Assignment on TOL Plasmid pWW0

Department of Biotechnology COMSATS Abbottabad

Subject: Genetic Engineering

Submitted to: Dr. Rafiq
Submitted by: Jawad Ahmed
MS 1st Sem.

FA15-R02-005
Date: 05/10/2015

Fig. 1. Functional map of the TOL plasmid pWWO. The locations of Xho I and
HindSL cleavage sites are taken from refs. 14 and 15. Solid triangles, Tn5 insertions that
inactivate all or part of the Xyl/Tol degradation pathway; open triangles, insertions that
have no influence on the catabolic functions. Locations of specific genes are based on
cloning studies. Rep/ Tra, determinants of autonomous replication and coqjugal transfer
functions. m-Tot, meto-toluate (Franklin et al., 1981).

Fig. 2. Map of pWW0 genetic features identified from the DNA

sequence. The black line represents the genome from co-ordinate0
to 116 580 kb. ORFs running left to right are shown as blocks above
the line; ORFs running right to left are shown below the line.
Promoters and proposed transcriptional units based on predicted
promoters and transcriptional terminators are shown as black
arrows. Predicted functions of encoded proteins are indicated by
colour: dark green, replication and stable inheritance; light green,
conjugative transfer; yellow, transposition; red, gene regulation;
magenta, toluene/xylene degradation genes; dark blue, additional
genes with predicted function; light blue, genes predicted to be
membrane associated but with no predicted function. Uncoloured
blocks

indicate

no

predicted

function

or

obvious

sequence

characteristics (Greated et al., 2002).

Host:
The plasmid is carried by Pseudomonas putida mt-2 (Rocha et al.,
2013)
This strain has received much attention since its isolation in the early
70s because of its fascinating ability to thrive on (the otherwise quite
unpalatable) m-xylene and toluene as sole C sources. Although many other
strains have been described to grow on the same or similar hydrocarbons,
the

complexity

of

the

regulatory

network

that

orchestrates

their

biodegradation in P. putida mt-2 is quite perplexing. If the problem were only

maximizing m-xylene biodegradation, an engineer (or a synthetic biologist)
would surely consider on arraying the genes encoding the necessary
enzymatic activities one after the other to form a single polycistronic operon
and place the whole under the control of a strong inducible promoter
responsive to the pathway substrate.
Promotor:

Polycistronic operons four promoters (Pr, Ps, Pu and Pm). The most
striking feature of the regulatory architecture of the TOL plasmid is the
interplay between the two regulators (XylR and XylS) and the way they
activate their cognate promoter. Expression of XylS is under the control of
the XylR-responsive promoter Ps. This means that the presence of m-xylene
triggers both transcription of the upper pathway promoter (called Pu) as well
as overproduction of XylS. What makes this system really extraordinary is
that such an overproduction suffices to activate Pm, the promoter of the
lower pathway, in the absence of the endogenous effecto of XylS (3MBz).
This unusual property of XylS results in the simultaneous activation of the
upper and the lower operons before the substrate of the lower route has the
time to materialize (Silva-Rocha et al., 2011). The rightward promoters all
consist of the putative -35 and -10 regions (TTGACT . . . N17 . . . GATACT)
(Greated et al., 2002).

Fig. 3. The TOL catabolic pathway is encoded in two main

operons, upper and lower, expressed from the Pu and Pm promoters
respectively. The XylR regulator is expressed from the Pr promoter,
whereas XylS is expressed from Ps. When active, XylR triggers the
expression of Pu and Ps while it represses Pr. In the case of XylS, is
active form triggers the expression of Pm (Silva-Rocha and de
Lorenzo, 2012).

Fig. 4. Promotor regions of the TOL Operon.

