Tartrate catabolism gene

DNA sequences which are capable of expressing a polypeptide with the ability to catabolize L-tartrate are incorporated into suitable vectors and used to transform both prokaryotic and eukaryotic hosts.

BACKGROUND OF THE INVENTION 
The ability to catabolize tartrate and utilize tartrate as a carbon source 
is very rare in microorganisms. It would be desirable to identify and 
isolate a gene which is able to confer the ability to utilize tartrate on 
a suitable host. For example, yeast used to ferment juice to alcohol are 
unable to utilize tartrate which is a byproduct of fermentation. 
Introduction of a functional tartrate gene into yeast would allow the 
organism to utilize this additional energy source, leading to the 
production of additional alcohol from a given amount of juice. The trait 
would also be useful when it is desired to produce wine free of tartrate. 
SUMMARY OF THE INVENTION 
DNA sequences expressing polypeptides having the ability to catabolize 
L-tartrate are provided. Vectors incorporating the DNA sequences are used 
to transform a susceptible host to utilize L-tartrate as a carbon source.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The DNA sequences of the present invention appear to be less than about 20 
kbp, probably less than about 10 kbp, and can be derived directly or 
indirectly from the TAR gene found in the pTAR plasmid of certain strains 
of Agrobacterium tumefaciens, as well as from the genome of a certain 
large cryptic plasmid (exceeding 200 megadaltons) found in certain biotype 
3 strains of A. tumefaciens, including the strains designated 9-3, 14-1, 
23-4, 24-2, 37G4, 52BA1, 56A2, 58-1, 70D4, 7384, and 77B2. Perry and Kado, 
Phytopath. (1981) 71:249. 
The TAR gene may be obtained from the appropriate strain of A. tumefaciens 
by conventional techniques. For example, messenger RNA (mRNA) can be 
isolated and complementary DNA (cDNA) synthesized using reverse 
transcriptase. Alternatively, endonucleases may be used to cleave 
available restriction sites on either side of the TAR gene in the pTAR 
plasmid, or other TAR-bearing plasmid. 
The pTAR plasmid may be isolated and purified from particular strains by 
conventional techniques. Conveniently, A. tumefaciens 1D1422 is lysed and 
the RNA hydrolyzed. This strain has been deposited at the A.T.C.C. for 
patent purposes on Dec. 23, 1980, and given designation 31779. The 
chromosomal DNA is fragmented, typically by mechanical shearing, and the 
resulting lysate extracted with organic solvents, conveniently phenol 
followed by chloroform having from about 2 to 5 volume percent isoamyl 
alcohol. The remaining aqueous solution of DNA is then gradient density 
centrifuged and the plasmid DNA isolated and subjected to conventional 
purification. Methods for isolating pure plasmid DNA from cleared lysates 
are described in Old and Primrose, Principles of Gene Manipulation, U. 
California Press, 1981, pp. 29-31. 
The DNA sequences of the present invention will often include regions other 
than the structural gene encoding the polypeptides responsible for the 
tartrate utilization. Depending on the contemplated host, various 
regulatory and other regions can be included in the sequence, typically 
including an origin of replication, a promoter region and markers for the 
selection of transformants. In general, such DNA sequences will provide 
regulatory signals for expression, amplification, and for a regulated 
response to a variety of conditions and reagents. 
It will often be desirable to provide for replication and regulation 
capability in both eukaryotic and prokaryotic hosts, which allows 
amplification of the vector in a prokryotic host while retaining the 
ability to be expressed in eukaryotic hosts. Such vectors are referred to 
as shuttle vectors. 
Various markers may be employed for selection of transformants, including 
biocide resistance, particularly to antibiotics, such as ampicillin, 
tetracycline, trimethoprim, chloramphenicol, and penicillin; toxins, such 
as colicin; and heavy metals, such as mercuric salts. Alternatively, 
complementation providing an essential nutrient to an auxotrophic host may 
be employed. The ability to utilize tartrate as the sole carbon source can 
also provide a selective marker. Often, different screening markers will 
be required for both the prokaryotic and the eukaryotic hosts, although in 
some cases both types of organisms may be able to express the same 
markers. 
Hosts which may be employed for the production of the polypeptides of the 
present invention include unicellular microorganisms, such as prokaryotes, 
i.e. bacteria; and eukaryotes, such as fungi, including yeasts, algae, 
protozoa, molds and the like. Specific bacteria which are susceptible to 
transformation include members of the Enterobacteriaceae, such as strains 
of Escherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis; 
Pneumococcus; Streptococcus; and Haemophilus influenzae. Specific yeasts 
which are of interest include Saccharomyces cerevisiae and Saccharomyces 
sacchrin. 
