Recombinant DNA encoding a desulfurization biocatalyst

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

BACKGROUND 
Sulfur contaminants in fossil fuels can create problems in refinery 
processes which can be costly to rectify. The sulfur contaminants that 
occur in fossil fuels fall into either of the following general classes: 
mineralized (inorganic, e.g., pyritic) sulfur and organic sulfur (sulfur 
that is covalently bound to carbonaceous molecules, referred to as 
organosulfur compounds). The presence of sulfur has been correlated with 
corrosion of pipeline, pumping and refining equipment, and with premature 
breakdown of combustion engines. Sulfur also poisons many catalysts which 
are used in the refining of fossil fuels. Moreover, the atmospheric 
emission of sulfur combustion products, such as sulfur dioxide, leads to 
the form of acid deposition known as acid rain. Acid rain has lasting 
deleterious effects on aquatic and forest ecosystems, as well as on 
agricultural areas located downwind of combustion facilities. Monticello, 
D. J. and W. R. Finnerty, (1985) Ann. Rev. Microbiol, 39:371-389. 
Regulations such as the Clean Air Act of 1964 require the removal of 
sulfur, either pre- or post-combustion, from virtually all coal- and 
petroleum-based fuels. Conformity with such legislation has become 
increasingly problematic due to the rising need to utilize lower grade, 
higher-sulfur fossil fuels as clean-burning, low-sulfur petroleum reserves 
become depleted, as well as the progressive reductions in sulfur emissions 
required by regulatory authorities. Monticello, D. J. and J. J. Kilbane, 
"Practical Considerations in Biodesulfurization of Petroleum", IGT's 3d 
Intl. Symp. on Gas, Oil, Coal, and Env. Biotech., (Dec. 3-5, 1990) New 
Orleans, La. 
One technique which is currently employed for the pre-combustion removal of 
organic sulfur from liquid fossil fuels, e.g., petroleum, is 
hydrodesulfurization (HDS). HDS is suitable for the desulfurization of 
fossil fuels wherein organosulfur compounds account for a significant, 
e.g., a major, proportion of all sulfur contaminants present. HDS is thus 
useful for treating crude oil or bitumen, petroleum distillate fractions 
or refining intermediates, liquid motor fuels, and the like. HDS is more 
particularly described in Shih, S.S. et al., "Deep Desulfurization of 
Distillate Components", Abstract No. 264B AIChE Chicago Annual Meeting, 
presented Nov. 12, 1990, (complete text available upon request from the 
American Institute of Chemical Engineers); Gary, J. H. and G. E. Handwerk, 
(1975) Petroleum Refining: Technology and Economics, Marcel Dekker, Inc., 
New York, pp. 114-120, and Speight, J. G., (1981) The Desulfurization of 
Heavy Oils and Residue, Marcel Dekker, Inc., New York, pp. 119-127. HDS is 
based on the reductive conversion of organic sulfur into hydrogen sulfide 
(H.sub.2 S) in the presence of a metal catalyst. HDS is carried out under 
conditions of elevated temperature and pressure. The hydrogen sulfide 
produced as a result of HDS is a corrosive gaseous substance, which is 
stripped from the fossil fuel by known techniques. Elevated or persistent 
levels of hydrogen sulfide are known to poison (inactivate) the HDS 
catalyst, complicating the desulfurization of liquid fossil fuels that are 
high in sulfur. 
Organic sulfur in both coal and petroleum fossil fuels is present in a 
myriad of compounds, some of which are termed labile in that they can 
readily be desulfurized, others of which are termed refractory in that 
they do not easily yield to conventional desulfurization treatment, e.g., 
by HDS. Shih, S.S. et al. Frequently, then, even HDS-treated fossil fuels 
must be post-combustively desulfurized using an apparatus such as a flue 
scrubber. Flue scrubbers are expensive to install and difficult to 
maintain, especially for small combustion facilities. Moreover, of the 
sulfur-generated problems noted above, the use of flue scrubbers in 
conjunction with HDS is directed to addressing environmental acid 
deposition, rather than other sulfur-associated problems, such as 
corrosion of machinery and poisoning of catalysts. 
Recognizing these and other shortcomings of HDS, many investigators have 
pursued the development of microbial desulfurization (MDS). MDS is 
generally described as the harnessing of metabolic processes of suitable 
bacteria to the desulfurization of fossil fuels. Thus, MDS typically 
involves mild (e.g., ambient or physiological) conditions, and does not 
involve the extremes of temperature and pressure required for HDS. It is 
also generally considered advantageous that biological desulfurizing 
agents can renew or replenish themselves under suitable conditions. 
Microbial desulfurization technology is reviewed in Monticello and 
Finnerty (1985), 39 ANN. REV. MICROBIOL. 371-389 and Bhadra et al. (1987), 
5 BIOTECH. ADV. 1-27. Hartdegan et al. (1984), 5 CHEM. ENG. PROGRESS 63-67 
and Kilbane (1989), 7 TRENDS BIOTECHNOL. (No. 4) 97-101 provide additional 
commentary on developments in the field. 
Several investigators have reported mutagenizing naturally-occurring 
bacteria into mutant strains with the acquired capability of breaking 
down, i.e., catabolizing, dibenzothiophene (DBT). Hartdegan, F. J. et al., 
(May 1984) Chem. Eng. Progress 63-67. DBT is representative of the class 
of organic sulfur molecules found in fossil fuels from which it is most 
difficult to remove sulfur by HDS. Most of the reported mutant 
microorganisms act upon DBT nonspecifically, by cleaving carbon-carbon 
bonds, thereby releasing sulfur in the form of small organic breakdown 
products. One consequence of this microbial action is that the fuel value 
of a fossil fuel so treated is degraded. Isbister and Doyle, however, 
reported the derivation of a mutant strain of Pseudomonas which appeared 
to be capable of selectively liberating sulfur from DBT, thereby 
preserving the fuel value of treated fossil fuels. U.S. Pat. No. 
4,562,156. 
Kilbane recently reported the mutagenesis of a mixed bacterial culture, 
producing a bacterial consortium which appeared capable of selectively 
liberating sulfur from DBT by an oxidative pathway. Resour. Cons. Recycl. 
3:69-79 (1990). A strain of Rhodococcus rhodocrous was subsequently 
isolated from the consortium. This strain, which has been deposited with 
the American Type Culture Collection under the terms of the Budapest 
Treaty as ATCC No. 53968 and also referred to as IGTS8, is a source of 
biocatalytic activity as described herein. Microorganisms of the ATCC No. 
53968 strain liberate sulfur from forms of organic sulfur known to be 
present in fossil fuels, including DBT, by the selective, oxidative 
cleavage of carbon-sulfur bonds in organic sulfur molecules. Kilbane has 
described the isolation and characteristics of this strain in detail in 
U.S. Pat. No. 5,104,801. 
SUMMARY OF THE INVENTION 
This invention relates in one aspect to a deoxyribonucleic acid (DNA) 
molecule containing one or more genes encoding one or more enzymes that, 
singly or in concert with each other, act as a biocatalyst that 
desulfurizes a fossil fuel that contains organic sulfur molecules. The DNA 
molecule of the present invention can be purified and isolated from a 
natural source, or can be a fragment or portion of a recombinant DNA 
molecule that is, e.g., integrated into the genome of a non-human host 
organism. The gene or genes of the present invention can be obtained from, 
e.g., a strain of Rhodococcus rhodochrous microorganisms having suitable 
biocatalytic activity. That is, the gene or genes of the present invention 
can be obtained from a non-human organism, e.g., a microrganism, that 
expresses one or more enzymes that, singly or in concert with each other, 
act as a desulfurizing biocatalyst. Biocatalysis, as described more fully 
below, is the selective oxidative cleavage of carbon-sulfur bonds in 
organosulfur compounds. The present invention is particularly useful for 
the desulfurization of fossil fuels that contain organosulfur compounds, 
e.g., DBT. 
The invention further relates to recombinant DNA vectors, recombinant DNA 
plasmids and non-human organisms that contain foreign (recombinant, 
heterologous) DNA encoding a biocatalyst capable of desulfurizing a fossil 
fuel which contains organosulfur compounds. Such organisms are referred to 
herein as host organisms. 
The invention described herein thus encompasses ribonucleic acid (RNA) 
transcripts of the gene or genes of the present invention, as well as 
polypeptide expression product(s) of the gene or genes of the present 
invention. The present polypeptide expression products, after such 
post-translational processing and/or folding as is necessary, and in 
conjunction with any coenzymes, cofactors or coreactants as are necessary, 
form one or more protein biocatalysts (enzymes) that, singly or in concert 
with each other, catalyze (promote, direct or facilitate) the removal of 
sulfur from organosulfur compounds that are found in fossil fuels. This is 
accomplished by the selective, oxidative cleavage of carbon-sulfur bonds 
in said compounds. The biocatalyst of the present invention can be a 
non-human host organism, viable (e.g., cultured) or non-viable (e.g., 
heat-killed) containing the DNA of the present invention and expressing 
one or more enzymes encoded therein, or it can be a cell-free preparation 
derived from said organism and containing said one or more biocatalytic 
enzymes. 
In another aspect, the present invention relates to a method of 
desulfurizing a fossil fuel using the above mentioned non-human organism, 
said organism expressing a desulfurizing biocatalyst. Alternatively, the 
present invention relates to a method of desulfurizing a fossil fuel using 
a biocatalyst preparation comprising one or more enzymes isolated from 
said organism. The process involves: 1) contacting said organism or 
biocatalyst preparation obtained therefrom with a fossil fuel that 
contains organic sulfur, such that a mixture is formed; and 2) incubating 
the mixture for a sufficient time for the biocatalyst expressed by or 
prepared from the organism to desulfurize the fossil fuel. The 
biocatalytically treated fossil fuel obtained following incubation has 
significantly reduced levels of organosulfur compounds, compared to a 
sample of the corresponding untreated fossil fuel. 
In yet another aspect, the invention relates to nucleic acid probes which 
hybridize to the recombinant DNA of the present invention. 
In still other aspects, the present invention relates to the production of 
new non-human organisms containing the recombinant DNA of the present 
invention and preferably expressing the biocatalyst encoded therein. 
Availability of the recombinant DNA of this invention greatly simplifies 
and facilitates the production and purification of biocatalysts for 
desulfurizing a fossil fuel. Costly and time consuming procedures involved 
in the purification of biocatalysts can be reduced, eliminating the need 
to generate the biocatalyst from one or more non-human organisms in which 
it is naturally present or has been produced by mutagenesis. More 
specifically, non-human host organisms can be generated which express the 
gene or genes of the present invention at elevated levels. In addition, 
the invention described herein furthers the discovery of genes encoding 
desulfurization biocatalysts in additional non-human organisms. This 
objective can be accomplished using the nucleic acid probes of the present 
invention to screen DNA libraries prepared from one or more additional 
non-human organisms in whom biocatalytic function is known or suspected to 
be present. Any genes present in such organisms and encoding 
desulfurization biocatalysts or components thereof can be replicated at 
large scale using known techniques, such as polymerase chain reaction 
(PCR). PCR advantageously eliminates the need to grow the non-human 
organisms, e.g., in culture, in order to obtain large amounts of the DNA 
of interest.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
In the petroleum extraction and refining arts, the term "organic sulfur" is 
generally understood as referring to organic molecules having a 
hydrocarbon framework to which one or more sulfur atoms (called 
heteroatoms) are covalently joined. These sulfur atoms can be joined 
directly to the hydrocarbon framework, e.g., by one or more carbon-sulfur 
bonds, or can be present in a substituent joined to the hydrocarbon 
framework of the molecule, e.g., a sulfonyl group (which contains a 
carbon-oxygen-sulfur covalent linkage). The general class of organic 
molecules having one or more sulfur heteroatoms are sometimes referred to 
as "organosulfur compounds". The hydrocarbon portion of these compounds 
can be aliphatic, aromatic, or partially aliphatic and partially aromatic. 
Cyclic or condensed multicyclic organosulfur compounds in which one or more 
sulfur heteroatoms are linked to adjacent carbon atoms in the hydrocarbon 
framework by aromatic carbon-sulfur bonds are referred to as 
"sulfur-bearing heterocycles". The sulfur that is present in many types of 
sulfur-bearing heterocycles is referred to as "thiophenic sulfur" in view 
of the five-membered aromatic ring in which the sulfur heteroatom is 
present. The simplest such sulfur-bearing heterocycle is thiophene, which 
has the composition C.sub.4 H.sub.4 S. 
Sulfur-bearing heterocycles are known to be stable to conventional 
desulfurization treatments, such as HDS. For this reason, they are said to 
be refractory or recalcitrant to HDS treatment. Sulfur-bearing 
heterocycles can have relatively simple or relatively complex chemical 
structures. In complex heterocycles, multiple condensed aromatic rings, 
one or more of which can be heterocyclic, are present. The difficulty of 
desulfurization increases with the structural complexity of the molecule. 
Shih et al. That is, refractory behavior is most accentuated in complex 
sulfur-bearing heterocycles, such as dibenzothiophene (DBT, C.sub.12 
H.sub.8 S). 
DBT is a sulfur-bearing heterocycle that has a condensed, multiple aromatic 
ring structure in which a five-membered thiophenic ring is flanked by two 
six-membered benzylic rings. Much of the residual post-HDS organic sulfur 
in fossil fuel refining intermediates and combustible products is 
thiophenic sulfur. The majority of this residual thiophenic sulfur is 
present in DBT and derivatives thereof having one or more alkyl or aryl 
radicals attached to one or more carbon atoms present in one or both 
flanking benzylic rings. Such DBT derivatives are said to be "decorated" 
with these radicals. DBT itself is accepted in the relevant arts as a 
model compound illustrative of the behavior of the class of compounds 
encompassing DBT and alkyl- and/or aryl-decorated derivatives thereof in 
reactions involving thiophenic sulfur. Monticello and Finnerty (1985), 
Microbial desulfurization of fossil fuels, 39 ANNUAL REVIEWS IN 
MICROBIOLOGY 371-389, at 372-373. DBT and radical-decorated derivatives 
thereof can account for a significant percentage of the total sulfur 
content of particular crude oils, coals and bitumen. For example, these 
sulfur-bearing heterocycles have been reported to account for as much as 
70 wt % of the total sulfur content of West Texas crude oil, and up to 40 
wt % of the total sulfur content of some Middle East crude oils. Thus, DBT 
is considered to be particularly relevant as a model compound for the 
forms of thiophenic sulfur found in fossil fuels, such as crude oils, 
coals or bitumen of particular geographic origin, and various refining 
intermediates and fuel products manufactured therefrom. Id. Another 
characteristic of DBT and radical-decorated derivatives thereof is that, 
following a release of fossil fuel into the environment, these 
sulfur-bearing heterocycles persist for long periods of time without 
significant biodegradation. Gundlach et al. (1983), 221 SCIENCE 122-129. 
Thus, most prevalent naturally occuring microorganisms do not effectively 
metabolize and break down sulfur-bearing heterocycles. 
A fossil fuel that is suitable for desulfurization treatment according to 
the present invention is one that contains organic sulfur. Such a fossil 
fuel is referred to as a "substrate fossil fuel". Substrate fossil fuels 
that are rich in thiophenic sulfur (wherein a significant fraction of the 
total organic sulfur is thiophenic sulfur, present in sulfur-bearing 
heterocycles, e.g., DBT) are particularly suitable for desulfurization 
according to the method described herein. Examples of such substrate 
fossil fuels include Cerro Negro or Orinoco heavy crude oils; Athabascan 
tar and other types of bitumen; petroleum refining fractions such as light 
cycle oil, heavy atmospheric gas oil, and No. 1 diesel oil; and 
coal-derived liquids manufactured from sources such as Pocahontas #3, 
Lewis-Stock, Australian Glencoe or Wyodak coal. 
Biocatalytic desulfurization (biocatalysis or BDS) is the excision 
(liberation or removal) of sulfur from organosulfur compounds, including 
refractory organosulfur compounds such as sulfur-bearing heterocycles, as 
a result of the selective, oxidative cleavage of carbon-sulfur bonds in 
said compounds by a biocatalyst. BDS treatment yields the desulfurized 
combustible hydrocarbon framework of the former refractory organosulfur 
compound, along with inorganic sulfur--substances which can be readily 
separated from each other by known techniques such as frational 
distillation or water extraction. For example, DBT is converted into 
hydroxybiphenyl or dihydroxybiphenyl, or a mixture thereof, when subjected 
to BDS treatment. BDS is carried out by a biocatalyst comprising one or 
more non-human organisms (e.g., microorganisms) that functionally express 
one or more enzymes that direct, singly or in concert with each other, the 
removal of sulfur from organosulfur compounds, including sulfur-bearing 
heterocycles, by the selective cleavage of carbon-sulfur bonds in said 
compounds; one or more enzymes obtained from such microorganisms; or a 
mixture of such microorganisms and enzymes. Organisms that exhibit 
biocatalytic activity are referred to herein as being CS+ or Dsz+. 