Operators
An inverted repeat (5- TAGTCAAA . . . N5 . . . TTTGACTA-3), which
may be the operator for whatever repressor regulates transcription of this
region, is present in each of these promoter regions. Of the orfs in the
rightward direction, the product of orf26 shows strong similarity to a
cytotoxin from a P. aeruginosa phage, phiCTX. The first two leftward ORFs (18
and 19) encode products showing strong similarity to two hypothetical
proteins from P. fluorescens plasmid pRA2, which are also encoded together.
Restriction Sites: Restriction sites for three restriction endonucleases
are present in the TOL Plasmid pWW0 namely Sst I, Xho I and HindIII. Tsuda
and Lino (1987) also showed restriction site fot Eco RI. In another map.
Restriction endonuclease fragmentation analysis of plasmids isolated from 90
transconjugant clones revealed that all insertions that caused the Xylphenotype were clustered in one of two distinct regions of the plasmid
genome, defined by the Xho I G and the Xho I J, I, E, and D fragments, that
are separated by a DNA segment 14-kb long. Tn5 insertions that mapped
within the intervening segment did not inactivate catabolic functions.
(Franklin et al., 1981).
Replicative zones
The sector of pWW0 from co-ordinates 113 kb to 2.5 kb aligns closely
with the mini-replicon derived from another IncP-9 plasmid pM3. Plasmid
replication depends on the presence of a rep gene. The predicted Rep protein

from pWW0 is 96.2% identical to that of pMT2 (the minireplicon of pM3),

forming a novel group of Rep proteins.
GC Contents
The natural host of pWW0, Pseudomonas putida, has a chromosomal G+C
content of 60%. Although the pWW0 genome has on average a G+C
content of 59%, certain segments differ significantly from this mean
Translation Products
A total of 210 open reading frames (ORFs) were identified by the
presence of an initiation (ATG, GTG or TTG) and a stop (TAA, TAG or TGA)
codon and an uninterrupted coding region usually at least 60 amino acids in
length.
TOL pWW0 as Expression vector
TOL plasmid pWW0 degrades the toxic Toulene into tricarboxylic acid.
The pathways is as follows

Fig. 5. Pathway for degradation of toluene encoded

by

Pseudomonas plasmid pWWO. Chemical intermediates are listed to

the left of the pathway, while the specific degradation genes and

the abbreviations of the enzymes that they encode are to the right
(Burlage et al., 1989).
Marker:
Resistance to Tellurite has been used as a Selection Marker for Genetic
manipulation by Romero et al., (1998). Antibiotic resistance marker
kanamycin has also been used as selection marker.The TOL plasmid pWWO
is able to directly mobilize and retromobilize a kanamycin resistance marker
integrated into the chromosome of other P. putida strains, a process that
appears to involve a single conjugational event.
Overall Organization of Plasmid and Mobile Elements
When the nucleotide sequences of known transposable elements are
added
picture,

into
an

transposition

interesting
functions

modular
lie

within

the

organization
the

emerges.

boundaries

of

All

the

identified
transposon

designated Tn4653(co-ordinates 2193292664), which is defined at one end

by tnpA/tnpR functions closely related to those of Tn501 and at the other end
by an inverted terminal repeat closely related to that of Tn501, preserving
the EcoRI sites characteristic of the ends of this transposon family. These
mobile elements are responsible for the selectable catabolic phenotype of
the plasmid as well as for the ability to mobilize or retromobilize
chromosomal genes (RamosGonzalez et al., 1994; Ronchel et al., 2000).
Tn4653 is bounded by 5 bp direct repeats implying a simple insertion event.
Tn4653 appears to be the product of at least two insertion events into what
can be imagined as an ancestral transposon, Tn4653A. The more major of
these was the insertion of Tn4651 (co-ordinates 2722583278), which itself
is based on a smaller transposon Tn4652, a 17 kb derivative of Tn4651 that
lacks the 39 kb region encompassed by the two copies of IS1246 (36836
38111 and 7587977153) and was identified in the chromosome of P. putida
PaW85 (Tsuda and Iino, 1987). Tn4652 has been sequenced by H. M. Tan,

