GB2220942A - Genetic modification of escherichia coli - Google Patents

Abstract

A method of modifying the characteristics of Escherichia Coli comprising manipulating a gene in its DNA that is responsible for determining its physical and morphological properties is described. The gene is the morgene (previously known as the fluB gene) or its equivalent and it may be deactivated or enhanced in its activity. Preferably the manipulation involves isolation of the morgene by cloning a segment of DNA containing it in order to form a plasmid.

Description

GENETIC MODIFICATION OF ESCIlERICIIIA COLI This invention relates to methods for the genetic modification of the microorganism Escherichia coli (herein abbreviated E.coli) and to genetic material, ie DNA sequences produced using the method.

Industrial microbiological fermentation is becoming an increasingly important process for the production of useful products.

One type of such fermentation employs microorganisms which have been "transformed" by the insertion of extraneous DNA which expresses the product.

In order to isolate the product in a pure form from the culture a number of 'purification steps are necessary "downstream" from the fermentation stage. These steps involve separation of tie cells from the medium, and if the product is to be extracted from the cells themselves, disruption of cell membranes.

Separation of cells is generally accomplished by one or more of a number of techniques, which are made easier if the cells have certain desirable characteristics. For example sedimentation, centrifugation and filtration are assisted if the cells are of a type which aggregate to form clumps. Electrical methods of separation, and liquid/liquid two-phase extraction are influenced by cell surface charge and hydrophobicity, for example a high hydrophobicity assists partitioning between aqueous and non-aqueous liquid phases. Disruption of cells to release their contents depends upon rupture of the cell membrane, either total lysis or rupture only of the outer membrane, and is obviously made easier by a cell membrane that is more susceptible to lysis by conventional techniques such as thennal or osmotic lysis.All of these cell characteristics are related to the nature of the cell membrane.

Whatever the characteristics of the cell are, it is highly desirable tllat they remain fixed and do not fluctuate prior to the separation anci/or disruption process.

E.coli is a particularly useful microorganism for industrial microbiological fermentations as it is widely available, relatively easy to culture and handle, and is amenable to transformation.

Certain useful strains of E.coli K12 are unstable in that they spontaneously switch between two states having different. properties as below: State 1 State 2 Colony morphology rough smooth Presence of pili no yes Cells aggregate yes no Hydrophobicity higher lower Surface charge lower higher This spontanous interconversion between two unstable states having different physical properties is clearly a disadvantage to separation of the cells from a culture medium.

Interconversion has been attributed (Diderichsen 1980) to the operation of a gene in the E.coli chromosome designated the flu gene, located between his and shi A on the E.coli chromosome.

Deletion of an extensive region of the chromosome in a region including the arg H gene was found to fix the E.coli in an aggregating form1 but no further characterisation of the E.coli to assess whether this extensive deletion has had any adverse effects on the microorganism was presented.

Later workers (Warne & Bowden 1987) identified a gene in the E.coli chromosome which is implicated in interconversion, and which lies in a different region of the chromosome from the flu gene. This gene is located between the arginine operon (arg) and the btu B gene.

It was initially designated flu B but for convenience is designated herein mor. Cells in which flu B were deleted flu B, were found to aggregate and have a significantly reduced surface charge (zeta potential). The technique used to isolate the flu B gene was however 'cosmid cloning', a technique that isolates large regions of the chromosome, up to about 50 kb. The results of cosmid cloning therefore do not enable the position of the flu B gene in the E.coli chromosome to be determined with sufficient accuracy to allow it to be genetically modified with precision whilst avoiding unpredictable effects on other E.coli genes in the chromosome.

It is a principal object of the present invention to control the E.coli mor gene whilst avoiding excessive and possibly disadvantageous deletion of regions of the chromosome. This has been achieved inter alia by the more precise location and structural analysis of the mor gene of E.coli reported herein. Other objects and advantages of this invention will be apparent from the following.

According to a first aspect of this invention, there is provided a method of modifying the characteristics of E.coli which includes the step of manipulation of the E.coli mor gene.

Manipulation may be to deactivate the mor gene, for example by replacing the mor gene in the E.coli chromosome with a DNA sequence in which either (a) a disrupted mor gene is included. or (b) the mor gene is wholly or partly deleted. Alternatively manipulation may be to increase the effect of the mor gene by introducing extra copies of the mor gene into E.coli or/and causing extra promoters to act upon the mor gene.

A preferred method of manipulation of the mor gene includes the steps of deactivating the E.coli mor gene by isolating a frag ment of the E.coli chromosome which contains the mor gene, then deactivating the mor gene in the fragment by either: a. disrupting the DNA sequence of the E.coli mor gene, or b. deleting all or part of the E.coli mor gene, then resinserting all or part of the said fragment into an E.coli chromosome, An alternative method of manipulation of the mor gene is to manipulate the mor gene in situ in the E.coli chromosome by chemical or transposon mutagenesis.