The DNA sequences can be introduced directly into the genome of the host or 
can be first incorporated into a vector which is then introduced into the 
host. Exemplary methods of direct incorporation include transduction by 
bacteriophages, transfection where specially treated host bacterial cells 
can be caused to take up naked phage chromosomes, and transformation by 
calcium precipitation. These methods are well known and need not be 
described further. Exemplary vectors include plasmids, cosmids and 
viruses. The availability and use of such vectors are described in Old and 
Primrose, supra., Chapters 3-5, which are incorporated herein by 
reference. 
When incorporated into a host according to the above-described techniques, 
the DNA sequences of the present invention are able to confer the ability 
to catabolize the L isomer of tartrate. This phenotypic trait is useful 
under a variety of circumstances. For example, transformed microorganisms 
or hosts can be used to resolve a racemic mixture of tartrate by removing 
substantially all the L isomer from the mixture. Alternatively, the 
polypeptides responsible for L-tartrate degradation can be obtained from 
suitably modified microorganisms, purified and used to achieve such 
resolution. 
The trait is particularly useful in that it allows a microorganism to 
utilize tartrate as a carbon source when previously it was incapable of 
such utilization. For example, tartaric acid is a byproduct in the 
fermentation of juice for the production of alcohol. Introduction of a 
functional tartrate gene into yeast responsible for fermentation would 
allow the organisms to convert the tartaric acid into additional alcohol. 
EXPERIMENTAL 
*All temperatures are .degree.C. and all percentages are by weight, unless 
otherwise indicated. 
1. Isolation of Plasmid DNA from Agrobacterium tumefaciens. 
Cells are grown in 1 liter of Luria broth for 15-20 hours at 23.degree. C. 
with aeration. The cells are then harvested by centrifugation 
(10,000.times.g, 10 min), washed once with TES buffer (50 mM Tris-Cl, pH 
8.0, 5 mM Na.sub.2 EDTA, 50 mM NaCl) and the cell pellet frozen at 
-20.degree. C. The cell pellet is thawed and resuspended in 28 ml of TNS 
buffer (10 mM Tris-Cl, pH 8.0, 100 mM NaCl, 20% sucrose) and 9 ml of 
freshly prepared egg white lysozyme (10 mg/ml in 120 mM Tris-Cl, pH 8.0, 
50 mM Na.sub.2 EDTA) is added. After 30 min incubation on ice, 3.8 ml of 
pancreatic ribonuclease (1 mg/ml, heat-treated at 85.degree., 10 min to 
remove any deoxyribonuclease) is added. After 15 min at room temperature, 
12 ml of 0.5 M Na.sub.2 EDTA, pH 8.0 and 27 ml of lysis solution (100 mM 
Tris-Cl, pH 8.0, 50 mM Na.sub.2 EDTA, 500 mM CaCl, 2% sarkosyl NL97) is 
added. The solution is incubated in ice for 20 min and the chromosomal DNA 
is sheared by passing the DNA solution through a no. 18 gauge needle using 
a 50 ml syringe twenty times. The lysate is extracted twice with phenol 
(distilled and equilibrated with TES before use). The mixture is 
emulsified by shaking and then centrifuged at 5000.times.g, 20 min. The 
aqueous phase is collected and extracted twice with an equal volume of 
chloroform-isoamyl alcohol (24:1, vol/vol). Each time the organic solvent 
phase is removed by filtration through Whatman 1 PS paper. CsCl and 
ethidium bromide are added to the aqueous phase to a final density of 
1.395 g/cm and 380 .mu.g ethidium bromide per ml of DNA solution. The 
mixture is placed in polyallomer centrifuge tubes prerinsed with ethanol 
and dried, and is centrifuged for 72 hours at 35,000 rpm, 20.degree. C. in 
a Spinco type 60 Ti rotor in a Beckman L350 ultracentrifuge. The plasmid 
DNA is withdrawn from the side of the centrifuge tube using a 3 ml syringe 
equipped with a no. 18 gauge hypodermic needle. The plasmid band 
visualized with ultraviolet light can be easily seen below the broad band 
composed of chromosomal and linear DNA. The plasmid DNA is subjected to a 
second round of centrifugation and purification in a Type 65 rotor. 