unpublished results (accession no. AF151431), and translation products are

identical to the equivalent putative proteins encoded by Tn4651. Insertion of
Tn4651 or Tn4652, depending on the sequence of events, appears to have
been a simple one, as it is again flanked by direct repeats of 4 or 5 bp
(depending on how you interpret the flanking sequences). Finally, an
additional putative insertion sequence of 3471 bp (defined by its terminal
inverted repeats), IStol (Shaw et al., 2002), runs from 84397 to 87768. It is
not flanked by identifiable direct repeats, so it may not be the result of a
simple insertion. One can therefore reconstruct the probable ancestral
sequence in Tn4653A. Insertion of the segment flanked by repeated copies of
IS1246 also appears to be a simple one, being flanked by 5 bp direct repeats
of ATAAA, so it is also possible to reconstruct the ancestral Tn4652 without
the IS1246 insertion.
Both Tn4653 and Tn4652 move independently from each other, and
both have characteristics of class II transposons (Grinsted et al., 1990; Tsuda,
1996). Tn4651 was shown to belong to a novel class II transposon subgroup
with a res region encoding two proteins, TnpS and TnpT, required for
resolution. Overexpression of tnpA is pre vented by the repressor protein
TnpC encoded directly downstream from the tnpA gene. Tn4651 can be
converted to Tn4652 when a 39 kb region, including the xyl genes, is lost as
a result of deletion by recombination between the two copies of IS1246.
Within both insertion sequences, an ORF was identified showing significant
identity to genes encoding transposases common to other IS elements
(Reddy et al., 1994). Removal of the 39 kb region does not affect the
transposition functions of the, now cryptic, transposon (Tsuda, 1996). Tn4653
encodes its own transposase and resolvase (TnpR). However, the res region
upstream of the tnpR of Tn4653 is defective because of the lack of one of the
three resolvase binding sites essential for co-integrate resolution (Allmeier et
al., 1992). Hence, Tn4653 shares tnpT, tnpS and res with Tn4651. On the

other hand, tnpR of Tn4653 is important for the ability of pWW0 to promote
retromobilization of chromosomal DNA (Ronchel et al., 2000).

References
Antoine, R., and Locht, C. (1992) Isolation and molecular characterisation of
a novel broad-host-range plasmid from Bordetella bronchiseptica with
sequence similarities to plasmids from Gram-positive organisms. Mol
Microbiol 6: 17851799.
Franklin, F.C.H., Bagdasarian, M., Bagdasarian, M.M., and Timmis, K.N. (1981)
Molecular

and

functional

analysis

of

the

TOL

plasmid

pWW0

from

Pseudomonas putida and cloning of genes for the entire regulated aromatic
ring meta cleavage. Proc Natl Acad Sci USA 78: 7458 7462.
Fullner, K.J. (1998) Role of Agrobacterium virB genes in transfer of T
complexes and RSF1010. J Bacteriol 180: 430434.
Greated, A., Titok, M., Krasowiak, R.M., Fairclough, R.J., and Thomas, C.M.
(2000) The replication and stable inheritance functions of IncP-9 plasmid
pM3. Microbiology 146: 22492258.
Ramos-Gonzalez, M.-I., Duque, E., Ramos, J.L., 1991. Conjugational transfer
of recombinant DNA in cultures and in soils: host range of Pseudomonas
putida TOL plasmids. Appl. Environ. Microbiol. 57, 3020 e3027.
Ronchel, M.C., Ramos-Diaz, M.A., and Ramos, J.L. (2000) Retrotransfer of DNA
in the environment. Environ Microbiol 2: 319323.
Silva-Rocha R, de Lorenzo V. (2012). Stochasticity of TOL plasmid catabolic
promoters sets a bimodal expression regime in Pseudomonas putida mt-2
exposed to m-xylene. Mol Microbiol; e-pub ahead of print 30 July 2012
Tsuda, M. (1996), Catabolic transposons in Pseudomonas. In Molecular
Biology of Pseudomonads. Nakazawa, T., et al. (eds). Washington, DC:
American Society for Microbiology Press, pp. 219228.