The said mor gene is the gene located in the region of the E.coli chromosome between the Bam H1 and Eco Ri sites that are in turn located between the arg EBCH and trm A loci in the E.coli chromosome and which includes the DNA sequence between the ATG (start) codon and the TAA (stop) codon shown in table 1 below, or a DNA sequence derived therefrom and having an equivalent function in the E.coli chromosome.

By 'derived therefrom' is meant a DNA sequence which contains codons which are riegenerate with those of that sequence (ie having a different DNA sequence but coding for the same amino acid), or a sequence containing some extra bases in addition to those listed, or lacking some bases relative to those listed provided the gene performs an equivalent function in the E.coli chromosome. 'Derived therefrom' is also meant to include generally a gene having a 70%o or greater conformity to that sequence, eg 80%, 90% or greater.

The structure of the region of the E.coli chromosome in which the mor gene is located, at around 89.5 minutes, is shown in Fig 1 of the accompanying drawings. A Sal I site lies approximately 1.S kb to the right (ie towards the btu B end) of the Eco R1 site at 6.0 kb in Fig 1. The position of any other Sal I sites, if any, in this region have not yet been determined. The DNA sequence of the approximately 2.3 kb Bam H1 to Eco R1 fragment which contains the mor gene of E.coli strain BD 1512 (supplied by B. Diderichsen, Novo Ltd. ) has been determined and is listed in Table 1 below, in which the start(ATG) and stop (TAA) codons are identified.It will be understood by those skilled in the art that in other E.coli strains, some degeneracy of this sequence may occur but without substantial alteration of the performance or position of the gene.

Of options a and b indicated above, option a , ie disruption of the mor gene is preferred. Disruption of the mor gene is preferably accomplished by inserting a disrupting DNA sequence into the mor gene. This disrupting sequence preferably has a genetic marker in it to assist selection of strains containing the disrupting sequence. Such a marker may be inserted into the disrupting sequence before or after insertion of the sequence into the mor gene.

The disrupting sequence should preferably be inserted into the DNA of the mor gene, preferably at a position between 0.2 and 1.1 kb from the Bam Hl end of the 2.3 kb Bam H1 to Eco R1 fragment which contains the mor gene.

The disrupting DNA sequence may be any DNA sequence which partly but preferably completely prevents the mor gene from operating, and which preferably does not adversely influence the E.coli chromosmme.

Suitable DNA sequences to achieve this will be known to those skilled in the art. The sequence may or may not contain codons which are transcribed in E.coli. Such a sequence may conveniently be inserted into the mor gene by cleavage and insertion of the sequence at restriction sites in the mor gene sequence which will be apparent to those skilled in the art from table 1.

An alternative method for inserting a disrupting DNA sequence into the mor gene is to use a transposon, ie a fragment of DNA that has the ability to insert itself into other DNA sequences such as chromosomal DNA. Many transposons are known, for example Tn 1737 Cm (Ubben & Schmitt, 1987), a transposon which contains the coding seq uence for -galactosidase at one end. Methods for insertion of tr- ansposons are known. Insertion of transposons generally occurs at random positions in the DNA sequence so it will normally be necessary to select for sequences in which the transposon has inserted itself into the mor gene in positions which cause disruption.

A further consequence of the use of an inserted transposon to disrupt the mor gene is that it is generally necessary to deactivate the DNA sequ ences of the transposon that are involved in transposition so that no further transposition takes place. This too may be achieved by the use of restriction enzyme cleavage and insertion of a disruption DNA sequence into appropriate positions in the transposon. Conveniently the disrupting sequence reinserted into the transposon may contain the genetic marker. In the case of Tn 1737 Cm for example the tnp R and tnp A genes may be removed and replaced by a marker.

A number of suitable genetic markers may be used, but a preferred form of marker is a gene which confers resistence to an antibiotic, so that strains into which the disrupting sequence has been inserted may be identified by their resistence to that antibiotic.

Convenient markers are the genes which confer resistence to kanamycin (kmr), ampicillin (ampr) or tetracycline tetr).

A further method of disrupting the DNA sequence of the mor gene is to use site directed mutagenesis to alter the sequence of the codons so that the mor gene is no longer functional. For example one of the codons between the stop and start codons in table 1 may be changed into an in-phase stop codon, to halt translation part way.

Site directed mutagenesis does not easily allow insertion of a marker so selection is less easy. For this reason it is not preferred.

