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 70’s 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 Xyl-

phenotype 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 ª 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 into the

picture, an interesting modular organization emerges. All identified transposition functions lie

within the boundaries of the transposon designated Tn4653(co-ordinates 21932–92664), 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 27225–83278),

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 75879–77153) 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

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range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from

Gram-positive organisms. Mol Microbiol 6: 1785–1799.

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.

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Ronchel, M.C., Ramos-Diaz, M.A., and Ramos, J.L. (2000) Retrotransfer of DNA in the

environment. Environ Microbiol 2: 319–323.

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

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Press, pp. 219–228.