and concomitant 14CO2 evolution occurred (Figure 2). In the
soil inoculated with MTB6 cells the initial rate of 14CO2
evolution was 10-fold higher than in soils in which MTB6
had not been introduced (not shown).
TABLE 4. Solvent Efflux by Solvent-Sensitive and
Solvent-Tolerant P. putida Strainsa
14C in cell membranes /DO
660
These results therefore support the hypothesis that
solvent-tolerant strains can be useful vehicles in bioreme-
diation.
strain
-toluene
+toluene
MTB5
MTB6
R1
6 ( 2 × 105
4 ( 1 × 105
8 ( 2 × 104
6 ( 1 × 105
1.5 ( 0.5 × 105
7 ( 1 × 105
Acknowledgments
a
Cells were grown on LB m edium and when the turbidity of the
This work was supported by grants from the European
Commission (BIO-CT4-97-2270) and from the CICYT (BIO97-
0657).
cultures was around 1 at 660 nm , 2 µCi of 1,2,4-[14C]-trichlorobenzene
was added, and accum ulation of 14C in the cell m em branes assayed.
Literature Cited
(1) Atlas, R. M. In Bioremediation; The Tokyo’94 Workshop. OECD
Documents; OCDE Press: Paris, 1995.
(2) Gibson, D. T.; Subramanian, V. Microbial degradation of
aromatic hydrocarbons. In Microbial degradation of organic
compounds; Gibson, D. T., Ed.; Marcel Dekker: New York, NY,
1984; pp 181-252.
(3) Sikkema, J.; de Bont, J. A. M.; Poolman, B. Microbiol. Rev. 1995,
59, 201-222.
(4) Gibson, D. T.; Hensley, M,; Yoshioka, H.; Mabry, T. J. Biochemistry
1970, 9, 1626-1630.
(5) Worsey, M. J.; Williams, P. A. J. Bacteriol. 1975, 124, 7-13.
(6) Olsen, R. H.; Kukor, J. J.; Kaphammer, B. J. Bacteriol. 1994, 176,
3749-3756.
(7) Shields, M. S.; Montgomery, S. O.; Chapman, P. J.; Cuskey, S.
M.; Pritchard, P. H. Appl. Environ. Microbiol. 1989, 55, 1624-
1629.
TABLE 5. Survival in Soil of P. putida MTB6, MTB5, and R1
a
after Toluene Shock
[toluene] % (vol/wt)
strain
0
0.1%
1%
10%
P. putida
MTB5
P. putida
MTB6
4 ( 1 × 107 6 ( 2 × 107 5 ( 1 × 104 <102
2 ( 1 × 107 7 ( 2 × 107 2 ( 1 × 106 9 ( 3 × 104
P. putida R1 2 ( 1 × 107 5 ( 2 × 107 4 ( 2 × 103 <102
a
About 107 CFU of each strain per g of soil was placed in separate
pots which were supplem ented with toluene at the indicated concen-
tration. Fifteen days later sam ples were rem oved and bacteria counted
on selective m edium . Assays were run in triplicate.
(8) Whited, G. M.; Gibson, D. T. J. Bacteriol. 1991, 173, 3017-3020.
(9) Yen, K.-M.; Karl, M. R.; Blatt, L. M.; Simon, M. J.; Winter, R. B.;
Fausset, P. R.; Lu, H. S.; Harcourt, A. A.; Chen, K. K. 1991. J.
Bacteriol. 1991, 173, 5315-5327.
(10) Egli, T.; Lendenmann, U.; Snozzi, M. Antonie Leeuwenhoek 1993,
63, 289-298.
(11) Møller, S.; Pedersen, A. R.; Poulsen, L. K.; Arvin, E.; Molin, D.
Appl. Environ. Microbiol. 1996, 62, 4632-4640.
(12) Ramos, J. L.; Duque, E.; Ramos-Gonza´lez, M. I. Appl. Environ.
Microbiol. 1991, 57, 260-266.
(13) van Loosdrecht, M. C. M.; Lyklema, J.; Norde, W.; Zehnder, A.
J. B. Microbiol. Rev. 1990, 54, 75-87.
(14) Cruden, D. L.; Wolfram, J. H.; Rogers, R. D.; Gibson, D. T. Appl.
Environ. Microbiol. 1992, 58, 2723-2729.
(15) Inoue, A.; Horikoshi, K. Nature 1989, 338, 264-266.
