US 3923603 A
A transfer plasmid (factor K) is transferred from a first Pseudomonas organism to a second Pseudomonas organism. Factor K induces the mobilization of genes from the chromosome of the second Pseudomonas organism and under proper conditions will transfer the mobilized genes to a recipient organism in which those particular genes have been deleted. The factor K and the mobilized genes enter the recipient and become established there as stable separate plasmids constituting a plasmid aggregrate. Non-transmissible artificial hydrocarbon degradative plasmids have been created in Pseudomonas organisms in this manner and (with factor K present in the host to accomplish the transfer) have been transmitted to other Pseudomonas recipients.
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United States Patent 1191 Chakrabarty et al.
[ Dec.2, 1975 DISCRETE PLASMID CONSTRUCTION FROM Cl-IROMOSOMAL GENES IN PSEUDOMONAS  Inventors: Ananda M. Chakrabarty, Latham;
Denise A. Friello, Schenectady, both of N.Y.
 Assignee: General Electric Company,
221 Filed: Aug. 1, 1974 211 App] No.: 493,691
 US. Cl 195/78; 195/30  Int. Cl. C12B 1/00  Field of Search 195/96, 28 R, 1, 3 H, 30,
 References Cited OTHER PUBLICATIONS Chakrabarty Dissociation of Degradative Plasmid Aggregate In Pseudomones" J. Bacteriol, 118, 815-820 (1974).
Primary ExaminerA. Louis Monacell Assistant Examiner-R. B. Fenland Attorney, Agent, or Firm-Jane M. Binkowski; Joseph T. Cohen; Jerome C. Squillaro  ABSTRACT A transfer plasmid (factor K) is transferred from a first Pseudomonas organism to a second Pseudomonas organism. Factor K induces the mobilization of genes from the chromosome of the second Pseudomonas organism and under proper conditions will transfer the mobilized genes to a recipient organism in which those particular genes have been deleted. The factor K and the mobilized genes enter the recipient and become established there as stable separate plasmids constituting a plasmid aggregrate. Non-transmissible artificial hydrocarbon degradative plasmids have been created in Pseudomonas organisms in this manner and (with factor K present in the host to accomplish the transfer) have been transmitted to other Pseudomonas recipients.
5 Claims, N0 Drawings DISCRETE PLASMID CONSTRUCTION FROM CHROMOSOMAL GENES IN PSEUDOMONAS BACKGROUND OF THE INVENTION The terminology of microbial genetics is sufficiently complicated that certain definitions will be particularly useful in the understanding of this invention:
Plasmid A hereditary unit that is physically separate from the chromosome of the cell; the terms extrachromosomal element and plasmid are synonymous; when physically separated from the chromosome, some plasmids can be transmitted at high frequency to other cells;
Transmissible plasmid A plasmid that carries genetic determinants for its own intercell transfer via conjugation;
DNA Deoxyribonucleic acid;
Bacteriophage A particle composed of a piece of DNA encoded and contained within a protein head portion and having a tail and tail fibers composed of protein;
Deletion The condition in which a substantial portion of the chromosomal DNA is missing;
Transducing phage A bacteriophage that carries fragments of bacterial chromosomal DNA and transfers this DNA on subsequent infection of another bacterium;
Conjugation The process by which a bacterium establishes cellular contact with another bacterium and the transfer of genetic material occurs;
Curing The process by which plasmids can be selectively eliminated from the microorganism;
Curing agent A chemical material or a physical treatment that enhances curing;
Degradative'pathway A sequence of enzymatic reactions (e.g. 5 to enzymes are produced by the microbe) converting the primary substrate to some simple common metabolite, a normal food substance for microorganisms;
Replication The process by which a piece of DNA divides itself into two identical copies;
Replicon A genetic unit that enables a piece of DNA to undergo replication;
Auxotroph A mutant organism that requires a food source containing a particular amino acid or vitamin for growth;
Recombinant A cell in which a portion of a genetic fragment has been substituted in the chromosome;
(Sole carbon source) --Indicative of a mutant incapable of growing on the given sole carbon source;
(Plasmid)" Indicative of cells from which the designated plasmid has been completely driven out by curing or in which no portion of the plasmid ever existed;
(Plasmid) Indicative of cells lacking in the designated plasmid or cells harboring a non-functional derivative of the given plasmid;
(Amino-acid) Indicative of a strain that cannot manufacture the designated amino acid;
(Vitamin) Indicative of a strain that cannot manufacture the designated vitamin and (Plasmid) Indicates that the cells contain the designated plasmid.
