BioSMART ISSN: 1411-321X

Volume 5, Nomor 2 Oktober 2003

Halaman: 78-80

Isolation of Transpositional Mutant of p-Toluic Acid Assimilating Bacteria

Producing Catechol

SHANTI RATNAKOMALA
Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong-Bogor 16911.

Received: 17 May 2003. Accepted: 17 August 2003.

ABSTRACT

The production of catechol by microorganisms has been investigated and has several advantages. Transposon mutagenesis was a method
to obtain catechol-accumulating mutants from p-toluic acid assimilating bacteria. In this experiment about 14 isolates from soil samples
could grow on 0.3% p-toluic acid medium (w/v) and 104 transpositional mutants were obtained. However, there were no catechol-
producing mutants. Only one colony, which accumulated yellow pigment, which might be probably due to production of HMS, was
observed. The transposon got inserted to the recipient plasmid at the other place where it blocked the action of HMS degrading enzymes
and the yellow pigment was accumulated. Limited duration time of study made it was difficult to obtain the potential p-toluic acid
accumulating mutant producing catechol.

Key words: transpositional mutant, transposon, p-toluic acid assimilating bacteria, catechol.

INTRODUCTION MATERIAL AND METHODS

Catechol and its derivates are important chemicals in Strain

the manufacturing of stains, photographic developers and
synthetic flavors. Although catechol is now synthesized by
oxidation of salicylaldehyde and the demethylation of
guaiacol, the production of catechol by microorganisms has
been investigated, because some bacteria metabolize
aromatic compound from catechol (Kodama et al., 1997).
The production of catechol by microorganisms has several
advantages in comparison with organic synthesis. Catechol
can be synthesized from inexpensive substrate such as
phenol, benzoic acid, and benzene at atmospheric pressure
and ordinary temperature (Kodama et al., 1996). Trans-
positional mutants of aniline-assimilating Pseudomonas sp.
AW-2 produced catechol and accumulated it in a cultural
medium. Strain B-9 produced a maximal amount of
catechol 0.97 mg/ml from 1 mg/ ml of aniline in a 13.5 h
growing culture.
In this study we carried out transposon mutagenesis to
obtain catechol-accumulating mutant from p- toluic acid
assimilating bacteria. The insertion of a transposon into a
target gene results in complete blockage of function of the
gene. Transposon mutagenesis brings the drop of function
of other gene constituting an operon besides the target
gene, because of s strong polar effect of the transposon
inserted (Harayama et al., 1984). In this report we describe
the isolation of transpositional mutants synthesizing
catechol from p-toluic acid.
Isolation of p-toluic acid assimilating bacteria
For the specific screening of soil bacteria, about 0.5
grams of ten soil samples from different places in Japan
were suspended in each of 5 ml of 0.8% Nail (w/v) and 0.5
ml of these soil suspension were poured into each of 10 ml
of liquid medium containing aromatic compound, p-toluic
acid 0.3%, as a sole C and N source and other mineral
supplements. Incubated at 30oC under shaking condition.
When the sufficient growth appeared, the inoculum was
streaked on a p-toluic acid 0.3% medium to obtain single
colony. Growing colonies from petridishes were transferred
on agar slant containing p-toluic acid 0.2% to obtain more
growth of the microorganisms capable of assimilating
higher concentration of the desired substrate.
Isolation and selection of antibiotic resistant p-toluic acid
assimilating bacteria
PT-14 a p-toluic acid assimilating strain isolated from
soil sample was experimented to obtained antibiotic
resistant strain and was used through out this study. Fresh
culture of PT-14 strain was spread on the solid p-toluic acid
medium with different concentration of Streptomycin
(Sm)-50 µg, 100 µg and 200 µg/ml and incubated at 30oC
for 2 days; isolated colonies of Sm-resistant PT-14 strain
were obtained. The having more resistance to Sm (200
µg/ml concentration of Sm) were transferred on agar slants
with the same concentration of antibiotics. This Sm-