Organisms that lack biocatalytic activity are referred to herein as being 
CS- or Dsz-. 
As summarized above, the invention described herein relates in one aspect 
to a DNA molecule or fragment thereof containing a gene or genes which 
encode a biocatalyst capable of desulfurizing a fossil fuel that contains 
organosulfur compounds. The present DNA molecule or fragment thereof can 
be purified and isolated DNA obtained from, e.g., a natural source, or can 
be recombinant (heterologous or foreign) DNA that is, e.g., present in a 
non-human host organism. The following discussion, which is not to be 
construed as limiting on the invention in any way but is presented for 
purposes of illustration, recounts the isolation of DNA encoding a 
desulfurizing biocatalyst from a strain of Rhodococcus rhodochrous, ATCC 
No. 53968, that is known to express suitable biocatalytic activity. This 
preferred strain of Rhodococcus rhodocrous is disclosed in U.S. Pat. No. 
5,104,801 (issued 1992), the teachings of which are incorporated herein by 
reference, and has been referred to in the literature as IGTS8. IGTS8 was 
developed by investigators at the Institute of Gas Technology in Chicago 
Ill. Other organisms that are known to express suitable biocatalytic 
activity and thus are viewed as suitable sources of the DNA of the present 
invention include the strain of Bacillus sphaericles described in U.S. 
Pat. No. 5,002,888 and deposited with the American Type Culture Collection 
as ATCC No. 53969 and the Corynebacterium strain described in Omori et al. 
(1992), Desulfurization of dibenzothiophene by Corynebacterium sp. strain 
SY1, 58 APPL. ENV. MICROBIOL. (No. 3) 911-915. The isolation of the DNA 
of the present invention from the ATCC No. 53968 microorganism is 
schematically depicted in FIG. 1, and will now be described. 
Mutant strains of R. rhodochrous that are incapable of cleaving 
carbon-sulfur bonds (CS- or Dsz-), are produced by exposing a strain of R. 
rhodochrous, e.g., ATCC No. 53968, that exhibits biocatalytic activity 
(that is CS+ or Dsz+), to a mutagen under appropriate conditions that are 
known to or readily ascertainable by those skilled in the art. Suitable 
mutagens include radiation, e.g., ultraviolet radiation, and chemical 
mutagens, e.g., N-methyl-N'-nitrosoguanidine (NTG), hydroxylamine, 
ethylmethanesulphonate (EMS) and nitrous acid. Mutants thus formed are 
allowed to grow in an appropriate medium and screened for carbon-sulfur 
bond cleavage activity. Mutants identified as lacking carbon-sulfur bond 
cleavage activity are termed CS-. Any method of screening which allows for 
an accurate detection of carbon-sulfur bond cleavage activity is suitable 
in the method of the present invention. Suitable methods of screening for 
this activity include exposing the different mutants to carbon-sulfur bond 
containing molecules (e.g., DBT) and measuring carbon-sulfur bond 
cleavage. In a preferred embodiment, the mutants are exposed to DBT, such 
that the breakdown product, hydroxybiphenyl (HBP), which fluoresces under 
short wave ultraviolet light, is produced. HBP can also be detected 
colorimetrically through its blue reaction product with Gibbs' reagent. 
Other methods include gas and liquid chromatography, infrared and nuclear 
magnetic resonance spectrometry. See Kodama, et al., Applied and 
Environmental Microbiology, pages 911-915 (1992) and Kilbane and Bielaga, 
Final Report D.O.E. Contract No. DE-AC22-88PC8891 (1991). Once CS- mutants 
are identified and isolated, clones thereof are propagated using standard 
techniques and subjected to further analysis. 
Concurrent with the mutagenesis of the above-described culture of the CS+ 
organism, R. rhodochrous, a second culture of the same CS+ organism (1) is 
maintained in culture. CS+ organism DNA (3) is extracted from this culture 
of R. rhodocrous. Various methods of DNA extraction are suitable for 
isolating the DNA of this organism. Suitable methods include phenol and 
chloroform extraction. See Maniatis et al., Molecular Cloning, A 
Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, page 16.54 
(1989), herein referred to as Maniatis et al.. 
Once the DNA is extracted from R. rhodochrous 1, the DNA (3) is cut into 
fragments of various kilobase lengths, which collectively make up DNA 
library 5. Various methods of fragmenting the DNA of R. rhodochrous to 
purify DNA therefrom, including the DNA of the present invention, can be 
used, e.g., enzymatic and mechanical methods. Any four-base recognition 
restriction endonuclease such as TaqI or Sau 3A is suitable for 
fragmenting the DNA. Suitable methods of fragmenting DNA can be found in 
Maniatis et al.. 
The various DNA fragments are inserted into several CS- mutant clones of R. 
rhodochrous (2), with the purpose of isolating the fragment of DNA that 
encodes the biocatalyst of the present invention. The transformation of a 
previously CS- mutant cell to a CS+ transformed cell is evidence that the 
inserted DNA fragment encodes a biocatalyst. Any method of inserting DNA 
into R. rhodochrous which allows for the uptake and expression of said 
fragment is suitable. In a preferred embodiment, electroporation is used 
to introduce the DNA fragment into R. rhodochrous. See Maniatis et al.. 
Once transformed, CS+ mutant R. rhodochrous 7 has been produced and 
identified, DNA fragment 9 encoding the CS+ biocatalyst can be identified 
and isolated. The encoded biocatalyst can then be produced using the 
isolated DNA in various methods that are well known and readily available 
to those skilled in the art. In addition, the isolated DNA can be 
sequenced and replicated by known techniques, e.g., the techniques 
described in Maniatis et al.. 
As noted previously, the above-described method for isolating the DNA of 
the present invention can be applied to CS+ organisms other than R. 
rhodocrous microorganisms, e.g., of the strain ATCC No. 53968. Thus, 
Bacillus sulfasportare ATCC No. 53969 or Corynebacterium sp. SY1 can be 
used as the source organism for the DNA of the present invention. 
Furthermore, once isolated, the DNA of the present invention can be 
transfected into a non-human host organism other than a CS- mutant of the 
source organism. Thus, the DNA of the present invention can be transfected 
into, e.g., a suitable strain of Escherichia coli bacteria. Other types of 
non-human host organism can also be used, including unicellular organisms 
(e.g., yeast) and cells established in culture from multicellular 
organisms. 
Other methods of isolating the DNA of the present invention, include 
variations on the rationale described above and depicted in FIG. 1. For 
example, it would be possible to randomly insert a CS- DNA plasmid into 
clones of a CS+ strain of R. rhodochrous. DNA encoding a CS+ biocatalyst 
could then be identified by screening for clones that have been 
transformed from CS+ to CS-. 
The recombinant DNA molecule or fragment thereof of the present invention 
is intended to encompass any DNA resulting from the insertion into its 
chain, by chemical or biological means, of one or more genes encoding a 
biocatalyst capable of selectively cleaving carbon-sulfur bonds, said gene 
not originally present in that chain. Recombinant DNA includes any DNA 
created by procedures using restriction nucleases, nucleic acid 
hybridization, DNA cloning, DNA sequencing or any combination of the 
preceding. Methods of construction can be found in Maniatis et al., and in 
other methods known by those skilled in the art. 
Procedures for the construction of the DNA plasmids or vectors of the 
present invention include those described in Maniatis et al. and other 
methods known by those skilled in the art. Suitable plasmid vectors 
include pRF-29 and pRR-6 depicted in FIGS. 2 and 3, respectively. The 
terms "DNA plasmid" and "vector" are intended to encompass any replication 
competent plasmid or vector capable of having foreign or exogenous DNA 
inserted into it by chemical or biological means and subsequently, when 
transfected into an appropriate non-human host organism, of expressing the 
product of the foreign or exogenous DNA insert (i.e., of expressing the 
biocatalyst of the present invention). In addition, the plasmid or vector 
must be receptive to the insertion of a DNA molecule or fragment thereof 
containing the gene or genes of the present invention, said gene or genes 
encoding a biocatalyst that selectively cleaves carbon-sulfur bonds in 
organosulfur compounds. Procedures for the construction of DNA plasmid 
vectors include those described in Maniatis et al. and others known by 
those skilled in the art. 
The plasmids of the present invention include any DNA fragment containing a 
gene or genes encoding a biocatalyst that selectively cleaves 
carbon-sulfur bonds in organosulfur compounds. The term "plasmid" is 
intended to encompass any DNA fragment. The DNA fragment should be 
transmittable to a host microorganism by transformation or conjugation. 
Procedures for the construction or extraction of DNA plasmids include 
those described in Maniatis et al. and others known by those skilled in 
the art. 
The transformed non-human host organisms of the present invention can be 
created by various methods by those skilled in the art. For example, 
transfection electroporation as explained by Maniatis et al. can be used. 
By the term "non-human host organism" is intended any non-human organism 
capable of the uptake and expression of foreign, exogenous or recombinant 
DNA, i.e., DNA not originally a part of the organism's nuclear material. 
The method of desulfurizing a fossil fuel of the present invention involves 
two aspects. First, a host organism or biocatalytic preparation obtained 
therefrom is contacted with a fossil fuel to be desulfurized. This can be 
done in any appropriate container, optionally fitted with an agitation or 
mixing device. The mixture is combined thoroughly and allowed to incubate 
for a sufficient time to allow for cleavage of a significant number of 
carbon-sulfur bonds in organosulfur compounds, thereby producing a 
desulfurized fossil fuel. In one embodiment, an aqueous emulsion is 
produced with an aqueous culture of the organism and the fossil fuel, 
allowing the organism to propagate in the emulsion while the expressed 
biocatalyst cleaves carbon-sulfur bonds. 
Variables such as temperature, mixing rate and rate of desulfurization will 
vary according to the organism used. The parameters can be determined 
through no more than routine experimentation. 
Several suitable techniques for monitoring the rate and extent of 
desulfurization are well-known and readily available to those skilled in 
the art. Baseline and timecourse samples can be collected from the 
incubation mixture, and prepared for a determination of the residual 
organic sulfur in the fossil fuel. The disappearance of sulfur from 
organosulfur compounds, such as DBT, in the sample being subjected to 
biocatalytic treatment can be monitored using, e.g., X-ray fluorescence 
(XRF) or atomic emission spectrometry (flame spectrometry). Preferably, 
the molecular components of the sample are first separated, e.g., by gas 
chromatography. 
The nucleic acid probes of the present invention include any nuclear 
material capable of hybridizing to at least a portion of the DNA of the 
present invention. The term "nucleic acid probe" includes any nuclear 
material capable of hybridizing to DNA. 
The invention will now be further illustrated by the following specific 
Examples, which are not to be viewed as limiting in any way. 
EXAMPLE 1 
Isolation of DNA Encoding a Desulfurization Active Biocatalyst 
As used herein, the term "Dsz+" refers to the ability of an organism to 
utilize thiophenic compounds such as dibenzothiophene (DBT) as the sole 
source of sulfur by the selective cleavage of carbon-sulfur bonds therein. 
Rhodococcus rhodochrous strain IGTS8 demonstrates the Dsz.sup.+ phenotype. 
The term "Dsz-" referrs to an organism's inability to utilize said 
thiophenic compounds as a sole source of sulfur by the selective cleavage 
of carbon-sulfur bonds therein. 
Materials 
Bacterial Strains and Plasmids 
Rhodococcus rhodochrous strain IGTS8 (ATCC No. 53968), obtained from the 
Institute of Gas Technology (Chicago, Ill.), was used as a parent strain 
for production of mutant strains which have lost the desulfurization 
phenotype (Dsz-). Strain IGTS8 was also used for isolation of DNA 
fragments capable of complementing said mutants to produce Dsz+ mutants 
therefrom. Rhodococcus vector pRF-29 was obtained from the Institute of 
Gas Technology. The construction of pRF-29 is described in Desomer, et al. 
(1990 ), Transformation of Rhodococcus fascians by High-Voltage 
Electroporation and Development of R. fascians Cloning Vectors, APPLIED 
AND ENVIRONMENTAL MICROBIOLOGY 2818-2825. The structure of pRF-29 is 
schematically depicted in FIG. 2. 
Escherichia coli strain JM109 was used as a host in transformation with 
plasmid constructs derived from the plasmids pUC18 and pUC19 (Bethesda 
Research Laboratories, Bethesda, Md.). 
Enzymes and Reagents 
Restriction endonucleases were purchased from Bethesda Research 
Laboratories (BRL) and New England Biolabs (Beverly, Mass.). T4 ligase and 
the Klenow fragment of E. coli DNA polymerase I were purchased from BRL. 
HK.TM. Phosphatase was purchased from Epicentre Technologies (Madison, 
Wis.). All enzymes were used in accordance with manufacturers 
recommendations. Enzyme assay substrates Dibenzothiophene (DBT), 
Dibenzothiophene 5-oxide (DBT sulfoxide) and Dibenzothiphene sulfone (DBT 
sulfone) were purchased from Aldrich (Milwaukee, Wis.). Gibb's Reagent, 
2,6-dicholoroquinone-4-chloroimide, was purchased from Sigma (St. Louis, 
Mo.). Chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was also 
purchased from Sigma. 
Growth Media and Conditions 
E. coli JM109 was grown in L-broth (Difco, Detroit, Mich.). Transformants 
were selected on L-plates supplemented with 1.5% agar and containing 125 
.mu.g/ml ampicillin. E. coli strains were grown at 37.degree. C. 
Rhodococcus strains were maintained on Rhodococcus Media (RM) composed per 
liter of: 8.0g Nutrient Broth (Difco), 0.5g yeast extract, 10.0g glucose. 
Transformants of Rhodococcus strains were selected on RM plates 
supplemented with 1.5% agar and containing 25 .mu.g/ml chloramphenicol. 
For expression of the Dsz+ phenotype, Rhodococcus strains were grown in 
Basal Salts Media (BSM) composed per liter of: 2.44g KH.sub.2 PO.sub.4, 
5.57g Na.sub.2 HPO.sub.4 2.0g NH.sub.4 Cl, 0.2 g MgCl.sub.2.6H.sub.2 O, 
0.001g CaCl.sub.2.2H.sub.2 O, 0.001g FeCl.sub.3 6H.sub.2 O, 0.004g 
MnCl.sub.2.4H.sub.2 O, 6.4ml glycerol. Optionally, BSM can be supplemented 
with 5.0g/liter glucose. Rhodococcus strains were grown at 30.degree. C. 
Methods 
Sulfur Bioavailability Assay 
The sulfur bioavailability assay, described in U.S. Pat. No. 5,104,801, 
examines an organism's ability to liberate organically bound sulfur from 
substrates (e.g., DBT, DBT sulfoxide, DBT sulfone) for use as the sole 
source of sulfur for growth. In the assay, BSM, which contains no sulfur, 
is supplemented with one or more sulfur containing substrates, e.g., DBT. 
The organism's ability to liberate sulfur therefrom is measured by its 
ability to grow with proper incubation, as monitored by optical density at 
600 nm. 
Gibbs Assay for 2-Hydroxybiphenyl 
The oxidative product of DBT, DBT sulfoxide and DBT sulfone incubated with 
strain IGTS8 is 2-hydroxybiphenyl (2-HBP). The Gibbs assay 
colorimetrically quantitates the amount of 2-HBP produced from DBT and its 
above-mentioned oxidative derivatives. The assay measures 2-HBP produced 
in culture supernatants after incubation with DBT. The media must be 
adjusted to pH 8.0 before the Gibb's reagent is added. Gibb's Reagent, 
2,6- dicholoroquinone-4-chloroimide (10mg/ml in ethanol), is added to 
culture supernatants at 1:100 (v/v). Color development is measured as 
absorbance at 610nm after a 30 minute incubation at room temperature. 
HPLC Assay for 2-Hydroxybiphenyl 
2-HBP production cultures incubated with DBT can also be detected by HPLC 
using instrumentation available from Waters, Millipore Corporation, 
Milford, Mass. Reagent alcohol is added to culture broth at 1:1 (v/v) in 
order to solubilize all remaining DBT and 2-HBP. Samples are agitated for 
5 min at 220 rpm. Extracted broth samples are removed and centrifuged to 
remove cellular mass. Clarified supernatants are then analyzed by HPLC 
with the following conditions: 
______________________________________ 
Column: Waters 4.mu. Phenyl Novapak 
Detection DBT 233 nm, 1.0 AUFS 
Parameters: 2-HBP 248 nm, 0.2 AUFS 
Quantitative DBT 10-250 .mu.M 
Detection Limits: 
2-HBP 6-60 .mu.M 
Mobile Phase: Isocratic 70% Acetonitrile 
1.5 ml/min 
Retention times: 
DBT 4.5 minutes 
2-HBP 2.9 minutes 
______________________________________ 
IGTS8 Mutagenesis 
In order to generate mutant strains of R. rhodochrous which did not 
metabolize DBT (Dsz- mutants), biocatalyst source strain IGTS8 (Dsz+) was 
subjected to mutagenesis by short-wave UV light and to chemical 
mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). With UV 
exposure mutagenesis, a kill rate of greater than 99% was targeted. 