The symbol [OCT] as used herein signifies the plasmid aggregate composed of OCT, factor K and MER.
Plasmidsare believed to consist of double-stranded 5 component of the host cell. Generally, plasmids are not essential for cell viability.
Plasmids may be compatible (i.e. they can reside stably in the same host cell) or incompatible (i.e. they are unable to reside stably in a single cell). Among the known plasmids, for example, are sex factor plasmids and drug-resistance plasmids.
As is shown in U.S. Pat. No. 3,813,316 Chakrabarty the capability of some Pseudomonas species to degrade complex organic compounds is due to the presence of one or more transmissible plasmids, each of :which specifies the entire enzyme complement of a particular degradative pathway. Such plasmids are designated degradative plasmids. As is described in the Chakrabarty patent unique Pseudomonas organisms have been developed by transferring a number of such degradative pathways to the same recipient. However, the application of this genetic engineering technique has, heretofore, been limited to the transfer of, naturallyoccuring degradative plasmids. However, a Pseudomonas organism may have a desired capability of degrading some given complex organic compound, but this ca-. pability is of chromosomal origin. If it be desired to transfer the desired capability to a recipient cell by the techniques described in the patent, these techniques will not suffice.
It would be particularly advantageous to be able to transfer an entire enzyme complement of a particular degradative pathway, regardless of whetheror not a naturally-occurring degradative plasmid is available therefor.
DESCRIPTION OF THE INVENTION By the practice of the instant invention, once a Pseudomonas organism is found containing genes that specify the entire enzyme complement of a particular degradative pathway, if this pathway is not already plasmidborne, the requisite genes can now be transferred. This is accomplished by mobilizing these genes from the chromosome and transferring them to a preselected recipient in which these particular genes have been deleted. The transferred genes then become established therein as a separate stable artificial degradative plasmid. Mobilization of genes for subsequent transfer in this manner is accomplished by utilizing a transfer plasmid (factor K).
MANNER AND PROCESS OF MAKING AND USING THE INVENTION In brief, the process for transforming chromosomal 3. determine whether the specifying genes are plas-' mid-bome or are chromosomal; 4. if the desired degradative pathway is specified by the chromosome, introduce factor K into the cell by selection techniques and 5. use the cell so modified genetically to transfer the genes for the degradative pathway to a recipient Pseudomonas cell (factor K will, of course, also be transferred simultaneously), the recipient organism selected being one in which those particular genes have beendeleted. Recipient cells surviving growth on a minimal plate now contain the transmissible factor K and, separate therefrom (but constituting a plasmid aggregate therewith) the nontransmissible degradative pathway. Thereafter, the plasmid aggregate can be transferred to other recipient cell s. Steps 1-5 can be repeated to in troduce multiple compatible plasmids into the same recipient. Also this process can be used to introduce an artificial plasmid into a Pseudomonas cell which already has one or more plasmids so long as the ultimate plasmid content is compatible.
By the practice of this invention the development of cell capability in a single strain for the degradation and conversion of complex hydrocarbons can be extended for-even greater efficiency to cope with massive oil spills or engage in the production of protein. Thus, for example, if a total of degradative pathways are required for the efficient breakdown of a given crude oil, all that is necessary is that all the microorganisms providing these pathways be of the same strain in order to insure compatibility. The total complement of organisms all of the same strain may include a concentration of cells with four degradative plasmids and six concentrations of cells, each containing a different degradative pathway.
To facilitate the practice of this invention in the best mode contemplated, a culture of microorganisms possessing the transfer plasmid, factor K, is now on deposit with the United States Department of Agriculture. This culture is identified as follows:
P. putida PRSl K (NRRL B-8043) Derived from Pseudomonas putida PRSl (ATCC No. 12633) by genetic transfer thereto of a transfer plasmid (factor K) from Pseudomonas putida strain PpGl (ATCC No. 17453).
A sub-culture of this strain can be obtained from the permanent collection of the Northern Marketing and Nutrient Research Division, Agricultural Service, U.S. Department of Agriculture, Peoria, 111., USA.
Morphological observations in various media, growth in various media, general group characterization tests, utilization of sugars and optimum growth conditions for the strains from which the above-identified organisms were derived are set forth in The Aerobic Pseudomonads: A Toxonomic Study" by Stanier, R. Y. et al. [Journal of General Microbiology 43, pp. 159-271 1966) The taxonomic properties of the above-identified organisms remain the same as those of the parent strains. P. putida strain PpGl (ATCC No. 17453) is the same as strain 77 in the Stanier et al. study.