Insertion of a disrupting DNA sequence is preferably car-ried out by First isolating a portion of an E.coli chromosome containing the mor gene by cloning to form a plasmid, disrupting the gene and inserting any marker into the mor gene whilst this is in the plasmid, and then reinserting the disrupted mor gene into another E.coii chromosome in place of the corresponding region of that chromosome which contains the nor gene.

Conveniently a large region of the chronosome containing tile mor gene is first isolated by the cosmid cloning technique into a suitable plasmid vector, eg pEMBL cos 4 (Hadfield, 1987). Smaller regions of the cloned E.coli chromosome containing the mor gene may then be sub@loned into t piasmid, preferably a plasmid which is capable of replication in E.coli. A number of such plasmids are known, for example pre322 and pACYC 184. It is preferred to subclone the approximately 6.1 kb Hind III to Sal I fragment which contains the mor gene. This 6.1 kb fragment contains at its ends part of the arg ECBn operon and part of the trm A gene.

The disrupting sequence may then be inserted into the mor gene contained in the plasmids prepared by subcloning referred to above, for example Tn 1737 Cm may be inserted by the method of Ubben & BR< Schmitt (op cit).

At this stage it is convenient to insert a genetic marker, eg Kmr, into the disrupting sequence if required, and if a transposon has been inserted to deactivate its genes involved in transposition.

The disrupted mor gene may then be reinserted into another, unmodified, E.coli chromosome. The preferred method of doing this is by the known technique of honologous recombination. The preferred 6.1 kb Hind III to Sal I fragment referred to above appears to be particularly suitable as it includes ends which are homologous with parts of the E.coli chromosome but is small enough to be fairly stable. Homologous recombination is conveniently carried out by first converting the plasmid containing the disrupted mor gene into a linear form by restriction enzyme cleavage at a point which does not cleave the chromosomal DNA in the plasmid. This linear DNA is then inserted by homologous recombination into the E.coli chromosome.

If homologous recombination is used then the unmodified chromosome is preferably one which contains the recBC and sbc B mutations, which inactivate exonucleases which degrade linear DNA fragments.

Chromosomes which do not contain these mutations may still be used, but homologous recombination is likely to take place at a low frequency in their absence.

Option b. indicated above, ie preparing an E.coli chromosome in which all or part of the mor gene has been deleted may be carried out by subcloning the approximately 6.1 kb Hind III ta Sal I fragment referred to above which contains the mor gene into a suitable plasmid, and then deleting the region corresponding to the mor gene using appropriate restriction methods.

Genetic markers, eg Icm , may be inserted into the plasmid. The plasmid may then be linearised and reinserted into an ,jtimodifed E. coli chromosome by homologous recombination as dscribed above.

E-coli chromosomes and strains in which the mor gene has been deactivated by disruption or deletion as described above are terined herein mor forms.

Whether deactivation has been via disruption or deletion, mor strains may be selected for mor characteristics, or marker characteristics, eg kmr. The mor mutation may be transferred into other E.coli strains by for example carrying out transduction with bacteriophage P1 and selection, eg for kmr.

Plasmids formed during tile cloning and/or subcloning procedures of the method of the above first aspect of the invention may also be used to introduce doric or more additional mor genes into E.coli.

Therefore according to a further aspect of this invention, a method of modifying the characteristics of E.coli includes the step of introducing one or more additional copies of the mor gene into E.coli. in addition to the mor gene already naturally present in the organism.

The one or more additional copies of the mor gene may be inserted into the E.coli chromosome, using suitable restriction and insertion metheds. A preferred method however is to insert the mor gene into a plasmid vector and to introduce this recombinant vector into a host E.coli. Suitable recombinant vectors are the plasmids referred to above in which a region of the E.coli chromosome conta ining the mor gene has been subcloned into a plasmid capable of replication in E.coli. A preferred recobinant. vector is p3R322 into which has been subcloned the approximately 2@3kb

figment which includes the mor gene.To increase the effectiveness of thn mor gene in this recombinant vector an additional promoter sequence may also be included in the vector upstream of the mor gene, eg trp. Insertion of the plasmid vector into the E.coli host may be via entirely conventional methods.

The precise identification of the position Or the mor gene in the .N.coli chromosome by the inventors enables another method or modifying the characteristics of E.coli, which includes the step of inserting one or more promoters into the E.coli chromosome, in addition to the promoters present naturally in the chromosome, the promoter(s) being inserted in a position in the chromosome such that it / they act upon the er gene.

Preferab]y the promoter(s) is inserted in such a position that there are no other genes between it and the mor gene, and preferably as little intervening chromosomal DNA. The additional promoter(s) may be upstream or downstream of the mor gene's own promoter present nxt- urally in the chromosome. Insertion may be into the aPproximately 6.1 kb fragment between the 1iind III and Sal I sites which contains the mor gene, for example at the Bam HI site almost immediately upstream of the mor gene. A suitable promoter for insertion is trp.