Continuously stirred R. rhodochrous cells at an optical density 
(A.sub.660) of 0.3 were subjected to UV exposure from a Mineralight Lamp 
Model UVG-254 (Ultra-violet Products, Inc., San Gabriel, Calif.) at a 
distance of 10 cm for 55 to 65 seconds to obtain this kill rate 
(97.9-99.9%). For NTG mutagenesis, cell suspensions were treated with 500 
.mu.g/ml NTG for a duration determined to achieve a kill rate of 30%-50%. 
Combination mutagenesis utilizing both NTG and UV was also done. For these 
an overall kill rate of greater than 99.9% was used. Colonies surviving 
mutagenesis were picked onto RM plates and screened for the Dsz- phenotype 
as described below. 
Screening Example A: Initially, a DBT-spray plate screen was used to select 
Dsz- mutants. Mutant colonies were replica plated onto Basal Salts Media 
(BSM) electrophoretic-grade agarose plates which contained no added 
sulfur. Colonies were allowed to grow at 30.degree. C. for 24hr. The 
plates were then sprayed with an even coating of 10% DBT dissolved in 
ether and incubated at 30.degree. C. for 90 minutes. The plates were then 
wiped clean and observed under short-wave UV light. The observed end 
product of DBT metabolism, 2-hydroxybiphenyl (2-HBP) fluoresces under 
short-wave UV light. Colonies that produce 2-HBP are thus identified by 
fluorescent spots on the agarose. Colonies that do not produce 2-HBP (that 
are Dsz-) do not produce fluorescent spots. 
Screening Example B: A simpler variation of screening involved replica 
plating surviving mutagenized colonies to BSM agarose plates supplemented 
with 1.2ml/liter of a saturated ethanol solution of DBT. After 24 hours, 
production of 2-HBP can be visualized under UV illumination as above. 
Mutants which did not appear to produce 2-HBP by the above-described 
screening methods were examined with the sulfur bioavailability assay, 
with DBT as the sole source of sulfur. Growth of potential mutants was 
examined in 1.25ml liquid fermentations in BSM plus DBT media dispensed in 
24-well plates (Falcon). After a one day incubation at 30.degree. C. 2-HBP 
production was monitored by the Gibbs colorimetric assay. Strains which 
continue to demonstrate the Dsz- phenotype were incubated in larger 
volumes of BSM plus DBT and analyzed for 2-HBP or intermediates by the 
HPLC method. Because BSM is a defined minimal medium, a duplicate control 
culture which contained supplemental inorganic sulfur was grown in order 
to distinguish true Dsz- mutants from auxotrophic mutants. Mutants which 
failed to grow in both the control and experimental media were assumed to 
be auxotrophic mutants. 
Of 1970 individually analyzed potential mutants, two were identified as 
Dsz-. One mutant, GPE-362, was generated by NTG mutagenesis. The other, 
CPE-648, was generated by combination NTG/UV mutagenesis. Both GPE-362 and 
CPE-648 grow slowly in sulfur bioavailability assays, presumably from 
trace amounts of sulfur on the glassware or in the media components. 
However, no detectable amounts of 2-HBP were produced by either mutant 
after an extended incubation of 6 to 10 days with DBT, as assessed with 
either the Gibbs assay or the HPLC assay. Thus, independently produced R. 
rhodocrous IGTS8 mutants GPE-362 and CPE-648 were Dsz- organisms. 
Vector Construction 
Vector constructs were derived from R. rhodochrous and confer 
chloramphenicol resistance. All constructs were developed in E. coli 
strain JM109. Transformation of JM109 was carried out with the Gene Pulsar 
(Bio-Rad Laboratories, Richmond, Calif.) according to manufacturer's 
recommendations. Plasmid isolation from JM109 was performed by standard 
methods (Birnboim and Doly (1979), A rapid alkaline extraction procedure 
for screening recombinant plasmid DNA, 7 NUCLEIC ACIDS RES. 1513-1523; 
Maniatis et al. (1982), MOLECULAR CLONING: A LABORATORY MANUAL (Cold 
Spring Harbor Laboratory Press). Transformants containing correct vector 
constructs were identified by restriction analysis. 
Vector Construct A: pRR-6 (FIG. 3) contains the Rhodococcus origin of 
replication and Chloramphenicol resistance marker (Cm.sup.R). The ori and 
Cm.sup.R have been removed from pRF-29 as a 6.9kb XhoI/Xba (partial) 
fragment. The ends were made blunt with Klenow and ligated to SaII/XbaI 
cut pKF39. pKF39 is pUC18 with the SmaII site replaced with a BgIII site. 
A unique NarI site is available for cloning in pRR-6. NarI ends are 
compatible with 4-base recognition endonuclease TaqI. 
Transformation of Rhodococcus rhodochrous 
Transformation of IGTS8 and Dsz- mutants thereof can be achieved by 
electroporation. The following conditions were used in all transformations 
of Rhodococcus rhodochrous. Cells were grown in RM to mid-log phase and 
harvested by centrifugation (5000xg), then washed three times in cold, 
deionized, distilled water and concentrated 50-fold in 10% glycerol. The 
resulting cell concentrate could be used for electroporation directly or 
stored at -80.degree. C. 
Electroporations were carried out with the Gene Pulser (Bio-Rad) apparatus. 
100 .mu.l cells were mixed with transformation DNA in a 2-mm gapped 
electrocuvette (Bio-Rad) and subjected to a 2.5 kV pulse via the pulse 
controller (25 .mu.F capacitor, 200 .OMEGA. external resistance). Pulsed 
cells were mixed with 400.mu. RM and incubated for 4 hours at 30.degree. 
C. with regular agitation. Cells were then plated to RM supplemented with 
proper antibiotic. 
When IGTS8 was transformed with pRF-29, chloramphenicol resistant colonies 
were cleanly selected at a frequency of 10.sup.5 -10.sup.6 /.mu.g DNA on 
plates containing 25 .mu.g/ml chloramphenicol. 
Small Scale Plasmid Preparation from R. rhodochrous 
A single colony of Rhodococcus rhodochrous was used to inoculate 2 to 7ml 
of RM plus 25 .mu.g/ml chloramphenicol. The culture was incubated for two 
days at 30.degree. C. with shaking. Cells were pelleted by centrifugation 
and resuspended in 300 .mu.l sucrose buffer (20% sucrose, 0.05M Tris-Cl pH 
8.0, 0.01M EDTA 0.05M NaCl, 10 mg/ml lysozyme) and incubated at 37.degree. 
C. for 1 hour. 300 .mu.l Potassium acetate-acetate solution, pH 4.8 (60 ml 
5M KOAc, 11.5 ml Glacial acetic acid, 28.5 ml dH.sub.2 O), was added and 
the mixture was gently mixed by inversion. The mixture was placed on ice 
for 5 minutes and then cellular debris was pelleted by centrifugation. 500 
.mu.l supernatant was removed to a fresh tube to which RNAse was added to 
0.05 .mu.g/.mu.l and incubated for 20 minutes at 37.degree. C. The sample 
was then phenol:chloroform extracted and the aqueous layer was 
precipitated at -80.degree. C. with an equal volume of isopropanol. DNA 
was pelleted by centrifugation and resuspended in 0.3M NaOAc pH 8.0. DNA 
was precipitated again at -80.degree. C. with an equal volume of 
isopropanol. DNA was pelleted by centrifugation and resuspended in 0.3M 
NaOAc pH 8.0. DNA was precipitated again at -80.degree. C. with two 
volumes of 95% EtOH. Pelleted DNA was washed with 70% EtOH and resuspended 
in 50 .mu.l TE (Tris EDTA). 
Isolation of Genomic DNA from R. rhodochrous Strain IGTS8 
IGTS8 genomic DNA was isolated as described. 20 ml RM was inoculated with a 
single colony of IGTS8 and incubated at 30.degree. C. for 48 hours with 
shaking at 220 rpm. Cells were harvested by centrifugation (5000xg). Cells 
were resuspended in 10ml TE (10 mM Tris Base, 1 mM EDTA) with 100 mg 
lysozyme and incubated for 30 minutes at 30.degree. C. Cells were lysed by 
adding 1 ml of 20% sodium dodecyl sulfate (SDS). 10 ml of TE-saturated 
phenol and 1.5 ml 5M NaCI were added immediately and the mixture was 
gently agitated for 20 minutes at room temperature. Phenol was removed by 
centrifugation, and the aqueous layer was extracted twice with an equal 
volume of chloroform. An equal volume of isopropanol was added to the 
aqueous layer to precipitate the DNA. DNA was spooled onto a pasteur 
pipette and redissolved in TE. DNA was then RNased with 20 .mu.g/ml RNA 
for 1 hour at 37.degree. C. The sample was made to a final concentration 
of 100 mM NaCl and 0.4% SDS and proteased with 100 .mu.g/ml protease K. 
The sample was then extracted with phenol and chloroform and precipitated 
with isopropanol as before. The purified genomic DNA, which included the 
DNA of the present invention, was resuspended in TE. 
Construction of Plasmid Library of IGTS8 
Genomic DNA from the Dsz+ source organism (IGTS8) was cut with TaqI in 
order to produce fragments 0.5-23 kb in length. Cut DNA was 
electrophoresed through 0.8% low melting temperature agarose and DNA 
fragments greater than 5 kb in length were isolated and purified by 
standard methods (Maniatis, T. et al. (1982), MOLECULAR CLONING: A 
LABORATORY MANUAL (Cold Spring Harbor Laboratory Press)). Vector pRR-6 was 
cut with NarI to completion. The vector ends were dephosphorylated with 
HK.TM. phosphatase to prevent religation of the vector. The 
size-fractioning genomic DNA was ligated to cut and dephosphorylated 
pRR-6. 
Molecular Complementation of Dsz- Mutant Strain CPE-648 
Plasmid library ligations (above) were used to transform Dsz- mutant strain 
CPE-648 by electroporation as described. Negative control transformations 
of CPE-648, which did not contain DNA (mock transformations), were also 
performed. After the four hour incubation in RM, the cells were spun out 
of suspension by centrifugation and the supernatant was removed. The cells 
were resuspended in BSM with no sulfur. These cells were used to inoculate 
250 ml of BSM supplemented with 300 .mu.l of a saturated ethanol solution 
of DBT. By this procedure, clones which are capable of complementing the 
Dsz- mutation will be selected by the sulfur bioavailability assay. 
Strains containing the complementing sequences (i.e., the DNA of the 
present invention) will successfully remove the sulfur from DBT and grow 
preferentially. 
After 6 days incubation at 30.degree. C., the cultures were assayed for 
2-HBP by HPLC. Accumulation of 2-HBP was detected in experimental cultures 
while no accumulation of 2-HBP was detected in control cultures. The 
culture producing 2-HBP was spread onto RM plates supplemented with 
chloramphenicol to obtain single colonies that were harboring plasmids. 
These plates were replica-plated to BSM agarose plates supplemented with 
1.2 ml/liter of a saturated ethanol solution of DBT. After 24 hours 
incubation at 30.degree. C., 2-HBP could be detected around some 
individual colonies under short wave UV illumination. These colonies 
presumably harbored plasmids which complemented the Dsz- mutant by 
restoring the former Dsz+ phenotype. 
Characterization of Clones Complementing Dsz- Mutant CPE-648 
Two independent plasmid libraries successfully complemented mutant CPE-648 
to Dsz.sup.+ as described above. Plasmid DNA was isolated from single 
colonies which demonstrated 2-HBP production on BSM plus DBT plates 
(above) from cultures transformed with each of the two libraries. This 
plasmid DNA was used to transform E. coli strain JM109. Plasmid DNA was 
isolated and cut with restriction endonucleases in order to build a 
restriction map of the clones. Each of the two libraries yielded a single 
complementing clone. By restriction pattern similarities, the two clones 
appear to have overlapping sequences. These clones have been designated 
pTOXI-1 (FIG. 4) and pTOXI-2, respectively. pTOXI-1 contains an insert of 
approximately 6.6kb. pTOXI-2 contains an insert of approximately 16.8kb. 
Complementation of Dsz.sup.- Mutant GPE-362 
Dsz- mutant GPE-362 was transformed with plasmids pTOXI-1 and pTOXI-2. As a 
control, GPE-362 was also transformed with vector pRR-6. Transformants 
containing plasmid DNA were selected on RM plus chloramphenicol plates. 
Cm.sup.R colonies were transferred to BSM agarose plates supplemented with 
DBT. After 24 hr. incubation at 30.degree. C., 2-HBP production could be 
seen around colonies containing either pTOXI-1 or pTOXI-2 by short wave UV 
illumination. No 2-HBP could be detected around colonies containing only 
vector pRR-6. 
Overexpression of the Dsz.sup.+ Trait Upon Reintroduction of Cloned DNA 
Plasmids pTOXI-1 and pTOXI-2 were transformed into Dsz- mutant strain 
CPE-648. Transformants containing plasmid DNA were selected on RM plus 
chloramphenicol plates. The specific activity of individual clones was 
examined by the following protocol. 
Single colonies of CPE-648 containing either pTOXI-1 or pTOXI-2 were used 
to inoculate 25 ml RM plus 25 .mu.g/ml chloramphenicol in a 250 ml flask. 
As a positive control, parent strain IGTS8 was also grown in 25 ml RM. 
After 48 hours of growth at 30.degree. C., 225 rpm shaking, 2.5 ml of the 
cultures were crossed into 25 ml BSM supplemented with 0.7 mM DMSO. 
Cultures were incubated for an additional 40 hours at 30.degree. C. The 
optical density of each culture was measured at 600 nm against an 
appropriate blank. DBT was added to a final concentration of 150 .mu.M and 
the cultures were incubated for 3 hours at 30.degree. C. An equal volume 
of Reagent Alcohol (Baxter, McGaw Park, Ill.) was then added to each 
culture to solubilize any remaining DBT or 2-HBP. A 1 ml sample was 
removed and cellular debris removed by centrifugation. The supernatant was 
analyzed for 2-HBP by the HPLC assay described above. The specific 
activity is calculated as mg of 2-HBP per liter/hours of 
incubation/OD.sub.600. The results of the above assay is listed in Table 
1. 
TABLE 1 
______________________________________ 
Biocatalytic Desulfurization Activity of Transformed Mutants 
2-HBP Specific Activity 
STRAIN OD.sub.600 
(mg/l) (mg/l/hr/OD.sub.600 
______________________________________ 
IGTS8 2.89 3.94 0.45 
GPE-362 1.53 0.00 0.00 
CPE-648 4.10 0.00 0.00 
CPE648 (pTOXI-1) 
3.84 15.84 1.37 
CPE648 (pTOXI-2) 
2.88 5.74 0.66 
______________________________________ 
EXAMPLE 2 
DNA Sequencing of a Desulfurization Active Biocatalyst by the Dideoxy 
Method from Plasmid pTOXI-1 
Materials 
Bacterial Strains and Plasmids 
Plasmid pTOXI-1 was used as the original source of DNA for sequencing. 
Escherichia coli strain JM109 was used as a host for subcloning and 
plasmid maintenance. Plasmids pUC18 and pUC19 were purchased from Bethesda 
Research Laboratories (Bethesda, Md.). 
Enzymes and Reagents 
Restriction endonucleases were purchased from Bethesda Research 
Laboratories (BRL) and New England Biolabs (Beverly, Mass.). T4 ligase was 
purchased from BRL. A Sequenase Version 2.0 DNA sequencing kit was 
purchased from United States Biochemical Corporation (Cleveland, Ohio). 
All enzymes and kits were used in accordance with manufacturer's 
recommendations. 
Growth Media and conditions 
E. coli strain JM109 harboring plasmids was grown in L-broth (Difco) 
containing 100 .mu.g/ml ampicillin. Transformants were selected on 
L-plates supplemented with 1.5% agar and containing 100 .mu.g/ml 
ampicillin. E. coli strains were grown at 37.degree. C. 
Methods 
Plasmid DNA Preparation from E. coli 
Plasmid DNA was prepared from E. coli via lysis by SDS (Maniatis, et al.). 
The DNA was further purified through a polyethylene glycol precipitation 
before use in sequencing reactions. 
