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 genetic 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 another 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).
Genetic 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 microbes or improve some of their
specific metabolic properties.
Genetic transfer in bioleaching microbes 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,
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bioleaching microbes grow slowly with very
low cell yields making them difficult to
culture.
Nevertheless, genetic transfer was made
possible in some bioleaching microbes
(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
Acidiphilium sp. electropermeabilization
conjugation
Acidithiobacillus conjugation
thiooxidans
Acidithiobacillus electropermeabilization
ferrooxidans
Acidithiobacillus conjugation
ferrooxidans
Reference
2; 3
1; 2; 10; 11
4; 12
5
6; 7; 8; 9
References:
1.
Bruhn, D. F. and F. F. Roberto (1993).
Maintenance and expression of enteric arsenic
resistance
genes
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 Acidiphilium 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
electroporation. Biosci Biotechnol Biochem 57: 17701.
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 (1992).
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 Thiobacillus 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
mutagenesis. J Bacteriol 182: 2269-2276.
8.
Peng, J., Yan, W. and Bao, X. (1994).
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Expression of heterologous arsenic resistance genes
in the obligately autotrophic biomining bacterium
Thiobacillus ferrooxidans. Appl. Environ. Microbiol.
60: 2653-2656.
9.
Peng, 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 acidophilic 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.
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 formed 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 the entrance of DNA
iv.
When the electric field is stopped, the
membrane heals
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