 RATNAKOMALA – Isolation of bacteria producing catechol
resistant mutant was used as a recipient in transposon
mutagenesis. The recipient strain was also checked for its
sensitivity to antibiotics Kanamycin and Neomycin with
concentration 25µg and 50 µg/ml. Escherichia coli S17-1
carrying the plasmid pSUP5011 with Tn5-Mob as an insert
was used as a donor strain.
Culture media
Screening medium containing 0.3 mg/ml of p-toluic
acid divided in solution A and B, were prepared separately.
Solution A containing 0.3% p-toluic acid, 0.75% KH2PO4,
1% Na2HPO4.12H2O, 0.5% NaCl, 0.02% (NH4)2SO4,
0.02% Yeast Extract, and 1.5% Agar. Solution B
containing 0.5% MgSO4.7H2O, 0.005% FeSO4.7H2O,
0.005% CaCl2.2H2O, 0.005% CuSO4.5H2O, and 0.05%
ZnCl2. All media were autoclaved for 15 min at 120oC and
then mixed together aseptically. Luria Bertani (LB)
medium contained Polypeptone 10 g, 5 g each Yeast
Extract and NaCl and deionized water to 1 liter.
Isolation of catechol producing transpositional mutants
Transposon mutagenesis was carried out to obtain
mutants deficient in catechol 2,3-dioxygenase. The
insertion of a transposon into a target gene of the desired
bacterial plasmid results in complete blockage of function
of that gene (Simon, 1984). This implies that transposon
mutagenesis is favorable for the isolation of mutants
synthesizing catechol and its accumulation.
The transposon Tn5-Mob from E. coli S17-1 was
introduced into PT-14 strain Sm-resistant mutant by
conjugal transfer. E. coli S17-1 were subcultured in Luria
Bertani (LB) medium with Kanamycin (Km) 50 µg/ml and
incubated at 37oC for 24 hours. A growing colony was
transferred to LB broth with the same antibiotics
concentration and incubated at 37oC for 15 hours with
shaking condition. Culture of E. coli was transferred to LB
broth medium with Km 50 µg/ml in a bent tube and
incubated for 4 hours at 37oC with standing culture, until
turbidity reached 0.4 at 660 nm. The cells were
concentrated by centrifugation at 10,000 rpm for 30 sec and
supernatant was completely removed.
PT-14 Sm-resistant mutant strain was inoculated into
0,1 % p-toluic acid with Sm 200 µg/ml liquid medium and
incubated in shaker at 30oC for 36 hours. Growing culture
was transferred to the fresh liquid medium with the same
Sm concentration. After over night shaking incubation at
30oC, the culture was diluted with fresh LB liquid medium
with Sm to get turbidity between 0.3 - 0.4 at 660 nm. This
culture was kept at 30oC without shaking for 3.5 hours and
continued with gentle shaking for 30 min. The cell were
concentrated by centrifugation at 10,000 rpm for 30 sec.
Supernatant was removed and the collected cell of PT-14
Sm- resistant mutant as recipient cell were suspended
gently into E. coli S17-1 as the donor cell.
A slow mixing by pipetting was carefully done to allow
mating between donor and recipient cells. These mixtures
were dropped onto nitrocellulose filter on the LB medium
without antibiotics to allow the faster growth of cells. The
mating continued at 30oC for 3 hours. The cells on the filter
were suspended in 2.4 ml of 0.8% NaCl and 0,3 ml aliquots
79
of the suspension were spread on LB solid medium
containing antibiotics Sm 200 µg/ml and Km 50 µg/ml.
Catechol production was confirmed by dropping 4-
aminoantipyrine reagents on the plate. This reagent gives
pink color in presence of catechol. Colonies that changed to
pink color were selected as catechol accumulating mutant.
For further confirmation, pigment formation was also
checked. Growing colonies were duplicates on 0.1 % p-
toluic acid medium with Sm 200 µg/ml and Km 50 µg/ml.
Colonies could not grow on p-toluic acid medium and
produced dark brown or black pigments around the
colonies were selected as catechol accumulating mutants.
The mutants metabolized p- toluic acid through catechol
only.
The colonies, which grew well on p-toluic acid
medium, indicated that was not catechol accumulating
mutants. The bacteria maybe has an inducible enzyme,
which can synthesize catechol to another compound.
RESULTS AND DISCUSSION
Amount 14 isolates from soil samples could grow on