Plasmid Subcloning 
The following subclones of pTOXI-1 were generated by standard techniques to 
aid in DNA sequencing: 
a) pMELV-1 (FIG. 5) was derived by isolating the 6.7kb HinddIII/NdeI 
fragment from pTOXI-1 (shown in FIG. 4) and ligating it to pUC-18 cut with 
HindIII/NdeI. JM109 cells harboring pMELV-1 were identified by plasmid 
isolation and restriction endonuclease analysis (Maniatis, et al.). 
b) pSMELV-1A (FIG. 6) contains the 1.6kb SphI/XhoI fragment of pMELV-1 
subcloned into pUC-18. 
c) pSMELV-2A (FIG. 6) contains the 0.7kb BamHI/SacI fragment of pMELV-1 
subcloned into pUC-18. 
d) pSMELV-3A (FIG. 6) contains the 3.5kb SacI/XhoI fragment of pMELV-1 
subcloned into pUC-18. 
e) pSMELV-4A (FIG. 6) contains the 1.5kb SphI/BamHI fragment of pMELV-1 
subcloned into pUC-18. 
Dideoxy Sequencing from Plasmid DNA 
a) Denaturation. Prior to sequencing reactions, plasmid DNA must be 
denatured. This was accomplished by treatment with NaOH. The denatured DNA 
is then recovered by addition of salt and EtOH precipitation. Preferably, 
2-5 .mu.g of denatured plasmid DNA is used in each sequencing reaction. 
See manufacturer's recommendations with Sequenase Version 2.0 DNA 
sequencing kit (United States Biochemical Corporation). 
b) Dideoxy sequencing. Chain termination dideoxy sequencing with Sequenase 
2.0 was performed as described by the manufacturer (U.S. Biochemical 
Corporation). Sequencing of the cluster was initiated by priming subclones 
pMELV-1A, pMELV-2A, pMELV-3A, pMELV-4A with the "-40 Universal Primer" 
defined as: 
5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO:6) and the "Reverse Primer" defined as: 
5'-AACAGCTATGACCATG-3 (SEQ ID NO:7). The sequence was extended by 
synthesizing overlapping oligonucleotides to previously read sequence 
using the Gene Assembler Plus (Pharmacia, Piscataway, N.J.). The 
synthesized oligonucleotides were used as primers for continuing sequence 
reactions. Plasmid pMELV-1 was used as the template for all of the 
remaining sequences. DNA sequence was read from both strands of the 
plasmid clone to increase fidelity. 
EXAMPLE 3 
Complementation Cloning of a Desulfurization Active Biocatalyst from a 
Cosmid Library; Transfection of Biocatalyst DNA into an R. Fascians Host 
Organism 
Materials and Methods 
Bacterial Strains, Media and Reagents 
Rhodococcus sp. Rhodococcus rhodochrous strain IGTS8, obtained from the 
Institute of Gas Technology (Chicago, Ill.) was used. UV1 is a mutant of 
IGTS8 that is unable to desulfurize DBT, described herein. R. fascians 
D188-5 (Desomer, et al., J. Bacteriol., 170:2401-2405, 1988) and R. 
rhodochrous ATCC13808 (type strain from ATCC) do not metabolize DBT. E. 
coli XL1-Blue (from Stratagene Cloning System, La Jolla, Calif.) is recA1 
lac thi endA1 gyrA96 hsdR17 supE44 re1A1 [F' proAB lacI.sup.q 
lacZ.DELTA.M15 Tn10]. E. coli CS109 is W1485 thi supE F. E. coli S17-1 is 
a derivative of E. coli 294 and is recA thi pro hsdR.sup.- res.sup.- 
mod.sup.+ [RP4-2-Tc::Mu-Km::Tn7] (Simon, et al., Plasmid vectors for the 
genetic analysis and manipulation of rhizobia and other gram-negative 
bacteria, p. 640-659. In A. Weissbach, and H. Weissbach (eds.), Methods in 
enzymology, vol 118, Academic Press, Inc., Orlando, 1986). 
Pseudomonas minimal salts medium (PMS) was prepared according to Giurard 
and Snell (Biochemical factors in growth, p. 79-111. In P. Gerhardt, R. G. 
E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. 
B. Phillips (eds.), Manual of methods for general bacteriology, American 
Society for Microbiology, Washington, DC., 1981) and contained 0.2% 
glycerol, 40 mM phosphate buffer (pH 6.8), 2% Hutner's mineral base, and 
0.1% (NH.sub.4).sub.2 SO.sub.4. PMS medium lacking sulfate was prepared 
with chloride salts in place of sulfate salts. Luria broth (LB) was 1% 
bactotryptone, 0.5% yeast extract, and 1% NaCl. All liquid medium 
incubations were performed with shaking in water baths (New Brunswick 
Scientific, Edison, N.J.). Ampicillin (50 .mu.g/ml) and tetracycline (12.5 
.mu.g/ml) were included as selective agents when required. 
Dibenzothiophene (DBT) was purchased from Fluka Chemical Corporation of 
Ronkonkoma, N.Y. DBT-sulfoxide was from ICN Bio-chemicals of Irvine, 
Calif., and DBT-sulfone was obtained from Aldrich Chemical Company of 
Milwaukee, Wis. Agarose was obtained from BRL. 
Plasmid Vectors 
pLAFR5 (Keen, et al., Gene 70:191-197, 1988) and pRF29 (Desomer, et al., 
1988) served as sources of the Rhodococcus plasmid origin of replication. 
Cosmid Library Construction 
High molecular weight DNA was isolated from IGTS8 by the method of 
Consevage et al, (J. Bacteriol., 162:138-146, 1985), except that cell 
lysis was accomplished in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) 
containing lysozyme (5 mg/ml) and SDS (2%). The DNA was partially digested 
with Sau3AI and fragments of approximately 20 kb were isolated after 
centrifugation through a sodium chloride gradient (Frischauf, et al., 
Digestion of DNA: size fractionation, p. 183-189. In S. L. Berge, and A. 
R. Kimmel (eds.), Methods in Enzymology, vol 152, Academic Press, Inc, San 
Diego, Calif., 1987). These fragments were ligated into the BamHI site of 
pLAFR5 using standard procedures. In vitro packaging was performed using 
Gigapack Plus (Stratagene). Packaged cosmids were transduced into E. coli 
S17-1. 
DBT Spray Plate Assay 
A spray plate assay for the identification of bacteria capable of modifying 
dibenzothiophene (DBT) was originally described by Kiyohara et al, (Appl. 
Environ. Microbiol., 43:454-457, 1982) and modified by Krawiec (Bacterial 
desulfurization of thiophenes: screening techniques and some speculations 
regarding the biochemical and genetic bases, p. 103-114. In G. E. Pierce 
(ed.), Developments in Industrial Microbiology, vol 31, Society for 
Industrial Microbiology, Columbus, Ohio, 1990). The assay was further 
modified for use with R. rhodochrous IGTS8 as follows. Cells from 
individual IGTS8 colonies were transferred to LB plates as small (0.5 cm) 
patches and were incubated at 30.degree. C. for 24 to 36 h. Large amounts 
of cells from these patches were transferred onto PMS-1% agarose plates 
that lacked a source of sulfur. These plates were immediately sprayed with 
a 0.1% DBT solution in ethyl ether. The PMS-DBT plates were incubated at 
30.degree. C. for a minimum of 18 hours and fluorescent products around 
the patches were detected by viewing under short-wave (254 nm) UV 
illumination. 
Sulfur Bioavailability Assay 
IGTS8 was incubated in PMS medium at 30.degree. C. for 24 to 48 h, the 
cells were pelleted by centrifugation, followed by two washes with 
sulfur-free PMS. Washed cells were inoculated into PMS that contained, as 
a sole source of sulfur, a 0.2% concentration of one of the following: 
DBT, DBT-sulfoxide, or DBT-sulfone. The inoculum was adjusted so that the 
beginning absorbance at 600 nm (A.sub.600) was 0.02. The culture was 
incubated at 30.degree. C. and growth was monitored at A.sub.600. For 
cultures incubated with DBT, the supernatant was viewed at various 
intervals under short wave UV light to check for production of fluorescent 
products. 
Plasmid Isolation and Hybridizations 
Cosmid DNA (pLAFR5) was isolated from E. coli as described by Ish-Horowicz 
and Burke (Nucl. Acids Res., 9:2989-2998, 1981), and from Rhodococcus 
species as described by Singer and Finnerty (J. Bacteriol., 170:638-645, 
1988). Large scale cosmid preparations were carried out according to 
Birnboim and Doly (Nucl. Acids Res., 7:1413-1423, 1979). DNA hybridization 
experiments were performed according to Southern (J. Molec. Biol., 
98:503-517, 1975). DNA was labelled with .sup.32 P-dCTP (Amersham), using 
the random primer method of Feinberg and Vogelstein (Anal. Biochem., 
137:266-267, 1984). 
UV Mutagenesis of IGTS8 
IGTS8 was incubated overnight in LB at 30.degree. C. and approximately 3000 
colony forming units were spread onto fresh LB plates. These plates were 
immediately exposed to short wave UV light (254 nm) for 5 to 20 s at a 
distance of 3.5 cm. Plates were incubated at 30.degree. C. for 48 h or 
until colonies developed. Colonies from plates exhibiting &gt;50% cell death 
were assayed for their ability to metabolize or desulfurize DBT, using the 
spray plate assay. 
Electrotransformation of Rhodococcus 
R. rhodochrous IGTS8 and the UV1 mutant were transformed with plasmid DNA 
via electroporation (Gene Pulser, Biorad Laboratories, Inc, Hercules, 
Calif.). The bacteria were grown in LB for 24 to 48 h at 30.degree. C., 
diluted to an A.sub.600 of 0.15 with fresh LB, and incubated at 30.degree. 
C. for an additional 4 h. Cells were collected by centrifugation and 
washed four to five times with 0.3M sucrose and finally resuspended to 
.about.5.times.10.sup.9 cells/ml in 0.5M sucrose. To an ice cold 0.2 cm 
electroporation cuvette (Biorad), was added 40 .mu.l of this bacterial 
solution. The cells were pulsed at 25 .mu.F and 2.5 kV with the Pulse 
Controller at 800 ohms and were immediately diluted with 1 ml of LB 
containing 0.5M sucrose. The cells were incubated at 30.degree. C. for 1 
h, plated on LB agar plates plus appropriate antibiotics, and incubated at 
30.degree. C. until colonies developed. When the plasmid carried the pRF29 
Rhodococcus plasmid origin of replication, colonies were visible after 48 
h. In the absence of the pRF29 origin, colonies appeared after 4 to 5 
days. 
R. fascians D188-5 was transformed by electroporation in a similar manner 
but, due to its slower growth rate, it was incubated in LB overnight until 
it reached an A.sub.600 of .about.2.0. The cells were washed and 
resuspended in distilled water instead of sucrose. The Pulse Controller 
was set at 400 ohms and the recovery period after electroporation was in 
LB for 4 h before plating onto selective media. Successful transformation 
of R. fascians D188-5 with E. coli plasmids required that the DNA be 
methylated in vitro beforehand, using the CpG methylase, SssI (New England 
Biolabs, Inc., Beverly, Mass.). 
Gas Chromatography and Mass Spectroscopy 
Cells were incubated overnight in LB medium at 30.degree. C. and 100 .mu.l 
was used to inoculate 50 ml of PMS minimal medium. The culture was 
incubated at 30.degree. C. for 4 days, washed twice with sulfur-free PMS 
and the pelleted cells were inoculated into 50 ml of PMS that contained 
0.1% DBT as the sole source of sulfur. These cells were incubated at 
30.degree. C. for 24 h and the supernatant was stored frozen at 
-20.degree. C. For assays involving R. fascians D188-5, incubation times 
were increased 2 to 3-fold. 
Sample preparation and chemical analyses were performed as described 
(Olson, et al., Energy & Fuels, submitted, 1993). Briefly, each sample 
supernatant (.about.50 ml) was thawed and residual insoluble material was 
removed by centrifugation. The cleared supernatant was acidified with HCl 
to pH 1.0 and then extracted three times with 50 ml of ethyl acetate. 
Insoluble material from the centrifugation step was also extracted with 
ethyl acetate. The ethyl acetate extracts were combined, dried over 
anhydrous calcium chloride, filtered, and ethyl acetate was removed by 
rotary evaporation. A known amount of internal standard (octadecane in 
chloroform solution) was added to the sample, which was then analyzed by 
GC/FID (gas chromatography/flame ionization detection) and GC/FTIR/MS (gas 
chromatography/Fourier transform infrared/mass spectrometry). In some 
samples, the acidic components in the ethyl acetate extract or in the 
post-extraction aqueous layer were methylated by treating with an ether 
solution of diazomethane. 
The analyses were performed on a serially interfaced GC/FTIR/MS system as 
previously described (Diehl, et al., Spectros. Int. J., 8:43-72, 1990, 
Olson and Diehl, Anal. Chem., 59:443-448, 1987). This system consisted of 
the Finnegan ion trap (ITD 800) operated with the AGC on and the Nicolet 
20SXB Fourier transform infrared spectrometer. Gas chromatography was 
conducted with a 30 m.times.0.32 mm DB5 column (1.0 .mu.m phase thickness) 
with a 2.0 ml/min helium carrier flow rate measured at 330.degree. C. 
On-column injections were utilized for sample introduction because the 
sulfoxides and sulfones are thermally unstable and they decompose in split 
or splitless injectors (Vignier, et al., J. High Resol. Chromatogr. & 
Chromatogr. Commun., 6:661-665, 1983). The oven temperature program was as 
follows: 40.degree. C. injection, followed by increases in temperature at 
rates of 20.degree. C./min to 80.degree. C., 5.degree. C./min to 
200.degree. C., 10.degree. C./min to 330.degree. C., and hold for 5 min. 
GC/FID analyses were performed with a HP 5880A with a similar column and 
program for flow rate and oven temperature. 
Results 
Isolation of a Dsz- Mutant of R. rhodochrous IGTS8 
When cloning from a foreign bacterial genus into E. coli, not all genes are 
expressed nor are all protein products active. To assure that cloned 
desulfurization genes would be expressed in the host cell, a mutant of R. 
rhodochrous IGTS8 that could no longer desulfurize DBT was isolated. Using 
this mutant as a cloning recipient would insure that the cellular 
environment was appropriate for gene expression and protein function, 
thereby allowing screening for cloned desulfurization genes by 
complementation. 
R. rhodochrous IGTS8 was mutagenized by exposure to UV light, and 1000 
survivors were screened for the ability to produce a UV fluorescent 
product in the DBT spray plate assay. Three potential desulfurization 
negative mutants were identified and then re-evaluated in the sulfur 
bio-availability assay. Two mutants (designated UV1 and UV23) could not 
use DBT or DBT-sulfone as sole sources of sulfur and thus appeared to be 
Dsz-. When grown in the presence of DBT, mutant UV1 could not metabolize 
DBT to 2-HBP or to any other potential intermediate, as measured by GC/MS 
analysis. Therefore, strain UV1 was considered to be Dsz- and was used as 
the host for complementation studies to identify clones that carried 
desulfurization genes. 
Cosmid Cloning of Desulfurization Genes 
DNA from Dsz+ source organism IGTS8 was used to construct a library in the 
cosmid vector, pLAFR5. This library was transduced into E. coli S17-1and 
plasmids were isolated from approximately 25,000 colonies. These cosmids 
were electroporated into R. rhodochrous UV1, a Dsz- mutant of IGTS8, with 
an efficiency of .about.300 transformants/.mu.g DNA. Various numbers of 
UV1 transformants were pooled and incubated for 18 hours at 30.degree. C., 
after which the cells were washed twice and resuspended in sulfate-free 
PMS. Approximately 7.times.10.sup.8 pooled cells were inoculated into 100 
ml of PMS with DBT as the sole source of sulfur. A predicted product of 
the DBT desulfurization reaction is 2-HBP, which is fluorescent when 
exposed to UV light. Therefore, batch cultures were grown at 30.degree. C. 
and the supernatants were observed for fluorescence. Approximately 3300 
UV1 transformants were screened in four separate batches. In one batch 
(representing .about.600 transformants) a UV fluorescent product appeared 
in the supernatant after five days' incubation. Individual colonies were 
isolated and twelve of these continued to produce a fluorescent product 
when exposed to DBT. 
Attempts to recover cosmid DNA from these isolates failed, so Southern 
hybridizations were performed to determine if the cosmids had become 
integrated into the chromosome of strain UV1. Chromosomal DNA was isolated 
from seven transformants and digested with EcoRI. After agarose 
electrophoresis and blotting, the fragments were hybridized with .sup.32 
P-labelled probes derived from pLAFR5. In all transformants tested, pLAFR5 
probes hybridized to a DNA fragment .about.20 kb in size. Vector derived 
probes did not hybridize to the control IGTS8 genome. Therefore, the 
desulfurization positive cosmid clones had apparently integrated into the 
chromosome of strain UV1. 