All the organisms referred to herein are non-pathogenic as is the general case with laboratory strains of Pseudomonas.
The capability for creating plasmid structures from chromosomal genes has been made possible by two discoveries. First, was the discovery that there exists for the Pse udomonas species a transfer plasmid, factor K, serving as a gene transfer vector in somewhat the same way as the F plasmid, the sex factor in E. coli. Next, was the discovery that factor K could be used to mobilize chromosomal genes and transfer the mobilized genes to a recipient cell in which those particular genes have been deleted, the mobilized genes thereupon becoming stabilized in the recipient as a separate inheritable plasmid.
Demonstration is given in the publication Dissociation of a Degradative Plasmid Aggregrate in Pseudomonas by A. M. Chakrabarty (J. Bacteriol. l 18 815820, 1974) that the transfer of the [OCT] plasmid from P. oleovorans to P. putida strain PpGl results in the acquisition by the recipient of three independent replicons: OCT (a non-transmissible plasmid specifying enzymes of the octane degradative pathway); MER (a transmissible plasmid, which confers resistance to high concentration of mercury ions) and factor K (the transfer plasmid). Factor K is shown as being responsible for the transfer of the OCT plasmid and some chromosomal genes. The chromosomal genes so substituted became part of the chromosome of the recipient cell (recombinant) as contrasted to the formation of a conjugatant in the instant invention. In this invention, however, the recipient cell is deliberately selected as one in which the genes for the given degradative pathway have been deleted and as a result the transferred chromosomal genes cannot undergo recombination with the recipient chromosome. These genes, therefore, replicate as a separate artificial plasmid.
The compositions of the synthetic mineral media for growth of the cultures were the same for all the Pseudomonas species employed. The mineral medium was prepared from:
m1. of 1 molar K I-lPO 50 ml. of 1 molar KH PO ml. of 1 molar Nl-l Cl 100 X Salts:
19.5 gm. MgSO 5.0 gm. MnSO .l-I O 5.0 gm. FeSO .7l-1 O 0.3 gm. CaCl .2H O
1.0 gm. ascorbic acid 1 liter H O Each of the above (PA Concentrate and 100 X Salts) was sterilized by autoclaving. Thereafter, one liter of the mineral medium was prepared as follows:
PA Concentrate 77.5 ml.
100 X Salts 10.0 ml.
Agar 15.0 gm.
H O to one liter (the pH is adjusted to 6.8 7.0). All experiments were carried out at 32C unless otherwise stated.
For experiments in the transfer of factor K a strain of P. putida PRSl was selected. The biochemical and enzymological characterization of the several aromatic degradative pathways discussed herein and transductional genetic analyses thereof has been set forth in the literature [Ornston, L. N. 1971. Regulation of Catabolic Pathways in Pseudomonas. Bacteriol. Rev. 35 87-116]. The origin, derivation and phenotypes of P. putida strains and their mutants used in this work are set forth in Table 1.