Ethidium bromide is removed from the final plasmid DNA solution by several 
extractions with water saturated n-butanol followed by dialysis against 
six changes of 3 liters of 10 mM Tris-Cl, pH 8.0, 1 mM Na.sub.2 EDTA, 
4.degree. C. 
2. pTAR Plasmid is Responsible for Catabolism of L-Tartrate 
A. tumefaciens SS18 and ID1119 were isolated from galls removed from field 
grapevines (Vitis vinifera). Strain SS18 is avirulent, harboring only the 
pTAR plasmid and is unable to grow on octopine or nopaline; ID1119, on the 
other hand, is a virulent strain that contains, in addition to an octopine 
type Ti plasmid, a smaller plasmid that shares DNA sequence homology with 
pTAR (see below). Both strains align with biotype I strains in that they 
will convert lactose to 3-ketolactose and utilize melezitose. Kerr and 
Panagopoulos, Phytopath. Zeitschr. (1977), 90:172-79. However, unlike most 
biotype I strains, ID1119 and SS18 will utilize L-tartaric acid as the 
sole carbon source in a basal salts medium. Other well characterized 
biotype I strains, e.g., 1D1, B.sub.6 (806), C58, ACH-5 and 1D135, are 
unable to grow on tartrate. As shown by agarose gel electrophoresis of the 
plasmid DNA, from strains SS18, 1D119, B.sub.6 (806), ACH-5, 1D1 (ATCC No. 
19599), C58 and 1D135, strain SS18 harbors only the 44 kb pTAR plasmid. It 
appears, therefore, that tartrate utilization is not correlated with any 
Ti plasmid coded functions such as octopine or nopaline utilization. 
Transfer of pTAR by mating SS18 (pTAR) with a plasmid-free A. tumefaciens 
recipient NT1RE could not be accomplished, even in the presence of 
L-tartrate, suggesting that pTAR might be transfer deficient. However, 
pTAR was successfully transferred into NT1RE by cotransformation with a 
mixture of purified pTAR DNA and pTiACH5 DNA. 
Because it was found that tartrate and octopine utilization are independent 
functions, octopine strain ACH5 was transformed with nopaline strain L58. 
Transformants obtained at a frequency of 5.times.10.sup.-7 were selected 
on the basis of growth on basal salts medium (Langey and Kado, Mutation 
Res. (1972), 14:277-286) containing 0.1% octopine 100 .mu.g/ml rifampicin 
and 150.mu.g/ml erythromycin. Each transformant was screened for growth on 
L-tartrate agar, and electrophoresis on agarose gel indicated that all 
L-tartrate utilizers harbored the large pTiACH5 plasmid and the smaller 
pTAR plasmid. They no longer harbored a large 280 mdal cryptic plasmid 
that is observed in the wild-type strain ACH5. 
Transformants unable to grown on L-tartrate contained this large cryptic 
plasmid and the pTiACH5 plasmid. These results suggested that pTAR carries 
the genetic information for L-tartrate utilization. Also, pTAR may be 
incompatible with the large cryptic plasmid. This observation is supported 
by the fact that transformable strains B.sub.6 806, C58 and NT1, which 
harbor very large cryptic plasmids (megacryptic plasmids), were refactory 
to transformation with pTAR DNA despite repeated attempts. It was found, 
however, that Ti plasmids from these strains are able to coexist with pTAR 
when inserted by transformation into SS18. 
To further establish that pTAR carries genetic information for L-tartaric 
acid utilization, the transposon Tn732, which confers gentamicin 
resistance, was inserted into pTAR. Insertion mutants were obtained by 
transferring Tn732, into strain SS18 by conjugation with Escherichia coli 
HB101 (RK2:Mu.sub.cts ::Tn732). The RK2::Mu.sub.cts vector plasmid DF210 
(Figurski et al., Gene (1976), 1:107-119) undergoes abortive replication 
at 37.degree. C., ensuring that it does not persist in the recipient 
cells. Tn732 is a large transposon measuring 10.9 kb (Datta et al., Cold 
Springs Harbor Symps. Quant. Biol. (1981), 65:45-51) and its size is 
sufficient to displace pTAR plasmid DNA from its normal position in 
agarose gels after electrophoresis. Thus, pTAR plasmid with Tn732 
insertions were easily detected by examining individual colonies by a 
rapid mini-screening procedure (Kado and Liu, J. Bacteriol. (1981), 
145:1365-73). Gentamicin resistant colonies of SS18(pTAR) obtained at a 
frequency of 10.sup.-6 of the donor Agrobacterium cells were screened for 
their inability to catabolize L-tartrate by replicating the colonies en 
masse onto minimal salts agar containing 0.3% tartrate and 100 .mu.g/ml 
gentamicin sulfate at 37.degree. C. Examination of more than 5000 colonies 
produced none that failed to grow on tartaric acid as the sole carbon and 
energy source. Subsequently, 200 of these colonies were lysed and the 
plasmids examined electrophoretically. 