Since the plasmids had integrated into the chromosome, the genomic DNA 
connected to either side of the plasmid cloning site must represent R. 
rhodochrous IGTS8 sequences that were able to complement the Dsz- mutation 
in strain UV1. (This would be true regardless of whether the mode of 
integration was by homologous or illegitimate recombination.) Sequences 
were recovered that flanked the inserted plasmid from three 
desulfurization positive transformants by digesting genomic preparations 
with EcoRI or BamHI. These enzymes cut pLAFR5 once in the polylinker 
region so that an intact sequence of pLAFR5 could be recovered, linked to 
a neighboring chromosomal fragment from IGTS8. The digested DNA was 
ligated to itself (at a concentration of .about.20 ng/.mu.l) and was 
transformed into E. coli S17-1. Sixteen tetracycline resistant colonies 
were obtained, seven from the BamHI digestion and nine from the EcoRI 
digestion. Restriction enzyme analysis revealed that all the EcoRI-rescued 
clones contained a 2.1 kb fragment of IGTS8 DNA. The BamHI-rescued clones 
contained a 1.65 kb fragment from IGTS8. 
The 2.1 kb IGTS8 DNA from the EcoRI rescue experiment was used as a 
template to make labelled DNA probes, which were hybridized to colony 
lifts of the original, intact cosmid library in E. coli. Of 5000 colonies, 
17 hybridized with the IGTS8 probes. Cosmid DNA was isolated from each 
clone and transformed into strain UV1. Three of the seventeen DNA 
preparations complemented the Dsz- phenotype. 
A restriction map for this region was constructed, using EcoRI and HindIII. 
Probes from the 2.1 kb IGTS8 DNA hybridized to the 4.5 kb EcoRI fragment. 
All cosmid clones that conferred the Dsz+ phenotype contained the entire 
4.5 kb EcoRI fragment and portions of the 4.5 kb EcoRI-HindIII and 18 kb 
EcoRI fragments. These results indicated that the desulfurization genes 
lay within a 15 kb region. 
Subcloning the Desulfurization Genes 
The 4.5 kb EcoRI and the 4.5 kb EcoRi-HindIII fragments were subcloned into 
pLAFR5, but neither fragment complemented the Dsz- mutation of strain 1. 
The 9.0 kb EcoRI fragment from GE1-H, the 15.0 kb EcoRI-HindIII fragment 
from GE1-C, and the 18 kb EcoRI fragment from GE1-K were subcloned into 
pLAFR5 to yield the plasmids pSAD60-28, pSAD48-12, and pSAD56-6, 
respectively. When transformed into UV1, all three produced UV fluorescent 
products from DBT in the spray plate assay, consistent with the 
localization of the Dsz+phenotype as determined by restriction mapping. 
Construction of additional subclones from this region narrowed the 
location of the relevant genes to a 6.5 kb BstBI fragment. 
Nature of the Mutation in Strain UV1 
Genomic blots of EcoRI digested IGTS8 and UV1 DNA were hybridized with 
probes produced from the 2.1 kb EcoRI-rescued fragment of IGTS8. No 
hybridization was detected to UV1 DNA, indicating that the UV1 mutation is 
a large deletion and not a simple point mutation. 
A Rhodococcus Plasmid Origin of Replication Increases Transformation of UV1 
Electroporation of UV1 with pSAD48-12 typically resulted in a low 
transformation efficiency (.about.550/.mu.g DNA) and only about 50% of the 
transformants exhibited the Dsz+ phenotype (presumably because DNA had 
been lost or rearranged during recombination with the chromosome). To 
improve the transformation efficiency, a 4.5 kb HindIII fragment from 
pRF29 was cloned into the HindIII site of pSAD48-12, resulting in 
pSAD74-12. This 4.5 kb fragment contains a Rhodococcus plasmid origin of 
replication, which allowed pSAD74-12 to replicate as a plasmid in strain 
UV1. This clone transformed UV1 with an efficiency of greater than 
10.sup.4 transformants/.mu.g DNA. Nearly 100% of these transformants 
exhibited the Dsz+ phenotype. Unfortunately, the yield of plasmid prepared 
directly from UV1 was so poor that DNA from minipreparations could not be 
visualized on agarose gels. However, plasmid isolated from UV1 could be 
used to transform E. coli S17-1, from which large amounts of the plasmid 
were prepared. 
The Dsz+ Phenotype is Not Expressed in E. coli S17-1 
E. coli S17-1 was transformed with pSAD48-12 and desulfurization activity 
was measured with the spray plate assay. No positive colonies were 
identified. It was possible that the E. coli polymerase could not 
recognize the IGTS8 promoter(s) in pSAD48-12, so the IGTS8 DNA was placed 
under control of the E. coli lac promoter. The 15 kb EcoRI-HindIII IGTS8 
fragment from pSAD48-12 was subcloned into the pBluescript vectors, 
SK.sup.- and KS.sup.-, so that the IGTS8 fragment was cloned in both 
orientations with respect to the lac promoter. Neither clone expressed the 
Dsz+ phenotype in E. coli XL1-Blue. It is not yet known whether this stems 
from poor transcription or translation of the cloned genes or whether the 
overproduced proteins are inactive in E. coli S17-1. 
The Dsz+ Gene or Genes are Expressed in R. fascians 
Since the cloned genes were either not expressed or produced inactive 
proteins in E. coli, efforts were initiated to express the genes in other 
Rhodococcus species. R. fascians D188-5 exhibited no desulfurization in 
the DBT spray plate assay or in the sulfur bioavailability assay. Initial 
attempts to transform R. fascians with the desulfurization positive 
plasmid, pSAD74-12 were unsuccessful. Other Rhodococcus species are known 
to have endogenous restriction systems that cleave DNA at SalI-like 
restriction sites. Since pSAD74-12 contained multiple SalI recognition 
sequences, CpG methylase, SssI, was used to methylate pSAD74-12 in vitro. 
With methylated pSAD74-12 DNA, transformants of R. fascians D188-5 were 
obtained with an efficiency of about 7.times.10.sup.3 transformants/.mu.g 
DNA. These transformants displayed the Dsz+ phenotype in the spray plate 
assay and GC analysis of liquid medium supernatant revealed the formation 
of 2-HBP from DBT. 
Efforts to transform pSAD74-12 into a second species, R. rhodochrous 
ATCC13808 were ineffective, despite the use of unmethylated or 
CpG-methylated plasmid. It is possible that the electroporation conditions 
for ATCC13808 were not optimal, though a wide range of conditions was 
tested. It seems more likely that ATCC13808 has a restriction system that 
is not inhibited by CpG methylation. 
2-HBP is the Major Desulfurization Product 
The predominant metabolite produced from DBT by R. rhodochrous IGTS8 is 
2-HBP, with small amounts of 2'-hydroxybiphenyl-2-sulfinic acid 
(DBT-sultine) and 2'-hydroxybiphenyl-2-sulfonic acid (DBT-sultone) also 
identified by GC/MS analysis (Olson, et al., Energy & Fuels in press, 
1993). These products were also produced by IGTS8 in this work (Table 2). 
Neither R. fascians D188-5 nor R. rhodochrous Dsz- mutant UV1 produced 
these products from DBT. However, when R. fascians D188-5 was transformed 
with plasmid pSAD74-12 and when the R. rhodochrous UV1 mutant was 
transformed with plasmid pSAD104-10, these bacteria produced products from 
DBT that were identical to those identified for R. rhodochrous IGTS8 
(Table 2). In particular, 2-HBP was produced in large quantities, 
indicating that carbon-sulfur bond specific desulfurization of DBT was 
mediated by products of genes cloned from IGTS8. 
One subclone, pSAD90-11, carried a DNA fragment that was supposedly 
identical to that cloned into pSAD104-10, but the two plasmids differed in 
the results they produced when introduced into R. rhodococcus UV1. In the 
plate assay, the surface film of DBT disappeared from the vicinity of 
colonies that contained pSAD104-10, producing a clear zone, and a 
fluorescent halo appeared around those colonies. On the other hand, when 
cells contained pSAD90-11, no fluorescent products were produced but a 
zone of DBT clearing did form around each colony. GC/MS analysis showed 
that no 2-HBP was produced by cells containing pSAD90-11, but that a 
significant amount of DBT-sultone did accumulate (Table 2). The sultone 
does not accumulate in the parent strain, UV1 (data not shown). These 
observations imply that when the 9.0 kb EcoRI fragment was subcloned into 
pSAD90-11 the DNA was damaged so as to inactivate the gene(s) encoding the 
enzyme(s) that convert the sultone to 2-HBP. This suggests that at least 
two enzymes are involved in desulfurization and that the sultone may be an 
intermediate in the pathway. This result is consistent with the kinds of 
metabolites detected in the original isolate, R. rhodochrous IGTS8 (Olson, 
et al., 1993). 
TABLE 2 
__________________________________________________________________________ 
Metabolites produced from DBT by Rhodococcus species transformed with 
subclones derived from R. rhodochrous IGTS8. 
Rhodococcus species (plasmid) 
R. fascians 
R. rhodochrous 
UV1 UV1 D188-5 (pSAD- 
Metabolite.sup.a 
IGTS8 (pSAD104-10).sup.b 
(pSAD90-11).sup.c 
74-12).sup.d 
__________________________________________________________________________ 
DBT ++++.sup.e 
++++.sup.e 
++++ ++ 
DBTO + 0 0 0 
DBTO.sub.2 
0 0 0 0 
DBT-sultone 
+ ++ ++ + 
DBT-sultine 
0 or trace 
0 trace + 
2-HBP +++++ +++++ 0 +++ 
__________________________________________________________________________ 
.sup.a Products are: DBT, dibenzothiophene; DBTO, dibenzothiophene 5oxide 
(sulfoxide); DBTO.sub.2, dibenzothiophene 5,5dioxide (sulfone); 
DBTsultone, 2'-hydroxybiphenyl2-sulfonic acid (detected as 
dibenz[c,e][1,2]-oxathiin 6,6dioxide); DBTsultine, 
21hydroxybiphenyl-2-sulfinic acid (detected as dibenz[c,e][1,2]-oxathiin 
6oxide); dibenzothiophene sulfone; 2HBP, 2hydroxybiphenyl (Krawiec, pg. 
103-114. In G. E. Pierce (ed.), Developments in Industrial Microbiology, 
vol 31, Society for Industrial Microbiology, Columbus, Ohio, 1990). 
.sup.b 9.0 kb EcoRI DNA fragment from IGTS8 subcloned into pLAFR5, plus 
the origin of replication from pRF29. 
.sup.c Mutated 9.0 kb EcoRI DNA fragment from IGTS8 subcloned into pLAFR5 
plus the origin of replication from pRF29. 
.sup.d 15.0 kb EcoRIHindIII DNA fragment from IGTS8 subcloned into pLAFR5 
plus the origin of replication from pRF29. 
.sup.e Presence of metabolites is reported in relative amounts from very 
large amounts (+++++) to very small (+), i.e., trace amounts. 
IGTS8 Cannot Use DBT-Sulfoxide as a Sulfur Source 
R. rhodochrous IGTS8 was incubated in minimal medium with one of the 
following as the sole source of sulfur: DBT, DBT-sulfoxide, or 
DBT-sulfone. IGTS8 was incapable of utilizing the sulfur supplied by 
DBT-sulfoxide but grew well in the presence of DBT or DBT-sulfone. 
DBT-sulfoxide was not toxic to cells when grown in a rich medium (LB). 
Therefore, either IGTS8 cannot transport or otherwise act on 
DBT-sulfoxide, or else DBT-sulfoxide is not a true intermediate of the 
desulfurization pathway. 
EXAMPLE 4 
DNA Sequencing of a 9763 Nucleotide EcoRI-Sau3AI Fragment Containing the 
Gene or Genes for the Desulfurization Biocatalyst of IGTS8 by the Method 
of Sanger et al. 
A 9763 nucleotide EcoRI-Sau3AI fragment containing the gene or genes 
responsible for the Dsz+ phenotype was isolated from the IGTS8 source 
organism. The DNA sequence of this fragment was determined from both 
strands of DNA using the dideoxy chain-termination method of Sanger et al. 
(1977), DNA sequencing with chain-termination inhibitors, 74 PROC. NATL. 
ACAD. SCI. USA 5463-5467, a modified T7 DNA polymerase (USB) and 
[.alpha.-.sup.35 S]-dCTP (Amersham). Deletion clones for DNA sequencing 
were constructed in pBluescript (Stratagene) using exonuclease III and the 
methods of Henikoff (1984), Unidirectional digestion with exonuclease III 
creates targeted breakpoints for DNA sequencing, 28 GENE 351-359. 
Sequences from 141 individual deletion clones were used to reconstruct the 
entire 9763 nucleotide fragment. Computerized sequence assembly was 
performed using DNA InspectorII (Textco, Hanover, N.H.). The DNA sequence 
was determined independently for each strand of DNA, but the entire 9763 
nucleotide fragment was not completely sequenced on both strands. The 
sequence determined from one strand of DNA covered 95% of the 9763 
nucleotide sequence. On the other DNA strand, 96% of the sequence was 
determined. The sequence was determined from at least two independent 
deletion clones for the entire 9763 nucleotide fragment. 
EXAMPLE 5 
Further Resolution of the Sequence of pTOXI-1 and Open Reading Frames 
(ORFs) Encoded Therein; Dsz+ Promoter Engineering; Expression of the Dsz+ 
Phenotype in a Heterologous Host Organism; Maxicell Analysis of 
Desulfurization Gene Expression Products 
Organization of the Desulfurization Cluster 
Sequencing of pTOXI-1, the results of which are set forth below in the 
Sequence Listing, predicted three nearly contiguous open reading frames 
(ORFs) on one strand of the clone (FIG. 7). The sizes of each ORF are 
predicated as 1359 bases (bps 786-2144) for ORF 1, 1095 bases (bps 
2144-3238) for ORF 2 and 1251 bases (bps 3252-4502) for ORF 3. Subclone 
analysis described below has revealed that ORFs 1, 2 and 3 are required 
for the conversion of DBT to 2-HBP and that all of the genes encoded by 
these ORFs are transcribed on a single transcript as an operon. All 
subclones described below are maintained in E. coli - Rhodococcus shuttle 
vector pRR-6. Activity of each subclone was determined by growing 
transformants of Dsz- strain CPE-648 in a rich media (RM) for 48 hours. 1 
ml of the culture was used to inoculate 25 ml BSM supplemented with 
greater than 100 .mu.M DBT or DBT-sulfone. Cultures were assayed for 
desulfurization products after 48-120 hours. A diagram of each of the 
subcloned fragments is shown in FIG. 8. 
In subsequent studies, the subclones were grown in rich media with 
chloramphenicol, then crossed into BSM supplemented with 100 .mu.M of 
either DBT or DBT-sulfone. Cultures were shaken at 30.degree. C. for 2-5 
days and assayed for desulfurization products by HPLC. 
A. pENOK-1: A subclone was constructed which contains the 4.0 kb SphI 
fragment of pTOXI-1. This fragment spans ORFs 1 and 2 but truncates ORF 3. 
Analysis of pENOK-1 containing transformants revealed the production of no 
products when incubated with DBT. However these transformants were capable 
of producing 2-HBP from DBT-sulfone. 
B. pENOK-2: A suclone which contains the 3.6 kb SacI fragment of pTOXI-1 
was constructed. This fragment contains ORFs 2 and 3 but truncates ORF 1. 
Analysis of pENOK-2 transformants revealed no production of any 
desulfurization products from either DBT or DBT-sulfone. The lack of any 
activity detectable from either ORFs 2 or 3 suggests that the ORFs are 
arranged as an operon with transcription mediated from a single upstream 
promoter. Presumable, this promoter has been removed in this subclone. 
C. pENOK-3: A 1.1 kb XhoI deletion mutation of pTOXI-1 was constructed. 
Both ORFs 1 and 2 are truncated. ORF 3 remains intact. Transformants 
harboring pENOK-3 show production of DBT-sulfone from DBT. No production 
of 2-HBP is detected from either DBT or DBT-sulfone. It should also be 
noted that at the nucleotide level, a deletion of this type would not 
result in a polar mutation. The sequence predicts an in-frame splicing of 
ORFs 1 and 2 which would produce a hybrid protein that is presumably 
inactive. However, by avoiding stop codons, the putative single mRNA 
transcript remains protected by ribosomes allowing for translation of ORF 
3. The ability of the ORF-3 product to produce DBT-sulfone from DBT 
demonstrates that DBT-sulfone is a true intermediate in the carbon-sulfur 
bond specific biocatalytic desulfurization pathway of IGTS8. 