TABLE l-continued mutants as shown in Table 2, equal volumes of overnight-grown (about 2-3 X cells/ml) cells of the 2;? donor (Met, OCT, K organisms and the respective griginal recipient organisms were mixed together and kept in a esi- Source or 5 gnation work Phenotypg Derivation stationary condltlon for 1 hour. Allquots of 0.1 ml were then plated on glucose-m1n1mal, or benzo1c-m1n1mal 2% $5 37 *AC 30 plates to score for chromosomal recombinants. AC 75 y n: oCT **AC 4 -+AC 9 TABLE 2 AC g f MDL+Y 28 3 10 l I Marker Frequenfcy of AC 107 Met: 0UP AC H7 Donor Rec1p1ent Selected Trans er AC 10 AC 75 AC 72 Ben 2 X 10-" AC 108 PE fl MAC (ocr ,1 Met) (Ilv', Ben) QUI AC 2 AC 72 11v 1 X 10' AC 109 Met, our gg 1187- lt vg. Ben) T 2 l0 7 X r AC 110 Met, K ****AC 107 15 (Try) W Put1da AC 105 1115* 5 X 10- PRSl WT m PRSZ AC 73 Mdl *PRSl AC 123 His* 2 x 10- PRS2158 AC 72 11v-, Ben *PRSl (His: our AC 74 llv, Ben **AC 75 Nic' 3 AC 72 AC 123 Ben* 1 10" PRS33l AC 78 Trp" *PRSl (Ben: PRS6O AC 79 Ben *PRSl Q Nic) AC 105 His *PRSl AC 116 llv Ben, **AC 75---) 1 AC 72 AC 21" 2g 5" This work (Table 2) has shown that factor K is inpMw159 AC 123 His-i our, p w 85 25 deed capable of mobilizing and transferring chromo- N B r AC 124 851+, AC 4* somal genes from one stram of Pseudomonas to an Qui-y AC 123 other strain and the resulting recombinants appear to be stable. About 50-80% of such recombinants acquire Treatment of Source or Derivation: tNflemymmmomitwmuanidinc factor K, thus 1nher1t1n g donorab1l1ty of chromosomal j gation genes to other suitable recipients making possible the *Qili fg 2:35:22 cufing transfer of factor K to dlfferent strains of Pseudomonas. The effort was made on two occasions to transfer bb chromosomal genes and thus factor K from P. putida A revlatlons employed are as follows' I Wlld strain PpGl to P. aeruginosa strain PAO without suczi i 82 qumate MDL cess. However, this is believed not to be insurmount- 6 T octaPer Q Trp ",YP- able and additional attempts will be made with restrictophan, Met meth1on1ne, Ilv --1soleuc1ne and vahne tion negative mutants of P aemgl-nosa FAQ and H18 hlstldme- The results of conjugational transfer of mandelate Fund Stfam PMW 15 9 descnbed the amcle and quinate genes mediated by factor K are shown in The Clustermg on the Pseudomonas putzda Chromo- 40 Table TABLE 3 Conjugatant Donor Recipient Selected Frequency Phenotype AC 116 AC 30 Mdl 3 X io- Manon- (K*llv*Ben) (PpGl WT) AC l0(Met) Met 2 X l0 Met Mdl'Qui AC 10 Md!" 5 x 10- Mdl Met'Qui(40%) AC 117 Md1 Mer*Qui-(%) (llv'Ben 'K AC 30 Qui 1 x 10' QuPMdl AC 10 our 1 x 10- Qui Mct'Mdl'(30%) Qui Met* AC 10 Met*' 4 x 10- MevQui-Mdr AC 73 Mdl 2 x 10-5 Mdl AC 78 Trp l X 10 Trp AC 79 Ben 1 X 10 Ben" AC 1415* 2 x 10- His AC 123 His* 5 X 10' His Ben'QuiNie AC 123 Ben 4 X 10' HisBenOuiNic' some of Genes Specifying Dissimilatory Functions Leidigh et al. (J. Molec. Evolution 2 235-242, 1973).
Two P. putida PRSl chromosomal recombinants are In Table l the phenotypes of known plasmids have 60 shown therein as genetic donors. It is known from the been capitalized, while those of the chromosomal genes are designated in lower case letters.
The results of determining the ability of factor K to mobilize chromosomal genes is shown in Table 2 wherein auxotrophic mutants of P. putida PRSl are used as recipients. Y
In carrying out the chromosomal genes transferfrom strain PpGl to a variety of strain PRSl auxotrophic literature that the genes specifying enzymes involved in the degradation of D or L mandelate by P. putida PRSl are totally absent in P. putida PpGl. Similarly, P. putida PpGl lacks some genes which specify enzymes for quinate metabolism. All Mdl PpGl conjugatants obtained were Qui while all Qui conjugatants obtained were Mdl". Co-transfer of these two (mdl and qui) gene clusters has not as yet been observed.
bers from strain PRSl to strain PpGl. 10
Since P. putida PpGl does not have any region of genetic homology corresponding to the mdl and qui genes, an investigation was made to determine whether these gene clusters were integrated at some site on the PpGl chromosome or whether these genes would replil5 cate as autonomously-replicating genetic fragments (plasmids). Table 4 sets forth the mandelate enzyme levels (specific activities in nanomoles of substrate consumed or product formed per minute per mg of protein) in certain conjugatants of PRSl, PpGl and Md1 PpG1 strains. An assay of the mandelate enzymes was made according to the method outlined in the article Synthesis of the Enzymes of the Mandelate Pathway by Pseudomonas putida (J. Bacteriol. 91
The enzyme level designations in Table 4 suggest that the mandelate genes are expressed normally inside strain PpGl (after transfer thereto of the mdl gene clusters from strain PRSl) and that the number of copies of such genes in both strains PRSl (AC 78) and Mdl PpGl (AC 104) is presumably the same.