Invention of the promoter into the chromosome is preferably achieved by first isolating a portion ot an E.coli chromosome containing the mor gene by cloning to form a plasmid, inserting the promoter(s) by restriction and ligation techniques. then reinserting the mor gene plus promoter(s) into another E.coli chromosome in place of a corresponding region or that chromosome. This may be done using the cosmid cloning, subcloning and homologous recombination techniques discussed above with reference to the first aspect of the invention.

B.coli strains which contain extra copies of the mor gene, and strains and chromosomes in which the mor gene has been amplified by insertion of one or more promoters which act upon the mor gene are termed herein mor The methods of the invention described above appear to be app- licable to all E.coli strains.

Sorne of the plasmid vectors produced in the course of the above methods may also be useful, for example to enable a' mor or mor chromosome to be produced in other E'coli strains.

The invention therefore also provides a novel plasmid, char acterisel in that it contains a DNA sequence identical to or der- ivel from that Of E.coli chromosomal DNA in the region between arg EBCII and trmA and containi ng-a DNA sequence identical to or derived from the E.coli mor gene, but containing no DNA identical to or derived from E.coli chromosomal DNA from outside this region.

The DNA cqence is preferably identical to or derived from the DNA sequence of the approximately 6.1 kb Hind III to Sal I fragment or the approximately 2.3 kb Bam HI to Eco RI in this region which contains a DNA sequence identical to or rlerived from the E.coli mor gene.

The invention also provides a novel plasmid, characterised in that it contains a DNA sequence identical to or derived from that of the E.coli mor gene except in that the DNA sequence is disrupted.

In this plasmid the said DNA sequence may be disrupted by insertion or a disrupting DNA sequence, or as a result of site-directel mutagenesis. The plasmid preferably contains a DNA sequence identicsl to or 1erived from E.coli chromosomal DNA in the approximately 6.1 kb Mind III to Sal I fragment or the approximately 2.' kb Bam Hl to Eco Pl fragment which contains the E.coli mor gene in the E.coli chromosome, except in that the DNA sequence of the mor gene is disrupted.

The invention also provides a novel plasmid characterised in that it contains a DNA sequence identical to or derived from that or the E.coli chromosome in the region of the mor gene, except in that all or part of the mor gene is absent.

This plasmid preferably contains a tSA sequence identical to or derived from the approximately 6.1 kb Hind III to Sal I fragment or the approximately 2.3 kb Bam HI to Eco R1 which contains the E.coli mor gene in the E.coli chromosome, except in that all or part or the mor gene is absent.

The invention also provides a novel nlasmiS characterised in that it contains a DNA sequence identical to or derived from that Or the E.coli mor gene and having one or more promoters positio -ned upstream of the said DNA sequence in addition to any promoter(s) normally included in the sequence.

In each of the above four classes of novel plasmid, the DNA sequence is preferably clone7 into a rlasmii which is capable of replication in E.coli, such as pBR322 or pACYC 184. These novel plasmids may also contain one or more genetic markers, eg an antibiotic resistance gene. eg kmr. These plasmids may be prepared using the methods outline above, and examples of them are plasmis pSRW 220 and pSPW 220.1 shown in figures 3 and 4 of the accompanying drawings.

A further type of novel genetic material produced in the methods of the invention described above is the modified E-coli chromosomes themselves. Accordingly the invention further provides an E.coli chromosome in which the DNA sequence of the mor gene is disrupted, or in whic!i the mor gene is absent, or which contains one or more additional promoters upstream of the or gene.

Finally the invention also provides novel strains of the microorganism .coli, characterised in that either: a. its mor gene has been disrupted or replaced by a mor gene which has been disrupted, or: b. its chromosome lacks the whole or part of the mor gene, or: 0. it contains two or more genes having a DNA sequence identical to or derived from the NA sequence or the mor gene. or: 1. it contains one or more promoters acting upon the mor gene in addition to the promoter normally acting on the mor gene in its chromosome.

Strains a and b are thus mor, c and d are mor . These four strains may be prepared by means of the methods described above.

Mor strains have a number of advantageous properties. They are generally stable, ie they do not switch between states l and 2 described above. They generally show stable characteristics of state 1, ic a rough colony morphology, aggregation, reduced surface charge, increased hydrophobicity, and non-formation of type 1 pili.

They also show an unexpected advantage in that their outer cell membrane is weakened relative to the state 2 form of E'coli, whilst in unmodified E coli there is no difference in cell membrane strength between the two states.

Mor strains also have the advantageous properties of stable state 2 characteristics, ie smooth colony morphology, non-aggregation, increased surface charge, reduced hydrophobicity and formation of type 1 pili in static.culture.