D. pENOK-11: The 3.4 kb NcoI fragment from pTOXI-1 was subcloned into a 
unique NcoI site of pRR-6. This fragment contains all of ORFs 2 and 3 but 
truncates the 5' end of ORF1. Transformants with pENOK-11 demonstrated no 
desulfurizing-specific enzymatic activity towards DBT or DBT-sulfone. This 
indicates essential coding regions bordering this fragment. This is 
consistent with the predication that the entire cluster is expressed on a 
single transcript as discussed for subclone pENOK-2. Again, the promoter 
for gene transcription is not present in this subclone. Subclone pENOK-13 
(below) corroborates this prediction. 
E. pENOK-13: A subclone of pTOXI-1 was constructed which had a 2.6 kb 
SphI-XhoI deletion. This subclone only contains an intact ORF 3. ORF 1 is 
lost completely and ORF 2 is truncated. This subclone showed no 
desulfurizing-specific enzymatic activity towards DBT or DBT-sulfone. This 
result should be compared with the phenotype of pENOK-3 which demonstrated 
production of DBT-sulfone from DBT. Because pENOK-13 differs from pENOK-3 
by the additional deletion of the smaller SphI/XhoI fragment, this would 
indicate an element in the 1.6 kb SphI/XhoI fragment which is essential 
for gene expression. Because sequencing has revealed no significant ORF's 
contained in this region, it is postulated that a promoter element may be 
present in this region. 
F. pENOK-16: A subclone of pTOXI-1 was designed which eliminates nearly all 
unnecessary sequences from the desulfurization cluster. This construct 
contains the 4 kb BstBI-SnaBI which presumably contains all essential 
sequence for complete desulfurization in that in contains all of ORFs 1, 2 
and 3 as well as 234 bases of upstream sequence. The 3' SnaBI site lies 80 
base pairs beyond the termination of ORF 3. CPE-648 harboring this plasmid 
was capable of converting DBT and DBT-sulfone to 2-HBP. pENOK-16 thus 
represents the smallest amount of the cluster yet observed which 
demonstrates the complete desulfurization phenotype. 
G. pENOK-18: This subclone contains a NsiI-BfaI fragment of pTOXI-1. The 
NsiI site is 23 bp downstream of the predicted start site of ORF 1. 
CPE-648 harboring this subclone lacks desulfurization activity on both DBT 
and DBT-sulfone. This subclone most likely eliminates the promoter region 
and truncates the first structural gene. 
H. pENOK-Nsi: To help further elucidate the start site of ORF 1, a subclone 
was made in which a 4 bp deletion is introduced at the unique NsiI site 
which is 23 bp downstream of the predicted start site of ORF1. The 
mutation was generated by cutting with NsiI and blunting the ends with T4 
DNA Polymerase. If the NsiI site is within the first structural gene this 
frameshift mutation would cause an early stop signal in ORF 1. 
Transformants of pENOK-Nsi were capable of producing DBT-sulfone from DBT. 
However, no production of 2-HBP was detected indicating that the mutation 
had disrupted an essential structural gene. 
In subsequent studies, due to the clear expression of the ORF-3 encoded 
oxidase, in this clone, it was considered likely that the ORF-2 product 
would also be expressed. Accordingly, ORF-2 alone is incapable of further 
metabolism of DBT-sulfone. 
I. pENOK-19: A subclone of pTOXI-1 was constructed which contains a 
deletion from the NotI site, which is in the earlier part of ORF 2, to the 
SnaBI which is after ORF 3. This subclone should demonstrate the activity 
of ORF 1 alone. CPE648 transformants harboring this subclone displayed no 
enzymatic activity towards DBT or DBT-sulfone. 
The results of pENOK-Nsi and pENOK-19, taken together, suggest that the 
ORF-I and ORF-2 products must be simultaneously expressed in order to 
further metabolize DBT-sulfone. 
J. pENOK-20: In order to evaluate the function of ORFs 2 and 3 separately 
from ORF 1, DNA spanning ORFs 2 and 3 was amplified by the Polymerase 
Chain Reaction (PCR). Primers RAP-1 (5'-GCGAATTCCGCACCGAGTACC-3' (SEQ ID 
NO:8), bps 2062-2082) and RAP-2 (5'-ATCCATATGCGCACTACGAATCC-3' (SEQ ID 
NO:9) bps 4908-4886) were synthesized with the Applied Biosystems 392 
DNA/RNA Synthesizer. Nucleotides in bold were altered from the template 
sequence in order to create restriction sites for subcloning; thus primer 
RAP-1 contains an EcoRI site, and primer RAP-2 contains an NdeI site. 
Amplification was carried out with the GeneAmp Kit (Perkin Elmer Cetus) 
which utilizes the Taq polymerase and the Perkin Elmer Cetus 9600 
Thermocycler. Parameters were as follows: 
______________________________________ 
Template: pMELV-l Plasmid DNA 
0.2 or 2.0 ng 
Primers: RAP-1 0.5 or 0.2 .mu.M 
RAP-2 0.5 or 0.2 .mu.M 
Cycles: 1.times. @ 
96.degree. C. 
2 min 
25.times. @ 
96.degree. C. 
30 sec 
52.degree. C. 
30 sec 
72.degree. C. 
2 min 
______________________________________ 
Amplification yielded the predicted 2846 bp fragment. In order to express 
the amplified fragment harboring ORFs 2 and 3, it was ligated to the 
XbaI/EcoRI fragment of the chloramphenicol resistance gene promoter from 
Rhodococcus fascians (Desomer et al.: Molecular Microbiology (1992) 6 
(16), 2377-2385) to give plasmid pOTTO-1. Ultimately, a blunt end ligation 
was used for the subcloning of the amplified product due to the fact that 
ligation using the engineered restriction sites was unsuccessful. This 
fusion was ligated to shuttle-vector pRR-6 to produce plasmid pENOK-20. 
CPE648 transformants of pENOK-20 were grown in the presence of DBT and 25 
.mu.g/ml chloramphenicol for promoter induction. All transformants 
converted DBT to DBT-sulfone presumably through the activity of the ORF 3 
as demonstrated in subclone pENOK-3. The inability to further process 
DBT-sulfone with the presence of ORF 2 suggests that the product of ORF 2 
alone is incapable of using DBT-sulfone as a substrate. This is consistant 
with results obtained from pENOK-Nsi, and suggests that ORF-2 alone is 
incapable of using DBT-sulfone as a substrate. 
Assignment of Gene Products of ORFs 1, 2 and 3 
Based on the foregoing subclone analyses, functions have been tentatively 
assigned to each of the ORFs present within the pTOXI-1 sequence. ORF 3 
can be identified as responsible for an oxidase capable of conversion of 
DBT to DBT-sulfone. Subclone pENOK-3 demonstrates this activity very 
clearly. ORFs 1 and 2 appear to be responsible for conversion of 
DBT-sulfone to 2-HBP. This aryl sulfatase activity is evidenced in 
subclone pENOK-1. However subclones pENOK-19 and pENOK-20 indicate that 
neither ORF 1 or ORF 2 alone is capable of any conversion of the 
intermediate DBT-sulfone. This suggests that the protein products of ORFs 
1 and 2 work together to cleave both of the carbon-sulfur bonds. 
Presumably, this is achieved through a heterodimer arrangement of the 
proteins, or through a regulatory function of one protein on the other. 
The results of paralell investigations, presented in Example 3, suggested 
that ORF-1 encodes an enzyme that converts DBT-sulfone to DBT-sultone. 
Lengthy incubations of CPE-648 harboring pENOK-19 (intact native promoter 
and ORF-I) have shown neither the depletion of DBT-sulfone nor the 
production of any new products. This is contrary to indications derived 
from Example 3. 
Alternative Promoter Screening 
Increasing the specific activity of desulfurization is a significant 
objective of the studies described herein. One approach to accomplishing 
this goal is to replace the original promoter with one that can produce 
both higher and constitutive expression of the desulfurization gene 
cluster. Because there are so few reported and characterized Rhodococcus 
promoters, random genomic libraries have been prepared and screened for 
promoter activity in two systems. In one, the reporter is the 
chloramphenicol resistance gene used in the above-discussed plasmid 
constructions. In the other, the desulfurization cluster itself is used as 
a reporter. 
Promoter Screening Example A. Chloramphenicol Resistance Reporter 
As also described below, partially digested Rhodococcus genomic DNA has 
been cloned upstream of a promoterless chloramphenicol resistance gene. 
The resulting libraries were then transformed into Rhodococcus which are 
subjected to chlorarnphenicol selection. Four apparent promoter elements 
were rescued by pRHODOPRO-2, although plasmid could be isolated from only 
one of these, possibly due to vector instability. The stable plasmid 
RP2-2A has been subjected to sequence analysis. Technical problems have 
been observed with restriction enzyme treatment of the NarI cloning site 
used in these vectors. Unfortunately, the NarI enzyme demonstrates severe 
site-selectivity and does not appear to digest the vector well. New 
vectors have been constructed in order to alleviate this problem, although 
a lack of convenient and unique restriction sites slowed the progress of 
these studies. A recent observation on the Rhodococcus replication origin 
will aid in constructing a more effective promoter probe, as discussed 
below. 
Recently, the 1.4kb BglIIfragment was removed from pRR-6, and the ends were 
blunted and religated to produce pRR-12 (FIG. 9), which contains no BglII 
sites. Desomer et al. (Molecular Microbiology (1992) 6 (16), 2377-2385) 
reported that this region was needed for plasmid replication. Thus, it was 
surprising that this construct was capable of producing Cm.sup.r 
transformants, indicating that this region was not essential for plasmid 
replication in the strain of organisms used for the present studies. This 
observation forms the conceptual basis for construction of a vector that 
will utilize a synthesized BglII site for cloning the random genomic 
fragments. BglII accepts DNA digested by Sau3A, an effective and frequent 
cutter of IGTS8 DNA. These constructs are expected to allow for the 
production of better, more representative random genomic libraries. 
Promoter Screening Example B: Desulfurization Cluster Reporter 
Vector pKAMI has been used as a second direct "shot-gun" approach to 
finding a suitable alternative promoter (FIG. 10). An NdeI site was 
engineered upstream of the promoterless Dsz cluster to serve as the site 
of insertion of random genomic DNA (from strains GPE-362, CPE-648 and 
IGTS8) fractionated by NdeI and the compatible 4bp cutters MseI and BfaI. 
Originally, this ligation mixture was directly transformed into GPE-362 
cells, which were then used en masse to inoculate 250 ml BSM+DBT. These 
efforts were undertaken with the goal of amplifying a superior Dsz+ strain 
due to its ability to utilize DBT as the sole source of sulfur. To date, 
14 transformations of this type have been done. Of these, all but 2 have 
resulted in producing Dsz+ cultures. Eleven individual clones have been 
isolated and characterized. These are capable of low-level (0.6-1.0 mg/L 
2-HBP/OD.sub.600 /hr), constitutive expression of the desulfurization 
trait. Restriction analysis of plasmids isolated from these eleven has 
revealed that all but one (KB4-3) are simple rearrangements of the pKAMI 
backbone resulting in gratuitous expression from vector borne promoters. 
Many of the rescued plasmids show identical restriction patterns although 
originating from separate ligations, suggesting an inherent vector 
instability. It appears as if, with this type of selection, rearrangements 
of pKAMI that utilize a vector promoter sequence are strongly selected. 
The above-described selection procedure has thus given way to a promoter 
screen geared to minimize the plasmid rearrangement. In this procedure, 
the pKAMI/genomic library is first amplified in E. coli, then the 
individual JM109 colonies are pooled together. The plasmids are extracted, 
and used to transform Dsz- strain GPE-362. Instead of using en masse 
enrichment, the GPE362 transformations are plated to Rich 
Media+chloramphenicol for selection of plasmid containing cells. Resulting 
colonies are replica-plated to BSM agarose+DBT plates, then checked for 
desulfurization activity by UV fluorescence production. Over 7,000 GPE-362 
transformants have been screened in this fashion. Thirty-six have been 
isolated from these which produce UV fluorescence on BSM+DBT plates. 
Current efforts focus on the identification and characterization of the 
engineered plasmids borne by these 36 transformants. 
Alternative Promotor Engineering 
The close physical arrangement of the three ORFs of pTOXI-1 does not 
provide sufficient space for promoters for either ORFs 2 or 3. This fact, 
coupled with the results of the subclone analysis in which intact ORFs 2 
and 3 provided no activity (see pENOK-2, pENOK-11, and pENOK-13), 
suggested that this cluster of genes is organized as an operon with only 
one promoter for expression of the three genes. Given that the 
desulfurization trait of IGTS8 is repressed by sulfate (Kilbane and 
Bielaga, Final Report D.O.E. Contract No. DE-AC22-88PC8891 (1991), it is 
possible that the operon promoter is tightly controlled by sulfur levels. 
With the elucidation of the molecular arrangement of the desulfurization 
cluster, alternative promoters can be rationally engineered to eliminate 
the sulfur repression, increase expression of the desulfurization genes 
and thereby increase the specific activity of the Dsz.sup.+ trait. 
Examples of potential alternative promoters include other known and 
described promoters such as the chloramphenicol resistance gene promoter 
from Rhodococcus fascians (Desomer et al.: Molecular Microbiology (1992) 6 
(16), 2377-2385), the nitrile hydratase gene promoter from Rhodococcus 
rhodochrous (Kobayashi, et al.: Biochimica et Biophysica Acta, 1129 (1991) 
23-33), or other strong promoters isolated from Rhodococcus sp. by 
"shot-gun" promoter probing. Other potential alternative promoters include 
those from other Gram positive organisms such as Corynebacterium, 
Bacillus, Streptomyces, and the like. 
Promoter Engineering Example A: Expression from the Chloramphenicol 
Resistance Gene Promoter from Rhodococcus fascians 
pSBG-2 (FIG. 11). The promoterless desulfurization cluster was isolated 
from pTOXI-1 as a 4.0 kb DraI/SnaBI fragment and ligated to a unique 
blunted AflII site of pRR-6. This ligation inserted the cluster downstream 
of the chloramphenicol resistance gene promoter and upstream of the 
resistance structural gene. Thus, messenger RNA (mRNA) transcription 
should proceed through the Dsz gene cluster and proceed on to the 
resistance gene. However, original selections of transformants on 
chloramphenicol did not yield transformants, suggesting poor 
transcriptional read-through. Dsz+ transformants harboring the plasmid 
were selected first through sulfur bioavailability assays and secondarily 
on chloramphenicol plates. Unlike IGTS8, pSBG-2 transformants are capable 
of converting DBT to 2-HBP in BSM media supplemented with 20 mM Na.sub.2 
SO.sub.4, which demonstrates the removal of sulfate repression by promoter 
replacement. Specific activity of transformants was measured between 0.9 
and 1.7 mg 2-HBP/1/OD.sub.600 /hr for a 16 hr culture in a rich media (RM) 
supplemented with 25 .mu.g/ml chloramphenicol. 
pSBG-3. The Rhodococcus origin of replication was removed from pSBG-2 by 
elimination of the 4.0 kb Xbal fragment. Without the origin, 
transformation is obtainable only through integration. CPE-648 
transformants with this plasmid were selected on RM+chloramphenicol and 
replica-plated onto BSM+DBT plates. Colonies were obtained which produced 
2-HBP, as detected by fluorescence after 18 hr of incubation at 30.degree. 
C. 
Individual Expression Of each ORF 
Recently, studies have been initiated to express the three ORFs separately, 
each engineered with an alternative promoter. These studies are expected 
to elucidate the following: First, any potential rate limiting steps in 
the desulfurization process will be identified and overcome. Potential 
polarity effects of operon expression, i.e. poorer expression of 
downstream ORFs 2 and 3, may be causing such rate limitations. Also, given 
the unresolved issue of the individual functions of ORFs 1 and 2, these 
studies are expected to demonstrate reconstitution of DBT-sulfone to 2-HBP 
conversion by the Separate expression of ORFs 1 and 2. 
All ORFs were isolated through PCR amplification and subsequent subcloning. 
A typical Shine-Dalgarno sequence and a unique cloning site for 
alternative promoters has been engineered upstream of each ORF. Stop 
codons in all reading frames have been engineered downstream of each ORF 
to prevent read-through. Additionally, convenient flankng restriction 
sites for mobilization of the promoter/ORF fusions have been added to each 
primer. The primers used for amplification of each ORF are listed below. 
In-frame stop codons are marked with an asterik (*). Sequences identical 
to pTOXI-I template DNA are shown in bold. 
##STR1## 
Each ORF has been successfully amplified and subcloned into pUC-19 NdeI as 
EcoRI fragments. Alternative promoters will be ligated into the unique 
Ndel sites, and the fusions will be moved to Rhodococcus-E. coli shuttle 
vector pRR-6 for expression in Rhodococcus. 