(16) Kim, K.; Lee, S.; Lee, K.; Lim, D. 1998. J. Bacteriol. 1998, 180,
3692-3696.
(17) Pinkart, H. C.; Wolfram, J. W.; Rogers, R.; White, D. C. Appl.
Environ. Microbiol. 1996, 62, 1129-1132.
(18) Ramos, J. L.; Duque, E.; Ro´ driguez-Herva, J. J.; Godoy, P.;
Ferna´ndez-Barrero, A. J. Biol. Chem. 1997, 272, 3887-3890.
(19) Weber, F. J.; Isken, S.; de Bont, J. A. M. Microbiology 1994, 140,
2013-2017.
FIGURE 2. Evolution of 14CO from 14C-toluene in soils. Agricultural
2
soil from the gardens of the Estacio´n Experimental del Zaid´ın were
inoculated with P. putida MTB6. 14CO evolved (b) and viable cells
2
(O) were determined at the indicated times. Values are the average
of two independent duplicate assays, with standard deviations below
15% of the given values. Other conditions are described in Materials
and Methods.
(20) Segura, A.; Duque, E.; Mosqueda, G.; Ramos, J. L.; Junker, F.
Environ. Microbiol. 1999, 1, 191-198.
(21) Weber, F. J. Ph.D. Thesis, University of Wageningen, Wagenin-
gen, The Netherlands, 1995; p 125.
(22) Isken, S.; de Bont, J. A. M. J. Bacteriol. 1996, 178, 6056-6058.
(23) Kieboom, J.; Dennis, J. J.; Zylstra, G. J.; de Bont, J. A. M. J. Bacteriol.
1998, 180, 6769-6772.
organic solvent from the cell membranes. Whether the
difference in toluene tolerance between MTB5 and MTB6
lies in the differential rates of toluene removal or in the rate
of expression of the different pumps under different growth
conditions remains to be explored.
(24) Ramos, J. L.; Duque, E.; Godoy, P.; Segura, A. J. Bacteriol. 1998,
180, 3323-3329.
(25) Ramos, J. L.; Ha¨ıdour, A.; Pin˜ ar, G.; Calvo, V.; Oliva, J. M. Nature
Biotechnol. 1996, 14, 320-322.
(26) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a
laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory:
Cold Spring Harbor, NY, 1989.
Tolerance to Toluene in Soils by P. putida MTB5, R1,
and MTB6. Mineralization of 14C-Toluene by MTB6. P.
putida MTB5, MTB6, and R1 were introduced at about 107
CFU per g of soil in soil from the garden of Estacio´ n
Experimental del Zaid´ın, and toluene was added to an initial
concentration of 0.1, 1, and 10% (vol/ wt) to determine the
response of these strains to solvent shock. The addition of
0.1% (vol/ wt) toluene had no significant effect on the survival
of any of the strains (Table 5). However, MTB5 and R1 were
more sensitive to toluene shocks of 1 and 10% (vol/ wt) than
was the toluene-tolerant MTB6 strain (Table 5). We also tested
whether MTB6 was an efficient toluene-degrading strain in
the soil. To this end, soil with 1% toluene (vol/ wt) was
supplemented with 20 µCi 14C-toluene and was inoculated
or not with MTB6 cells, and we observed that upon an initial
drop in the number of viable MTB6 cells, growth took place
(27) Ramos, J. L.; Wasserfallen, A.; Rose, K.; Timmis, K. N. Science
1987, 235, 593-596.
(28) Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911-
917.
(29) Morrison, W. R.; Smith, L. M. J. Lipid Res. 1964, 5, 600-608.
(30) Ramos-Gonza´lez, M. I.; Ru´ız-Cabello, F.; Brettar, I.; Garrido, F.;
Ramos, J. L. J. Bacteriol. 1992, 174, 2978-2985.
(31) Wagner-Do¨ bler, I.; Bennasar, A.; Vancangnent, M.; Scro¨ mpl,
C.; Brummer, E.; Eichner, C.; Grammel, L. Appl. Environ.
Microbiol. 1999, 64, 3014-3022.
(32) Maidak, B. L.; Cole, J. R.; Parker, Ch. T., Jr.; Garrity, G. M.; Larsen,
N.; Li, B.; Lilburn, T. G.; McCaughey, M. J.; Olsen, G. J.; Overbeek,
9
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