Mor and mor strains of E'coli therefore have increased usefulness in the cell separation and disruption stages of industrial fermentations, for the reasons discussed above. Although mor strains are likely to be the more useful, there are situations in which the alternative characteristics of the mor+ strain are preferred.

These novel mor and mor strains of E.coli may be subjected to additional genetic modification, for example further modifications to the chromosome, or transformation by suitable plasmid vectors, which may be known or novel, which result in furtber improved strains of F..coli and/or expression of a useful expression product.

The invention will now be described by way of example only with reference to the following figures: Fig 1. Shows the structure of the E.coli chromosome in the region in which the mor gene lies.

Fig 2. Shows the positions and effect of a number of independent insertions of Tn. 1737 Cm into the 2.3 kb EcoRl - Bam Hl fragment of the E.coli chromosome.

Fig 3. Shows the structure of plasmid pSRW 220 Fig 4. Shows the structure of plasmid pSRW 220.1 Fig 5. Shows the structure of plasmid pSRW 231 Fig 6. Illustrates the homologous recombination procedure.

Experimental details (i) Cloning of the mor gene The mor gene was initially cloned by the cosmid cloning technique using the vector pEMDL cos 4 (Hadfield.C (1987).

A 6.1 kg Hind III - Sal I fragment was then subcloned from the cosmid isolate into pBR322 (Bolivar et al (1977)). This 6.1 kb fragment was siown to contain tie mor gene. Tlie plasmid formed by inserting the 6.1 kb fragment into pBR322 was designated pSRW 220.

Further sub-cloiiing from pSRW220 revealed that the mor gene could be isolated on a 2.3 kb Eco nl to Bam III fragment. The plasmid formed by Inserting this 2.3 kb fragment into pBR322 was designated pSRW226. The structure of pSRW 220 is shown in rig 3.

The presence of the mor gene on botli pSRW220 and pSnW22G was demonstrated by transforming these plasmids into the mor strain BDL302 (D@derichien 1980). It was shown that either of these plasmids converted this aggregating strain into one that was fixed in a non-aggregating form. Aggregation was measured in terms of the ability of cells grown in Miller's LD-broth (ingredients per litre: 10g Peptone 140, 5g Yeast extract, 10g Sodium Chloride) to sediment from a static suspension. In cases where cells settled out to form a sediment in 1 to 3 hours aggregation was taken to Iiave occurred.In addition it was found that plasmid containing strains were fixed in a glossy colony morphology when grown on Miller's LB agar (Ingredients per litre: log Peptone 140, 5g Yeast extradt, lOg Sodium chloride, 12g Agar) or MacConkey agar (supplied by Oxoid Ltd) as compared to tie rough colony morphology of the mor strain.

(ii) Precise mapping of the mor gene.

The precise location of tile mor gene within the 2.3 kb Bam 111 Eco ni fragment was determined by transposon mutagenesis of both pSRW 220 and PSRW 226 with Tn 1737 Cin (Ubben and Schmitt, 1987) which hos Lie coding sequence for /3 - galactosidase located at one end.

There is no promoter for the ss- galactosidase coding sequence within the transposoll and it only becomes activated if the transposon becomes inserted downstream from a promoter in the correct orientation.

Using this transposon it is possible to not only map the position of a particular gene by insertional inactivation but also to determine its direction of transcription by measuring ss - galactosidase activity.

The results of the transposon Inutagenesis are summarised in Figure 2 which indicates the positions of a number of independent insertions of Tn 1737 Cm into the 2.3 kb EcoR1 - Bam ill fragment.

Transposon inserts which brought about insertional inactivation are marked with a /. Tile position of the ss- galactosidase open reading frame is shown for each insert. Those cases where read through transcription from the chrornosomal DNA gave rise to galactosidase activity are marked with a +. Those cases where no ss galactosidase activity was observed are marked with -.

it can be seen rrom Figure 2 that inserts pSRw226.6, pSRW226.3, pSRW226.5 and pSRW220.1 brought about insertional inactiva tioti of the mor gene, of these only inserts pSRW220.1 and pSRW226.3 were positive for ss - galactosidase. These results therefore demoustrated that the mor gene is located towards the Bam lil end of the 2.3 kb fragment and that transcription proceeds from tiiis end towards the EcoRl end. Inserts pSRW226.6 and pSRW226.3 were particularly interesting since although they both caused insertional inactivation and were in the same orientation only insert pSRW226.3 was positive for ss - galactosidase.This indicated that insert pSRW226.6 lay within the promoter sequence of the mor gene.

Tie sequence of the 2.3 kb Econl - Dam 111 fragment has been determined and from the transposon mutagenesis data it has been possible to readily identify the coding sequence corresponding to tlie mor gene. This coding sequence encodes a protein of 305 amino acids. Tie sequence is presented in Table 1 and tie stop and start codons are ringed.