Curing degradative pathways from each strain with mitomycin C was accomplished by preparing several test tube ofL broth [Lennox, E. S. (1955), Virology, 1, ]containing varying concentrations of mitomycin C and inoculating these tubes with suitable dilutions of early stationary phase cells of the given strain to give concentrations 10 to 10 cells/ml. These tubes were incubated on a shaker at 32C for 2-3 days. Aliquots from tubes that showed some growth were then diluted and plated on glucose minimal plates. After growth at 32C for 24 hours, individual colonies were split and respotted on glucose-minimal and degradative pathway-minimal plates to give the proportion of MDL and QUI in order to determine the frequency of curing. It was, therefore, shown that in each instance the degradative pathway genes are plasmid-borne.
Several of the mandelate negative cured derivatives, when analyzed in the manner referred to above for the presence of mandelate enzymes, showed loss of all the mandelate enzymes, suggesting that curing leads to the loss of the entire gene clusters. This, in turn, indicates that these genes appear to replicate independently as plasmids.
Further proof for establishing the plasmid nature of mdl and gui gene clusters in P. putida PpGl (to which these geneshave been transferred from strain PRSl) is the high rate of transmissibility of such genes to other suitable recipients. The capability for transfer of chromosomal genes and MDL and QUI plasmids is shown in Table 6. The AC 104 organism, which was a Met mutant of strain PpGl that had received the mandelate ge- Table 5 above sets forth the result of curing of MDL netic region from strain PRSl, proved to be a potent and QUI plasmids from P. putida PpGl strains. Neither 5O donor for chromosomal markers as well as for mdl of these degradative pathways are curable from strain genes.
TABLE 6 Transfer Conjugatant Donor Recipient Select Frequency Phenotype PRSl. However, both the mandelate and quinate genes in the PpGl conjugatants (AC 104 and AC 107) can be cured at a very low frequency.
Several chromosomal recombinants (selection on glucose-minimal) acquired the mandelate plasmid (MDL) by secondary infection. Transfer of the MDL plasmid (selection on mandelate-minimal supplemented with from the K PRSl strain (AC 116, Table 3) to PpGl recipient was accompanied by a simultaneous transfer of factor K to the respective recipient.
Autonomous replication of mdl and qui genes as plasmids in strain PpGl was verified by transfer of such plasmids by transduction. Transducing phage, pf 16, is known to be effective for the transfer of plasmids between organisms of strain PpGl [Genetic Basis of the Biodegradation of Salicylate in Pseudomonas Chakrabarty (J. Bacteriol. 112 815-823, 1972)]. Table 7 shows the results of transductional transfer of both MDL and QUI plasmids to PpGl cells deleted for these genes. Transductants were selected on mandelate and quinate-minimal plates supplemented with required amino acids. Unlike conjugational transfer, there was no associated transfer of chromosomal genes during transduction of such plasmids. The transductants harbor the mdl or qui gene clusters as plasmids, since they can be cured as explained hereinabove from the transductants at low frequency. Unlike the conjugatants, however, the MDL or the QUI transductants are incapable of transferring the MDL or QUI plasmids by conjugation to other recipients.
Results set forth in Table 8 below indicate that P. putida PpGl auxotrophic mutant AC 110, which is a QUI segregant of AC 107, still retains the donorability for chromosomal markers. This suggests that factor K was still present in the AC 110 organism, which had been cured of the QUI plasmid. However, AC 109, which had received the QUI plasmid from AC 107 by transduction, could not transfer either chromosomal genes or the QUI plasmid. Thus, QUI plasmid appeared to be non-transmissible, per se, and any transmission thereof, was because of the presence of factor K.