Heterologous Expression of the Dsz+ Trait 
In order to determine whether plasmid pTOXI-1 contained all of the genetic 
material necessary for the Dsz+ trait, heterologous expression of pTOXI-1 
was attempted in Rhodococcus fascians, a related organism which does not 
metabolize DBT (Dsz-) and in E. coli, a non-related organism which is also 
Dsz-. 
A. Rhodococcus fascians (ATCC 12974), a Dsz- strain, was transformed with 
pTOXI-1. A single transformant demonstrated UV fluorescence on BSM+DBT 
plates, and further analysis by HPLC clearly indicated production of 2-HBP 
when DBT was provided as a substrate. Thus pTOXI-1 contains sufficient 
information to convert a heterologous Dsz- strain to the Dsz+ phenotype. 
B. E. coli strain JM109 was also transformed with pTOXI-1 and was incubated 
with each of the substrates DBT and DBT-sulfone in either a minimal media 
(BSM) or a rich media (Luria Broth). In no case was production of 2-HBP 
observed by HPLC analysis. The inability of E. coli to express the 
desulfurization genes was not unexpected as gram positive genes are not 
universally expressible in E. coli without promoter replacement. 
In order to replace the promoter Of the desulfurization cluster, a 4.0 kb 
DraI/SnaBI fragment Was isolated from pTOXI-1. This fragment contains all 
of the necessary structural genes but lacks the promoter sequences. This 
promoterless desulfurization cluster was ligated to E. coli expression 
vector pDR540 (Pharmacia, Piscataway, N.J.) cut with BamHI and ends made 
blunt with Klenow. The construction fuses the tac promoter to the 
desulfurization cluster. The tac promoter is under control of the lactose 
repressor and is repressed in a lacI.sup.q host such as JM109. Expression 
from the tac promoter is inducible by the addition of isopropyl 
.beta.-D-thiogalactopyranoside (IPTG). Transformants of JM109 harboring 
pDRDsz grown in Luria Broth at 30.degree. C. demonstrate the Dsz+ 
phenotype when incubated with DBT and induced with IPTG. A specific 
activity as high as 1.69 mg 2HBP/1/OD.sub.600 /hr has been observed with 
pDRDsz. Activity is greatly diminished when transformants are grown at 
37.degree. C. The highest level of activity has been observed at 1 hr post 
induction. 
The above-described expression of the Dsz+ trait in both a related and 
non-related heterologous host indicates that pTOXI-1 carries all of the 
genetic information required for conversion of DBT to 2-HBP. 
Successful expression in E. coli provided a workable system in which the 
proteins encoded by the desulfurization cluster could be identified and 
characterized. Total protein from Dsz+ cells of JMIO9 (pDRDsz) was 
isolated and examined on denaturing acrylamide gels. No novel bands could 
be detected with Coomassie stain. Cellular fractionation of proteins into 
periplasmic, cytosolic and membrane components were also analyzed by 
Coomassie stained gels. Again, no novel bands were detected. Without any 
purification, the newly expressed proteins were apparently levels too low 
to easily detect and resolve from background. 
Maxicell Analysis of E. coli Harboring pDRDsz 
Proteins encoded by genes on plasmid DNA can be specifically radiolabeled 
in UV-irradiated cells of E. coli (Sancar, et al. Journal of Bacteriology. 
1979, p. 692-693). This technique is known as Maxicell Analysis. Briefly, 
a recA strain of E. coli e.g. JM109 which harbors a plasmid is grown in 
M9CA medium (Maniatis et al.) to a density of 2.times.10.sup.8 cells/ml. 
Continuously stirred cells were then subjected to UV exposure from a 
Mineralight Lamp Model UVG-254 (Ultrovilet Products, Inc., San Gabriel, 
Calif.) at a distance of 10 cm for a fluence rate of 0.5 
Joules.multidot.m.sup.-2 s.sup.-1. Cells were exposed for either 60, 90 or 
120 seconds. The cells were then incubated at 37.degree. C. for 16 hours 
after which they were then washed with M9 buffer and suspended in minimal 
medium lacking sulfate. After 1 hour of starvation at 37.degree. C., 
[.sup.35 S]methionine (&gt;1000 Ci/mmol) (NEN Research Products, Boston, 
Mass.) was added at a final concentration of 5 .mu.Ci/ml and incubation 
was continued for 1 hour. Cells were collected by centrifugation and 
proteins isolated through a boiled cell procedure (Maniatis, et al.). 
Proteins were separated on an acrylamide gel. After the run, the gel was 
dried and subjected to autoradiography for 3 days. 
Maxicells of JM109 harboring vector pDR540 showed only vector marker 
galactokinase protein. Maxicells of JM109 harboring vector pDRDsz showed 
the presence of three novel protein bands of sizes which correlated well 
with the predicted molecular weights of the three proteins responsible for 
the Dsz+ trait, as predicted by open reading frame analysis (see Table 3). 
TABLE 3 
______________________________________ 
Open Reading Predicted Measured 
Frame Size (kDa) 
Size (kDa) 
______________________________________ 
ORF-1 49.5 49.5 
ORF-2 38.9 33.0 
ORF-3 45.1 45.0 
______________________________________ 
Data obtained from Maxicell analysis thus indicated that the three 
predicted open reading frames of pTOXI-1 encode three structural genes 
which constitute the desulfurization phenotype. 
The relative intensity of the three novel bands is reflective of both the 
number of methionine residues and the level of translation for each of the 
proteins. Clearly, ORF-2 with only 1 Met gives the faintest band. In 
addition to the incorporation of only a single Met residue, E. coli may 
process the single terminal methionine, further reducing the amount of 
labelled protein. Therefore, the low intensity of the ORF-2 band most 
likely does not strictly suggest a low level of protein translation. 
Interestingly, the ORF furthest from the promoter (ORF-3) appears to be 
present at levels comparable to ORF-1, indicating no polar effects in this 
operon when expressed in E. coli. It is expected that more significant 
information regarding protein levels will be obtained from a similar 
Maxicell analysis of a Rhodococcus sp. host containing plasmid pTOXI-I. 
Additionally, the presence of an ORF-I/ORF-2 heterodimer, postulated 
above, may be observable under non-denaturing conditions. 
As required by 37 C.F.R. Section 1821(f), Applicant's Attorney hereby 
states that the content of the "Sequence Listing" in this specification in 
paper form and the content of the computer-readable form (diskette) of the 
"Sequence Listing" are the same. 
EQUIVALENTS 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. These and all other such 
equivalents are intended to be encompassed by the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 16 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5535 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A ) NAME/KEY: CDS 
(B) LOCATION: 790..2151 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 3256..4506 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCATGCACGTCGCGCCGACGCATTTGCGCGCACGGCTCCGGGCAGTTCTCGCGGCGCTGG60 
AGGCACGGATGGGCACCCTCAACGAACTCACCCAAAC CACGCCGATAGCGATCCTCGCCG120 
AAACCCTCGGCTACAGCCCTCAGACATTGGAAGCTCATGCGCGACGCATCCGGATCGACC180 
TTTGCACGCTACGTGGCGACGCGGCTGGACTGACGCTGGAGGTCCGACCCGACGTGTGTG240 
GTGTAGCGCCGCTT AACGGGTGCGCACGGCGGGACATCGGCCAGCTGGCTTGCCCCTCCT300 
CCGCAGGTAGTCGACCACCCCTTCCCGCAGCGGTCGGAGGTGATCGACCGTTAGGGTCAT360 
TTGCTCGCAGATCGGCTGATGTTGCCGATCGACGTGGTCGACGGGACACGCTCGCGATTG 420 
GCATGGCGTCCGGTGCATACACGACGATCTAACCAGATCGACGGTTTTGAGCGTCGGTCA480 
ACGTCGACTCGATGCGCCGTGCGAGTGAGATCCTTTGTGGTGCTTGGCTATTGACCTCGA540 
CAAGGATAGAGATTCGAAGGACCTCGGATCGACCCAA ATGCGGACGGCCGGCAGCGGCGA600 
AGGCGGCCAAGTCATCGGCACCGTCACCGTCACCTTGACCCGACGTGCCCCGTGGTTCAA660 
GGCCTGAATTTGGCTGGTGGAGCATTGAAATCAGGTGAAGTTTAACGGTGGGCACACCCC720 
GGGGGTGGGGGTGA GACTGCTTAGCGACAGGAATCTAGCCATGATTGACATTTAAAGGAC780 
GCATACGCGATGACTCAACAACGACAAATGCATCTGGCCGGTTTCTTC828 
MetThrGlnGlnArgGlnMetHisLeuAlaGlyPhePhe 
1510 
TCGGCCGGCAATGTGACTCATGCACATGGGGCGTGGCGGCACACGGAC876 
SerAlaGlyAsnValThrHisAlaHisGlyAlaTrpArgHisThrAsp 
15 2025 
GCGTCGAATGACTTTCTGTCGGGGAAGTACTACCAACACATCGCCCGT924 
AlaSerAsnAspPheLeuSerGlyLysTyrTyrGlnHisIleAlaArg 
303 54045 
ACTCTGGAGCGCGGCAAGTTCGATCTGTTGTTTCTGCCTGACGGGTTG972 
ThrLeuGluArgGlyLysPheAspLeuLeuPheLeuProAspGlyLeu 
505560 
GCCGTCGAGGACAGCTACGGGGACAACCTGGACACCGGTGTCGGCCTG1020 
AlaValGluAspSerTyrGlyAspAsnLeuAspThrGlyValGlyLeu 
65 7075 
GGCGGGCAGGGTGCAGTCGCCTTGGAGCCGGCCAGTGTGGTCGCAACC1068 
GlyGlyGlnGlyAlaValAlaLeuGluProAlaSerValValAlaThr 
80 8590 
ATGGCCGCGGTGACCGAGCACCTGGGTCTTGGGGCAACCATTTCGGCG1116 
MetAlaAlaValThrGluHisLeuGlyLeuGlyAlaThrIleSerAla 
9510 0105 
ACCTACTATCCCCCGTATCACGTTGCTCGGGTGTTCGCGACGCTCGAT1164 
ThrTyrTyrProProTyrHisValAlaArgValPheAlaThrLeuAsp 
110115 120125 
CAGTTGTCAGGGGGTCGGGTGTCCTGGAACGTCGTCACCTCGCTCAAC1212 
GlnLeuSerGlyGlyArgValSerTrpAsnValValThrSerLeuAsn 
130 135140 
GACGCTGAAGCGCGCAACTTCGGCATTAATCAGCATCTGGAACACGAC1260 
AspAlaGluAlaArgAsnPheGlyIleAsnGlnHisLeuGluHisAsp 
145 150155 
GCCCGCTATGACCGCGCCGATGAGTTCTTGGAAGCGGTCAAGAAACTC1308 
AlaArgTyrAspArgAlaAspGluPheLeuGluAlaValLysLysLeu 
160165 170 
TGGAACAGCTGGGACGAGGACGCCCTCGTGCTGGACAAGGCGGCCGGC1356 
TrpAsnSerTrpAspGluAspAlaLeuValLeuAspLysAlaAlaGly 
175180 185 
GTGTTCGCCGATCCCGCGAAGGTGCACTACGTCGATCACCACGGGGAG1404 
ValPheAlaAspProAlaLysValHisTyrValAspHisHisGlyGlu 
190195200 205 
TGGCTGAATGTGCGCGGACCTCTGCAGGTACCGCGTTCACCTCAGGGT1452 
TrpLeuAsnValArgGlyProLeuGlnValProArgSerProGlnGly 
210215 220 
GAGCCGGTGATCCTGCAGGCCGGCCTGTCGCCCCGGGGTCGGCGCTTC1500 
GluProValIleLeuGlnAlaGlyLeuSerProArgGlyArgArgPhe 
225230 235 
GCCGGGAAGTGGGCCGAGGCCGTCTTCAGTCTTGCACCCAACCTCGAG1548 
AlaGlyLysTrpAlaGluAlaValPheSerLeuAlaProAsnLeuGlu 
2402452 50 
GTGATGCAGGCCACCTACCAGGGCATCAAAGCCGAGGTCGACGCTGCG1596 
ValMetGlnAlaThrTyrGlnGlyIleLysAlaGluValAspAlaAla 
255260265 
GGGCG CGATCCCGATCAGACGAAAATCTTCACCGCCGTGATGCCGGTA1644 
GlyArgAspProAspGlnThrLysIlePheThrAlaValMetProVal 
270275280285 
C TCGGCGAAAGCCAGGCGGTGGCACAGGAACGACTGGAATATCTCAAC1692 
LeuGlyGluSerGlnAlaValAlaGlnGluArgLeuGluTyrLeuAsn 
290295300 
AGTCTGGTCCATCCGGAAGTGGGACTGTCGACGCTATCCAGTCACACC1740 
SerLeuValHisProGluValGlyLeuSerThrLeuSerSerHisThr 
305310315 
GGC ATCAACCTGGCGGCGTACCCTCTCGACACTCCGATCAAGGACATC1788 
GlyIleAsnLeuAlaAlaTyrProLeuAspThrProIleLysAspIle 
320325330 
CTGCGGGA TCTGCAGGATCGGAATGTCCCGACGCAACTGCACATGTTC1836 
LeuArgAspLeuGlnAspArgAsnValProThrGlnLeuHisMetPhe 
335340345 
GCCGCCGCAACGCACA GCGAAGAGCTCACGCTGGCGGAAATGGGTCGG1884 
AlaAlaAlaThrHisSerGluGluLeuThrLeuAlaGluMetGlyArg 
350355360365 
CGCTATGGAACC AACGTGGGGTTCGTTCCTCAGTGGGCCGGTACCGGG1932 
ArgTyrGlyThrAsnValGlyPheValProGlnTrpAlaGlyThrGly 