(iii) lnscrtion of an antibiotic resistance marker into the chromosomal mor gene.

This was done by making use of one of the transposon insertion derivatives described in section (li) above.

A diagram of this insertion derivative, pSRW220.1 is shown in Fig 4 in which chromosomal DNA is indicated by the shaded area, T@ 1737 Cm DNA is indicated by the unshaded box and pBR322 DNA is indicated by the thin black line.

The kanamycin resistance gene of Tn 5 was inserted into pSRW220.1 by inserting the 1.2 kb Sma I fragment of pUC4-K Barang.F. (1985)) between the two Stu I sites of PSRW220.1.

The resulting plasnid was designated pSRW231 and is shown in Fig 5 t in which the source of the DNA is denoted in the same way as in Fig4, the 1.2 kb fragment from pUC4-K1XX containing the Kmr gene from Tn 5 is indicated by striped striped shading.

(iv) insertion of the inactivated mor gene into the E. coli chromosome.

This was done by first converting the plasmid pSRW231 to a linear form by cutting it with the restriction enzyme Tth III I and lien transferring tie linear DNA into the strain N2026 (obtained from Dr R Lloyd, University of Nottingbam) containing the recBC and @bc B mutations. This method for the integration of modified DNA into chromosome has previously bten described by Jasin and Schimmel (1984) and Orndorff et al (1985). Integration takes place by homologous recombination between the unmodified DNA at either side or the genetically engineered segment.

This is shown schematically in Fig 6, tEle source of tile components of pSRW 231 being identified by shading as in Fig 5. Homologous recombination occurs between the regions indicated X in Fig 6, to form an mor chromosome.

(v) Properties of tile mor E-coli strains strains of E. coli which had an mor chromosome were grown on Miller's LB broth and agar. They showed the following properties: a. The cells no longer showed any tendency to switch between two states.

b. A rough colony morphology was exhibited on solid media, ie the colonies were all dull and no smooth colonies with glossy surfaces that reflected incident light were formed.

c. The cells were fixed in an aggregating form. The cell clumps which are formed sediment from a suspension d. Type 1 pili were not formed when cells were grown in static culture.

e. TI0e cells had.an increased surface hydrophobicity and a reduced surface charge compared to the nonaggregating form. The increased hydrophobicity was demonstr- ate@ by both observing partitioning of the cells between a hexadecane / water two phase system (Rosenberg, 1984) and by hydrophobic interaction chromatography (Faris et al 1983).

Surface charge was measured using a Zeta Sizer (supplied by Malvern Instruments Ltd.) r. rue cells had an increased susceptibility to treatments wi,ici released internal protein. For example to totally lyse normal E-coli via thermal lysis a temperature of about 90 C is required (Watson et al.

1987). Mor strains of E.coli could be lysed at about 60 C. Rupture of the outer membrane of E.coli whilst leaving leaving the inner cytoplasmic membrane intact, to release periplasmic protein, by osmotic shock was also studied using the method of Neu and Ileppel . 1965. In this case. under identical conditions the yield of periplasmic proteins from mor strains of E.coli was about three times that from normal E.coli.

The advantageous properties of mor strains of E.coli are therefore demonstrated.

(vi) Properties of the mor E.coli strains.

Transformation of pSRW220 or pSnW226 into E.coli strain BD102 using conventional methods produced an mor strain (see (i! above. These strains had the following characteristics: a. All colonies are smooth with glossy surfaces when grown on solid media.

b The cells were fixes in a non-aggregating form with reduced surface hydrophobicity and increased surfaced charge relative to the corresponding aggregating form (see (v) e above).

c. Type 1 pill are formed when cells are grown in static culture.

References Warne & Bowden (1986) "Separation for Biotechnology" Ed. by Verrall M.S and Hudson M. J, Pub. Ellis Horwood Ltd. Chichester UK.

Rosenberg M. FEMS Microbiology Lett. (1984) 22 p289-295 Faris A. Lindall M. Ljungh A. Old D. C, Wadstrom T. J. Appl.

Bacteriology (1983) 55 97-100.

Neu R. C. and Heppel L. A, j. Biol. Chem (1965) 240 @635-3692.

Dierichsen.B, J. Bact. (Feb 1980), 14 (2). 858-867 Ubben & Schmitt, Gene (1987), 53, 127-134.

Hadfield.C, Sene Cloning and Analysis- a Laboratory Guide, (ed. G.Boulnois, pub. Blackwell Scientific) (1987) Jasin & Schimmel, J-Bact. (1984), 159, 783-786.

orndorff et al, J.l3act. (1985), 1G4, 321-330.

Table 1.