0.3% p-toluic acid medium. The color in some culture
medium was changed to brown/ black pigment or yellow
pigment. This might be due to conversion of p-toluic acid
by bacteria to catechol (brown/ black pigment) and 2-
hydroxymuconic 6-semi aldehyde (HMS) the intermediate
of p-toluic acid metabolisms, showed with yellow pigments
(Aoki et al., 1997).
About 104 transpositional mutants of strain PT-14 were
obtained. However, there were no catechol-producing
mutants, which confirmed by putting 4-aminoantipyrine
reagents on the pales. This reagent did not give pink color
at the mutant colonies in presence of catechol. By further
screening, brown/ black pigments formation was not
observed, which showed that there was no catechol-
producing mutants among the isolated mutants. The results
indicated that the insertion of the transposon to the
recipient plasmid did not take place at the proper place to
block the function of a gene responsible for the catechol
2,3-dioxygenase and due to which catechol could not be
accumulated.
Only one colony, which accumulated yellow pigment,
which might be probably due to production of HMS, was
observed. Further studies should be done on this mutant.
The transposon got inserted to the recipient plasmid at the
other place where it blocked the action of HMS degrading
enzymes and the yellow pigment was accumulated. The
isolation of HMS accumulating mutant needed further
confirmation. HMS is a useful chemical for synthesizing
suitable derivatives used in the production of many
chemicals such as pharmaceuticals, herbicides and dyes.
Catechol can be synthesized from inexpensive substrate
such as phenol, benzoic acid, and benzene, which be
generated by microbial metabolisms. Nitrobenzene is
degraded by Comamonas sp. JS765 via catechol to 2
hydroxymuconic 6-semialdehyde (He and Spain, 1999). 2-
hydroxymuconate, the product of the dehydrogenation of 2
80 B i o S M A R T Vol. 5, No. 2, Oktober 2003, hal. 78-80
hydroxymuconic 6-semialdehyde was degraded to pyruvate
and acetaldehyde by crude extract of Comamonas sp.
JS765, indicated the operation of a classical catechol meta-
cleavage pathway. Previously reported on the production of
catechol from aniline by transpositional mutant of the
aniline assimilating Pseudomonas sp. AW-2. Resting cells
of a mutant, strain B-9, convert 1 mg/ml (11 mM) of
aniline into catechol at conversion rate of 93% within 2 h
of incubation (Kodama et al., 1997).
Meanwhile, Solyanikova et al. (1999) reported catechol
1,2-dioxygenases of the ordinary ortho-cleavage pathway
have been isolated from strains Rhodococcus rhodnii 135
and Rhodococcus rhodochrous 89 which grown on phenol
as the sole source of carbon and energy. The activities of
the catechol 1,2-dioxygenase with 3- and 4- methylpyro-
catechols were 1.3-1.5 times higher than those with
pyrocatechol. Briganti et al., (1998) have also been purified
two catechol 1,2-dioxygenases from Pseudomonas sp. B13,
one specific for catechol and the other active towards a
broad range of halogen substitute catechol.
It is widely known that microorganisms, especially soil
microorganisms, have many sources of bioactive and
natural products. However, it is becoming difficult to
discover commercially useful secondary metabolites from
this organism. This research needs to be further continued
to discover some potential bacteria as source of various
bioactive and natural products. We believe that instead of
having scientific value, outcome of the research will also
have an economic value for industrial purposes.
CONCLUSION
The experiment was concluded with there was no
catechol-producing mutant among the isolated mutants.
The isolation of catechol accumulating mutants need long
time and much more soil source to obtain the potential p-
toluic acid assimilating mutant producing catechol.
ACKNOWLEDGEMENT
I am very grateful to Prof. Dr. K. Aoki and Dr. S.
Murakami for their valuable guidance during my laboratory
work, which is a part of Training Course on Biotechnology
at Laboratory of Applied Microbiology, Faculty of
Agriculture, Kobe University, Japan sponsored by JICA.
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