This conclusion is further supported by examination of the characteristics of a number of chromosomal recombinants using AC 107 as a donor. As noted in Table 6, 4-5% of chromosomal recombinants inherited the QUI plasmid under the conditions employed while nearly 50% of the chromosomal recombinants inherited the donorability. However, recombinants that acquire the QUl plasmid may or may not inherit factor K. AC 108, a recombinant, which had inherited QUl but not factor K was incapable (less than 1 X 10" transfer frequency) of transferring either chromosomal genes or the QUI plasmid to suitable recipients. However, if factor K were to be introduced into organism AC 108, this transfer plasmid would enable AC 108 to act as a donor for both QUI and other chromosomal genes. Thus, the presence of factor K is essential for the mobilization of QUI as well as the chromosome. Data for gansmissibility of the MDL plasmid was the same as for TABLE 8 Marker Transfer Donor Recipient Selected Frequency AC 107 AC 2 'lrp 4X 10: QUI K our 2 x 10- AC 109" AC 2 Trp 1 X 10" (QUPK') QUI 1 x 10"? J AC AC 2 Trp 2 X 10' (QUI'K*) QUP 1 X 10". AC 108 AC 10 Met 1 X 10' V (QUY'K) AC 10 QUl 1 x 1'0 AC 109 is a QUI transductant of Ac 10 from AC 107 as donor. Out of 10 transduct-ants tested none transferred either QUI or chromosomal markers.
AC 110 is a Mitomycln C-induced QUl'segregant of AC 107. r
AC 108 is a Trp'llv" recombinant of AC 20 which has acquired QUI but not factor K (donor used with AC 107). r
in Pseudomonas when factor K is utilized to mobilize chromosomal genes and transfer them to a recipient from which the chromosomal genes being transferred were deleted, the mobilized genes and factor K take the form of a plasmid aggregrate. The presence of factor K assures transmissibility of the artifically created plasmids to other cells, although the frequency of transfer for such plasmids is usually 50-100 fold lower than that of most chromosomal genes, as would be expected.
In the best mode contemplated for the practice of this invention, therefore:
1. the primary substrate to be degraded is determined,
2. a Pseudomonas strain is identified and isolated that exhibits the capability for converting the primary substrate to some common metabolite (i.e. the organism exercises the requisite degradative pathy),
3. the strain of Pseudomonas (different from the organism in step 2) actually to be used to accomplish the substrate degradation is selected, this strain being one in which the genes sepcifying the requisite degradative pathway are missing,
4. the nature of the genes (in the organisms of step 2) specifying the requisite degradative pathway (e.g. plasmid-home or chromosomal) is determined; this is accomplished by curing and conducting independent conjugal transmissibility tests (if the capability for the specific degradative pathway is transferred, the degradative pathway is plasmid-home and transmissible in which case this invention is not needed; if the specific capability is not cured from the donor organism, factor K is employed by the exercise of the following steps:
a. prepare a multiple auxotrophic mutant in the strain harboring the degradative pathway to be transferred as described in the textbook, Genetics of Bacteria and Their Viruses by William Hays [John Wiley & Sons, Inc. (1965)];
b. introduce factor K into this strain by using an auxotrophic mutant of P. putida PRSl K as donor and the multiple auxotroph as recipient; selection is made for the repair of any one of the requirements of the recipient auxotrophs (about 50% of the recombinants will have acquired factor K); this is followed by purification of 10-12 recombinants by single colony isolation and sub sequent analysis for the presence of factor K;
c. transfer the desired degradative pathway to the Pseudomonas isolated in step 2 using the auxotrophis recombinant harboring factor K as the donor; transfer is by conjugation in the manner 1 1 described in the Chakrabarty patent (column 7, lines lO-38 and d. purify the conjugatants; these conjugatants contain the desired degradative pathway in the form I of a plasmid that is non-transmissible in the absence of factor K.
Herein where reference is made to the quinic pathway it should be noted that this pathway applies for both quinic acid and p-hydroxybenzoic acid.
What we claim as new and desire to secure by Letters Patent of the US. is: Y
1. A process for transferring chromosomal genes specifying a hydrocarbon degradative pathway for a given substrate from a first strain of Pseudomonas and imparting said chromosomal genes as part of a plasmid aggregate into a second strain of Pseudomonas which does not contain said chromosomal genes comprising the steps of:
12 a. introducing factor K, a transfer plasmid, into at least one organism of said first strain of Pseudomonas which mobilizes said chromosomal genes and forms a plasmid aggregate therewith,
b. admixing the resulting first strain of Pseudomonas with said second strain of Pseudomonas transferring said plasmid aggregate by conjugation,
c. placing the mixture on minimal plates containing said given substrate as the sole source of carbon and d. purifying the resulting conjugatants.
2. The process of claim 1 wherein the source of factor K is P. putida PRSl K".
3. The process of claim 1 wherein the hydrocarbon pathway is for quinic acid degradation.
4. The process of claim 1 wherein the hydrocarbon degradative pathway is for mandelate degradation.
5. The process of claim 1 wherein the first strain is P.
putida PRSl and the second strain is P. putida PpGl.