1 GGATCCTGGA GATCCGCAAA AGTTCACGTT GGCTTTAGTT AT@CGAG@@@ 51 AGAAACTCTC GAAACGGGCA GTGACTTCAA GGGTTAAAAG AGGTGCCGCT 101 CCGTTTC@GT GAGCAATTAT CAGTCAGAAT GCTTGATAGG GATAA@CGTT 151 CATTGCTATT CTACCTATCG CCATGAACTA TCGTGGCGAT GGAGGATGGA 201 TA@TG@A@AT TCGTGATCTT GAGTACCTGG TGGCATTGGC TGAAC@CCGC 251 CATTTT@GGC GTGCGGCAGA TTCCTGCCAC GTTAGCCAGC CGACGCTTAG 301 CGGGCAAATT CGTAAGCTGG AAGATGGGCT GGGCGTGATG TTGCTGGAGC 351 GGACCAGCCG TAAAGTGTTG TTCACCCAGG CGGGAATGCT GCTG@@GGAT 401 CAGGCGCGTA CCGTGCTGCG TGAGGTGAAA GTCCTTAAAG AGATGGCAAG 451 CCAGCAGGGC GAGACGATGT CCGGACCGCT GCACATTGGT TTGATTCCCA 501 CAG@@GGACC GTACCTGCTA CCGCATATTA TCCCTATGCT GCACCAGACC 551 TT@CCAAAGC TGGAAATGTA TCTGCATGAA GCACAGACCC ACCAGTTACT 601 GGCGCAACTG GACAGCGGCA AACTCGATTG CGTGATCCTC GCGCTGGTGA 651 AAGAGAGCGA AGCATTCATT GAAGTGCCGT TGTTTGATGA GCCAATGTTG 701 CTGGCTATCT ATGAAGATCA CCCGTGGGCG AACCG@GAAT GCATACCGAT 751 GG@@GATCTG GCAGGGGAAA AACTGCTGAT GCTGGAAGAT GGTCACTGTT 801 TGCGCGATCA GGCAATGGGT TTCTGTTTTG AAGC@GGGGC GGATGAAGAT 851 ACACACTTCC GCGCGACCAG CCTGGAAACT CTGCG@AACA TGGTGGCGGC 901 AGGTAGCGGG ATCACTTTAC TGCCAGCGCT GGCTGTGCCG CCGGAGGCCA 951 AACGCGATGG GGTTGTTTAT CTGCCGTGCA TTAAGCCGGA ACCACGCCGC 1001 ACTATTGGCC TGGTTTATCG TCCTGGCTCA CCGCTGCGCA GCCGCTATGA 1051 GCAGCTGGCA GAGGCCATCC GCGCAAGAAT GGATGGCCAT TTCGATAAAG 1101 TTTTAAAACA GGCGGTTTAA ACCGTTTAAC GCAGCTACCC GATAGCTTCG 1151 CCATCGTCGG GTAGTTAAAG GTGGTGTTGA CGAAGTACTC AATAGTGTTG 1201 CCGCCA@CTT TCTGTTCCAT AATCGCCTGA CCGATATGAA TAATTTCCGC Table 1 (contd.) 1251 AGCG@GCTCG CCAAAGCAGT GAATACC@AG AATCTCTTTT GTTTCCCGAT 1301 GGAACAAAAT TTTCAGCGTG CCCACGTTCA TGCCGA@GAT TTGTGCGCGT 1351 GCCAGATGTT TAAACTGGGC GCGGCCCACT TCATATGGCA CTTTCATTGC 1401 GGTCAGCTGC TGTTCGGTTT TGCCCACAGA GCTGATTTCC GGGATGGTGT 1451 AAATACCGGT AGGGATATCT TCAATCAGAT GTGCGGTGGC TTCGCCTTTT 1501 ACCAGCGCCT GCGCGGCAAT GCGCCCCTGG TCATAGGCCG CCGACGCCAG 1551 GCTCGGATAA CCAATCACGT CGCCCACCGC GTAAACGTGT GGCTGTGCGG 1601 TCTGATACAT GCTGTTGACC TTCAGCTGTC CGCGGCTGTC AGTTTCTAGC 1651 CCAATGTTCT GTAACGCCAG CGAATCGGTA TTACCGGTGC GACCGTTGGC 1701 ATAGAG@AGG CAGTCAGCTT TCAGTTTTTT ACCCGACTTC AGATGCATGA 1751 TCACACCATC GTCACAG@CT TCGATCTTCT CGTACTCTTC GTTGTGACGA 1801 ATCACTACGC CACTGTTCCA GAAGTGATAG GAGAGAGAAT CTGACATCTC 1851 TTGATCGAGA AATGCCAGCA GGCGATCGCG GGTGTTGATC AGATCCACTT

Claims (24)

1 A method of modifying the characteristics of Escherichia Coli comprising manipulating a gene in its DNA responsible for determining its physical and morphological properties wherein the gene is the Escherichia Coli mor gene as herein before defined or a gene derived therefrom or having an equivalent function in the Escherichia Coli chromosome.