The surprising result of this study was that up to 50% of the transposants 
contained pTAR in addition to a pTAR::Tn732 cointegrate molecule. These 
two plasmid species appear to exist in harmony and are inseparable at the 
single colony level. However, four colonies were detected that harbored 
only a 55 kb plasmid. Purification of these plasmids revealed that they 
were indeed the result of an insertional event involving Tn732, and that 
three of the four were identical at the level of resolution obtained by 
EcoRI digestion. Reference to the physical map of the wild-type pTAR 
plasmid (FIG. 1) shows that these mutants had Tn732 inserted into or 
adjacent to PstI fragment E and that it is also present in Xhol fragment B 
and KpnI fragment A. Transfer of these pTAR::Tn732 plasmids into a 
plasmid-free strain by transformation produced transformants at a 
frequency of 10.sup.-7 that displayed the TAR.sup.- phenotype. 
L-Tartrate proved to be useful as a selective marker in transformation 
experiments with wild-type pTAR DNA and the plasmid-free recipient A. 
tumefaciens strain 12D12. The co-existence of pTAR and pTi indicates a 
compatible relationship in Agrobacterium. DNA sequence homologies between 
pTAR DNA and octopine and nopaline Ti plasmids were not detected by 
reciprocal blot hybridization and analysis (Southern). This further 
substantiates the distinction between the two plasmids and also supports 
their compatible existence in Agrobacterium. 
The procedure for the analysis was as follows: SmaI restricted pTi DNA of 
strains 15955, C58 and SS18 were each probed with pTi15955 DNA labelled by 
nick translation (Rigby et al., J. Mol. Biol. (1977), 113:237-51) with 
[.sup.32 P]-triphosphate deoxyribonucleosides: dCTP and dATP. SmaI 
restricted pTi DNA of strains C58, 19599 and pTAR were probed with 
labelled pTiC58 DNA. In both hybridizations, pTAR DNA was digested with 
PstI ad SalI. PstI restricted pTi DNA of strains SS18, C58 and 15955 were 
probed with labelled pTAR DNA. Blot hybridizations were performed 
according to Wahl et al., PNAS USA (1979), 76:3683-87 at 42.degree. C. 
Plasmid DNA blot hybridization analysis of virulent wild-type tartrate 
utilizing strain 1D1119 revealed DNA sequence homologies between pTAR DNA 
and the DNA of a plasmid of identical size in 1D1119. Quantitative 
estimates indicated that 20% of the pTAR DNA sequences were present. Thus, 
pTAR-like plasmids and the pTi plasmid seem to coexist in nature. 
Analysis of large numbers of freshly isolated A. tumefaciens may prove this 
to be the case in certain ecological niches beneficial to A. tumefaciens. 
One such niche may be defined by the host in which the particular A. 
tumefaciens strain invades and multiplies during infection to produce 
crown gall tumors. Strain SS18 and 1D1119 were isolated from grapevine 
galls originating from Hungary and California respectively. A survey of A. 
tumefaciens strains freshly isolated from grapevine galls in California 
showed that they all utilized L-tartrate. On the other hand, strains 
originally isolated from other types of host plants such as plum, cherry, 
peach, and tomato which were the original hosts of strains ACH5, C58, 
1D135 and B6(806), were unable to utilize L-tartrate. 
It was also shown that biotype 3 strains of A. tumefaciens, usually 
associated with grapevines, do not carry a plasmid similar in size to pTAR 
but they all harbor large cryptic plasmids &gt;200 megadalton. Nevertheless, 
they all utilize L-tartaric acid as the sole source of carbon. This 
suggests that TAR genes, of either megacryptic plasmid of chromosomal 
origin, may also be operating in pTAR-free strains. Because pTAR seems 
incompatible with the megacryptic plasmid, the possibility exists that 
they may have certain genetic homologies. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.