. B., W. M. Yan, and X. Z. Bao
(1994). Plasmid and transposon transfer to
Thiobacillus ferrooxidans.
J Bacteriol

176: 2892
-
7.

10.

Quentmeier, A. and C. G. Friedrich (1994).
Transfer and expression of degradative and antibiotic
resistance plasmids in a
cidophilic bacteria.
Appl
Environ Microbiol

60: 973
-
8.

11.

Roberto, F. F., Glenn, A. W., Bulmer, D. and
Ward, T. E. (1991). Genetic transfer in acidophilic
bacteria which are potentially applicable in coal
beneficiation.
Fuel

70: 595
-
598.

12.

Tian, K. L.,
J. Q. Lin, X. M. Liu, Y. Liu, C.
K. Zhang and W. M. Yan (2003). Conversion of an
obligate autotrophic bacteria to heterotrophic growth:
expression of a heterogeneous phosphofructokinase
gene in the chemolithotroph Acidithiobacillus
thiooxidans.
Biotechnol
Lett

25: 749
-
54.


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Figure 1. Transduction




i.

Phage infection





ii.

DNA hydrolysis





iii.

DNA packaging in phage capsid





iv.

transducing phage infection to a new microbial cell





v.

Recomibination between the microbe chromosome and the DNA
transduced by the

phage
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Figure 2. Conjugation






i.






ii.

A conjugation tube forms between the donor cell
and the recipient cell. A single strand from the
plasmid DNA is transferred throught the tube
from the donor to the recipient cells.




iii.

A double
-
stranded DNA is for
med from the single
strand in both the donor and the recipient cells




iv.





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Figure 3: Electrotransformation






i.








ii.

When the electric fiel is applied, the ions
move according to their charge









iii.

Pathways are formed across the
membrane allowing th
e entrance of DNA








iv.

When the electric field is stopped, the
membrane heals