2 A method according to Claim 1 wherein the Escherichia Coli is of strain type K12.

3 A method according to Claim 1 or Claim 2 wherein the manipulation involves: (a) deactivating the mor gene in the Escherichia Coli chromosome by replacing it with a DNA sequence in which a disrupted mor gene is included or in which the mor gene is wholly or partially deleted or (b) increasing the effect of the mor gene by introducing extra copies of it into the Escherichia Coli or by causing extra promotors to act upon it.

4 A method according to Claim 3 wherein the mor gene is manipulated by isolating a fragment of the Escherichia Coli chromosome containing it and then deactivating it by disruption before reinserting all or part of said fragment into an Escherichia Coli chromosome.

5 A methcd according to Claim 3 wherein the mor gene is manipulated by isolating a fragment of the Escherichia Coli chromosome containing it and deleting the mor gene from it before reinserting all or part of said fragment Lnto an Escherichia Coli chromosome.

6 A method according to Claims 4 or 5 wherein the fragment of tfic chromosome is of approximately 6.1 kilobases, lies between Hind III and Sal I restriction sites and has as its ends part of the argECSH operon and part of the trma gene.

7 A method according to Claims 4 or 5 wherein the fragment of the Escherichia Coli chromosome is of approximately 2.3 kilobases and lies between Bam H1 and Eco R1 restriction sites, the whole fragment lying within the fragment described in Claim 6.

8 A method according to any one of Claims 4 to 8 wherein the deactivation is performed by chemical or transposon mutagenesis.

9 A method according to any one of Claims 4 to 8 wherein the deactivation is performed by disrupting the mor gene by the insertion of a disrupting DNA sequence.

10 A method according to Claim 9 wherein the disrupting sequence is inserted at a position between 0.2 and 1.1 kilobases from the Bam H1 end of the 2.3 kilobase Bam H1 to Eco R1 fragment containing the mor gene.

11 A method according to Claim 10 or 11 wherein the mor gene is disrupted by insertion of DNA using a transposon.

12 A method according to Claims 10, 11 or 12 therein the fragment of the Escherichia Coli chromosome containing the mor gene is isolated by cloning to form a plasmid, the gene sequence is disrupted and a marker is inserted into it before the fragment is reinserted into an Escherichia Coli chromosome in place of the original sequence.

13 A method according to Claim 12 wherein a large region of the chromosome containing the mor gene is isolated by the cosmid cloning technique into a plasmid vector from which smaller regions are subcloned into a plasmid capable of replication in Escherichia Coli.

14 A method according to Claim 13 wherein the cosmid is pEFIBL cos4.

15 A method according to Claim 13 wherein the plasmid is pBR 322 or PACYC 184.

16 A method according to Claim 13 wherein the large region of the chromosome is the 6.1 kilobase Hind III to Sal I fragment.

17 A method according to any one of the Claims 10 to 15 wherein the disrupting sequence is Tn 1737 Cn.

18 A method according to Claim 3 wherein the characteristics of the Escherichia Coli are modified by introducing one or more additional copies of the mor gene into its DNA.

19 A method according to Claim 3 wherein the characteristics of the Escherichia Coli are modified by causing extra promotors of the gene to act upon it.

20 A novel plasmid containing a DNA sequence identical to or derived from Escherichia Coli chromosomal DNA in the region between argEBCH and trma and containing a DNA sequence identical to or derived from the Escherichie Coli mor gene and no other Escherichia Coli DNA.

21 A novel plasmid characterised in that it contains a DNA sequence identical to or derived from that of Escherichia Coli mor gene except in that the DNA sequence is disrupted.

22 A novel plasmid characterised in that it contains a DNA sequence identical to or derived from that of the Escherichia Coli chromosome in the region oF the mor gene except in that all or part or the gene is absent.

23 A novel plasmid characterised in that it contains a DNA sequence identical to or derived from that of Escherichia Coli mor gene and having one or more promoters positioned upstream of the DNA sequence in addition to any promoters normally included.

24 Novel strains of the organism Escherichia Coli characterised in that either: (a) their mor genes have been disrupted or replaced by a mor gene which has been disrupted or (b) their chromosomes lack the whole or part of the mor gene or (c) they contain two or more genes having a DNA sequence identical to or derived from the DNA sequence of the mor gene or (d) they contain one or more promoters acting upon the mor gene in addition to the promoter normally acting on the mor gene in its chromosome.