370375380 
GAGCAGATCGCT GACGAGCTGATCCGCCACTTCGAGGGCGGCGCCGCG1980 
GluGlnIleAlaAspGluLeuIleArgHisPheGluGlyGlyAlaAla 
385390395 
GATGGTTTCATCAT CTCTCCGGCCTTCCTGCCGGGCTCCTACGACGAG2028 
AspGlyPheIleIleSerProAlaPheLeuProGlySerTyrAspGlu 
400405410 
TTCGTCGACCAGGTGGTTC CGGTTCTGCAGGATCGCGGCTACTTCCGC2076 
PheValAspGlnValValProValLeuGlnAspArgGlyTyrPheArg 
415420425 
ACCGAGTACCAGGGCAACACTCTGCGC GACCACTTGGGTCTGCGCGTA2124 
ThrGluTyrGlnGlyAsnThrLeuArgAspHisLeuGlyLeuArgVal 
430435440445 
CCACAACTGCAAGGACAACCTTCA TGACAAGCCGCGTCGACCCCGCAAACCCCG2178 
ProGlnLeuGlnGlyGlnProSer 
450 
GTTCAGAACTCGATTCCGCCATCCGCGACACACTGACCTACAGCAACTGCCCGGTACCCA2238 
ACGCTCTGCTCACGGCATCGGAATC GGGCTTCCTCGACGCCGCCGGCATCGAACTCGACG2298 
TCCTCAGCGGCCAGCAGGGCACGGTTCATTTCACCTACGACCAGCCTGCCTACACCCGTT2358 
TTGGGGGTGAGATCCCGCCACTGCTCAGCGAGGGGTTGCGGGCACCTGGGCGCACGCGTC2418 
TA CTCGGCATCACCCCGCTCTTGGGGCGCCAGGGCTTCTTTGTCCGCGACGACAGCCCGA2478 
TCACAGCGGCCGCCGACCTTGCCGGACGTCGAATCGGCGTCTCGGCCTCGGCAATTCGCA2538 
TCCTGCGCGGCCAGCTGGGCGACTACCTCGAGTTGGATCCCTGGCGGC AAACGCTGGTAG2598 
CGCTGGGCTCGTGGGAGGCGCGCGCCTTGTTGCACACCCTTGAGCACGGTGAACTGGGTG2658 
TGGACGACGTCGAGCTGGTGCCGATCAGCAGTCCTGGTGTCGATGTTCCCGCTGAGCAGC2718 
TCGAAGAATCGGCGACCGTCAAGGG TGCGGACCTCTTTCCCGATGTCGCCCGCGGTCAGG2778 
CCGCGGTGTTGGCCAGCGGAGACGTTGACGCCCTGTACAGTTGGCTGCCCTGGGCCGGGG2838 
AGTTGCAAGCCACCGGGGCCCGCCCAGTGGTGGATCTCGGCCTCGATGAGCGCAATGCCT2898 
AC GCCAGTGTGTGGACGGTCAGCAGCGGGCTGGTTCGCCAGCGACCTGGCCTTGTTCAAC2958 
GACTGGTCGACGCGGCCGTCGACGCCGGGCTGTGGGCACGCGATCATTCCGACGCGGTGA3018 
CCAGCCTGCACGCCGCGAACCTGGGCGTATCGACCGGAGCAGTAGGCC AGGGCTTCGGCG3078 
CCGACTTCCAGCAGCGTCTGGTTCCACGCCTGGATCACGACGCCCTCGCCCTCCTGGAGC3138 
GCACACAGCAATTCCTGCTCACCAACAACTTGCTGCAGGAACCCGTCGCCCTCGATCAGT3198 
GGGCGGCTCCGGAATTTCTGAACAA CAGCCTCAATCGCCACCGATAGGAACATCCGC3255 
ATGACACTGTCACCTGAAAAGCAGCACGTTCGACCACGCGACGCCGCC3303 
MetThrLeuSerProGluLysGlnHisValArgProArgAspAlaAla 
1 51015 
GACAACGATCCCGTCGCGGTTGCCCGTGGGCTAGCCGAAAAGTGGCGA3351 
AspAsnAspProValAlaValAlaArgGlyLeuAlaGluLysTrpArg 
202530 
GCCACCGCCGTCGAGCGTGATCGCGCCGGGGGTTCGGCAACAGCCGAG3399 
AlaThrAlaValGluArgAspArgAlaGlyGlySerAlaThrAlaGlu 
35 4045 
CGCGAAGACCTGCGCGCGAGCGCGCTGCTGTCGCTCCTCGTCCCGCGC3447 
ArgGluAspLeuArgAlaSerAlaLeuLeuSerLeuLeuValProArg 
50 5560 
GAATACGGCGGCTGGGGCGCAGACTGGCCCACCGCCATCGAGGTCGTC3495 
GluTyrGlyGlyTrpGlyAlaAspTrpProThrAlaIleGluValVal 
6570 7580 
CGCGAAATCGCGGCAGCCGATGGATCTTTGGGACACCTGTTCGGATAC3543 
ArgGluIleAlaAlaAlaAspGlySerLeuGlyHisLeuPheGlyTyr 
85 9095 
CACCTCACCAACGCCCCGATGATCGAACTGATCGGCTCGCAGGAACAA3591 
HisLeuThrAsnAlaProMetIleGluLeuIleGlySerGlnGluGln 
100 105110 
GAAGAACACCTGTACACCCAGATCGCGCAGAACAACTGGTGGACCGGA3639 
GluGluHisLeuTyrThrGlnIleAlaGlnAsnAsnTrpTrpThrGly 
11512 0125 
AATGCCTCCAGCGAGAACAACAGCCACGTGCTGGACTGGAAGGTCAGC3687 
AsnAlaSerSerGluAsnAsnSerHisValLeuAspTrpLysValSer 
130135 140 
GCCACCCCGACCGAAGACGGCGGCTACGTGCTCAATGGCACGAAGCAC3735 
AlaThrProThrGluAspGlyGlyTyrValLeuAsnGlyThrLysHis 
145150155 160 
TTCTGCAGCGGCGCCAAGGGGTCGGACCTGCTGTTCGTGTTCGGCGTC3783 
PheCysSerGlyAlaLysGlySerAspLeuLeuPheValPheGlyVal 
165170 175 
GTCCAGGATGATTCTCCGCAGCAGGGTGCGATCATTGCTGCCGCTATC3831 
ValGlnAspAspSerProGlnGlnGlyAlaIleIleAlaAlaAlaIle 
180185 190 
CCGACATCGCGGGCTGGCGTTACGCCCAACGACGACTGGGCCGCCATC3879 
ProThrSerArgAlaGlyValThrProAsnAspAspTrpAlaAlaIle 
195200 205 
GGCATGCGGCAGACCGACAGCGGTTCCACGGACTTCCACAACGTCAAG3927 
GlyMetArgGlnThrAspSerGlySerThrAspPheHisAsnValLys 
210215220 
GT CGAGCCTGACGAAGTGCTGGGCGCGCCCAACGCCTTCGTTCTCGCC3975 
ValGluProAspGluValLeuGlyAlaProAsnAlaPheValLeuAla 
225230235240 
TTCATACAATCCGAGCGCGGCAGCCTCTTCGCGCCCATAGCGCAATTG4023 
PheIleGlnSerGluArgGlySerLeuPheAlaProIleAlaGlnLeu 
24525025 5 
ATCTTCGCCAACGTCTATCTGGGGATCGCGCACGGCGCACTCGATGCC4071 
IlePheAlaAsnValTyrLeuGlyIleAlaHisGlyAlaLeuAspAla 
260265270 
GCCAGGGAGTACACCCGTACCCAGGCGAGGCCCTGGACACCGGCCGGT4119 
AlaArgGluTyrThrArgThrGlnAlaArgProTrpThrProAlaGly 
275280285 
ATTCA ACAGGCAACCGAGGATCCCTACACCATCCGCTCCTACGGTGAG4167 
IleGlnGlnAlaThrGluAspProTyrThrIleArgSerTyrGlyGlu 
290295300 
TTCACCATCGCAT TGCAGGGAGCTGACGCCGCCGCCCGTGAAGCGGCC4215 
PheThrIleAlaLeuGlnGlyAlaAspAlaAlaAlaArgGluAlaAla 
305310315320 
CACCTGCTG CAGACGGTGTGGGACAAGGGCGACGCGCTCACCCCCGAG4263 
HisLeuLeuGlnThrValTrpAspLysGlyAspAlaLeuThrProGlu 
325330335 
GACCGCGGC GAACTGATGGTGAAGGTCTCGGGAGTCAAAGCGTTGGCC4311 
AspArgGlyGluLeuMetValLysValSerGlyValLysAlaLeuAla 
340345350 
ACCAACGCCGC CCTCAACATCAGCAGCGGCGTCTTCGAGGTGATCGGC4359 
ThrAsnAlaAlaLeuAsnIleSerSerGlyValPheGluValIleGly 
355360365 
GCGCGCGGAACACATC CCAGGTACGGTTTCGACCGCTTCTGGCGCAAC4407 
AlaArgGlyThrHisProArgTyrGlyPheAspArgPheTrpArgAsn 
370375380 
GTGCGCACCCACTCCCTGCACGAC CCGGTGTCCTACAAGATCGCCGAC4455 
ValArgThrHisSerLeuHisAspProValSerTyrLysIleAlaAsp 
385390395400 
GTCGGCAAGCACACCTTGAAC GGTCAATACCCGATTCCCGGCTTCACC4503 
ValGlyLysHisThrLeuAsnGlyGlnTyrProIleProGlyPheThr 
405410415 
TCCTGAGGATCTGAGGCGCTGAT CGAGGCCGAGGCCACCGCGCGGCCGAGTCG4556 
Ser 
CGAATCGCCCGCCGATACTCAGCTTCTCCATACGTACGGGTGCACACAAGGAGATATTGT4616 
CAAGACCTGTGGATGAGGGTGTTTCAGGCGACCTCCGTTTCGCTTGATTCGTCGGGCTCA467 6 
GCGGGTGAGATGTCGATGGGTCGTTCGAGCAGCTTGCCTTTGTGGAACACCGCGCCGGCA4736 
CGGACCAGCGCGACCAGATGGGGGGCGTTGACCGCCGCCAGCGGGCTTGTGCGGCGTCGA4796 
TCAGCTTGTAGGCCATGGCAATCCCGCTGCGACGTGACCCAG GGCCCTTGGTGACCTTGG4856 
TTCGCAACCGCACGGTCGCAAACGTCGATTCGATCGGATTCGTAGTGCGCAAGTGGATCC4916 
AGTGCTCGGCCGGGTACCGGTAGAACTCCAGGAGCACGTCGGCGTCGTCGACGATCTTGG4976 
CGACCGCCTTGGGGTACTTC GCGCCGTAATCTACCTCGAAGGCCTTGATCGCGACCTGGG5036 
CCTTGTCGATGTCCTCGGCGTTGTAGATTTCCCGCATCGCCGCGGTCGCACCTGGATGAG5096 
CCGACTTGGGCAGCGCAGCAAGCACATTGGCCTGCTTGTGAAACCAGCAGCGCTGTTCAC515 6 
GGGTATCCGGAAACACCTCCCGCAGTGCCTTCCAGAACCCCAGCGCCCCATCACCGACGG5216 
CCAGCACCGGGGCGGTCATCCCGCGGCGTCGGCATGAGCGCAGCAGATCAGCCCACGACT5276 
CTGTGGACTCCCGGAACCCATCGGTGAGCGCGACGAGCTCCT TGCGGCCGTCGGCGCGGA5336 
CGCCGATCATCACGAGCAAGCACAGCTTCTCCTGCTCCAGGCGGACATTGAGATGGATGC5396 
CGTCGACCCATAGGTACACGAAATCGGTGCCCGAGAGATCCCGGTCGGCGAAGGCCTTCG5456 
CCTCGTCCTGCCATTGCGCG GTCAGCCGGGTGATCGTCGAGGCCGACAGCCCGGCACCAG5516 
TGCCGAGGAACTGCTCCAA5535 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 453 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetThrGlnGlnArgGlnMetHisLeuAlaGlyPhePheSerAlaGly 
151015 
AsnValThrHisAlaHisGlyAlaTrp ArgHisThrAspAlaSerAsn 
202530 
AspPheLeuSerGlyLysTyrTyrGlnHisIleAlaArgThrLeuGlu 
3540 45 
ArgGlyLysPheAspLeuLeuPheLeuProAspGlyLeuAlaValGlu 
505560 
AspSerTyrGlyAspAsnLeuAspThrGlyValGlyLeuGlyGlyGln 
65 707580 
GlyAlaValAlaLeuGluProAlaSerValValAlaThrMetAlaAla 
859095 
ValThrGluHisLeu GlyLeuGlyAlaThrIleSerAlaThrTyrTyr 
100105110 
ProProTyrHisValAlaArgValPheAlaThrLeuAspGlnLeuSer 
115120 125 
GlyGlyArgValSerTrpAsnValValThrSerLeuAsnAspAlaGlu 
130135140 
AlaArgAsnPheGlyIleAsnGlnHisLeuGluHisAspAlaArgTyr 
145150155160 
AspArgAlaAspGluPheLeuGluAlaValLysLysLeuTrpAsnSer 
165170175 
TrpA spGluAspAlaLeuValLeuAspLysAlaAlaGlyValPheAla 
180185190 
AspProAlaLysValHisTyrValAspHisHisGlyGluTrpLeuAsn 
195 200205 
ValArgGlyProLeuGlnValProArgSerProGlnGlyGluProVal 
210215220 
IleLeuGlnAlaGlyLeuSerProArgGlyArgArg PheAlaGlyLys 
225230235240 
TrpAlaGluAlaValPheSerLeuAlaProAsnLeuGluValMetGln 
245250 255 
AlaThrTyrGlnGlyIleLysAlaGluValAspAlaAlaGlyArgAsp 
260265270 
ProAspGlnThrLysIlePheThrAlaValMetProValLeuGlyGlu 
275280285 
SerGlnAlaValAlaGlnGluArgLeuGluTyrLeuAsnSerLeuVal 
290295300 
HisProGluValGlyLeuSerThrL euSerSerHisThrGlyIleAsn 
305310315320 
LeuAlaAlaTyrProLeuAspThrProIleLysAspIleLeuArgAsp 
3253 30335 
LeuGlnAspArgAsnValProThrGlnLeuHisMetPheAlaAlaAla 
340345350 
ThrHisSerGluGluLeuThrLeuAlaGluMetGlyArg ArgTyrGly 
355360365 
ThrAsnValGlyPheValProGlnTrpAlaGlyThrGlyGluGlnIle 
370375380 
AlaAspGluLeuIl eArgHisPheGluGlyGlyAlaAlaAspGlyPhe 
385390395400 
IleIleSerProAlaPheLeuProGlySerTyrAspGluPheValAsp 
405 410415 
GlnValValProValLeuGlnAspArgGlyTyrPheArgThrGluTyr 
420425430 
GlnGlyAsnThrLeuArgAspHisLeuG lyLeuArgValProGlnLeu 
435440445 
GlnGlyGlnProSer 
450 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 417 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetThrLeuSerProGluLysGlnHisValArgProArgAspAlaAla 
151015 
AspAsnAspProValAlaValAlaArgGlyLeuAlaGlu LysTrpArg 
202530 
AlaThrAlaValGluArgAspArgAlaGlyGlySerAlaThrAlaGlu 
354045 
ArgGluA spLeuArgAlaSerAlaLeuLeuSerLeuLeuValProArg 
505560 
GluTyrGlyGlyTrpGlyAlaAspTrpProThrAlaIleGluValVal 
6570 7580 
ArgGluIleAlaAlaAlaAspGlySerLeuGlyHisLeuPheGlyTyr 
859095 
HisLeuThrAsnAlaProMetIleGlu LeuIleGlySerGlnGluGln 
100105110 
GluGluHisLeuTyrThrGlnIleAlaGlnAsnAsnTrpTrpThrGly 
115120 125 
AsnAlaSerSerGluAsnAsnSerHisValLeuAspTrpLysValSer 
130135140 
AlaThrProThrGluAspGlyGlyTyrValLeuAsnGlyThrLysHis 
145 150155160 
PheCysSerGlyAlaLysGlySerAspLeuLeuPheValPheGlyVal 
165170175 
ValGlnAspAspSerP roGlnGlnGlyAlaIleIleAlaAlaAlaIle 
180185190 
ProThrSerArgAlaGlyValThrProAsnAspAspTrpAlaAlaIle 
195200 205 
GlyMetArgGlnThrAspSerGlySerThrAspPheHisAsnValLys 
210215220 
ValGluProAspGluValLeuGlyAlaProAsnAlaPheValLeuAla 
225230235240 
PheIleGlnSerGluArgGlySerLeuPheAlaProIleAlaGlnLeu 
245250255 
IlePh eAlaAsnValTyrLeuGlyIleAlaHisGlyAlaLeuAspAla 
260265270 
AlaArgGluTyrThrArgThrGlnAlaArgProTrpThrProAlaGly 
275 280285 
IleGlnGlnAlaThrGluAspProTyrThrIleArgSerTyrGlyGlu 
290295300 
PheThrIleAlaLeuGlnGlyAlaAspAlaAlaAlaA rgGluAlaAla 
305310315320 
HisLeuLeuGlnThrValTrpAspLysGlyAspAlaLeuThrProGlu 
325330 335 
AspArgGlyGluLeuMetValLysValSerGlyValLysAlaLeuAla 
340345350 
ThrAsnAlaAlaLeuAsnIleSerSerGlyValPheGluValIleGly 
355360365 
AlaArgGlyThrHisProArgTyrGlyPheAspArgPheTrpArgAsn 
370375380 
ValArgThrHisSerLeuHisAspPr oValSerTyrLysIleAlaAsp 
385390395400 
ValGlyLysHisThrLeuAsnGlyGlnTyrProIleProGlyPheThr 
40541 0415 
Ser 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5535 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 2148..3245 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCATGCACGTCGCGCCGACGCATTTGCGCGCACGGCTCCGGGCAGTTCTCGCGGCGCTGG60 
AGGCACGGATGGGCACCCTCAACGAACTCACCCAAACCACGCCGATAGCGATCCTCGCCG120 
AAACCCTCGGCTACAGCCCTCAGACATTGGAAGCTCATGCGCG ACGCATCCGGATCGACC180 
TTTGCACGCTACGTGGCGACGCGGCTGGACTGACGCTGGAGGTCCGACCCGACGTGTGTG240 
GTGTAGCGCCGCTTAACGGGTGCGCACGGCGGGACATCGGCCAGCTGGCTTGCCCCTCCT300 
CCGCAGGTAGTCGACCACCC CTTCCCGCAGCGGTCGGAGGTGATCGACCGTTAGGGTCAT360 
TTGCTCGCAGATCGGCTGATGTTGCCGATCGACGTGGTCGACGGGACACGCTCGCGATTG420 
GCATGGCGTCCGGTGCATACACGACGATCTAACCAGATCGACGGTTTTGAGCGTCGGTCA480 
ACGTCGACTCGATGCGCCGTGCGAGTGAGATCCTTTGTGGTGCTTGGCTATTGACCTCGA540 
CAAGGATAGAGATTCGAAGGACCTCGGATCGACCCAAATGCGGACGGCCGGCAGCGGCGA600 
AGGCGGCCAAGTCATCGGCACCGTCACCGTCACCTTGACCCGA CGTGCCCCGTGGTTCAA660 
GGCCTGAATTTGGCTGGTGGAGCATTGAAATCAGGTGAAGTTTAACGGTGGGCACACCCC720 
GGGGGTGGGGGTGAGACTGCTTAGCGACAGGAATCTAGCCATGATTGACATTTAAAGGAC780 
GCATACGCGATGACTCAACA ACGACAAATGCATCTGGCCGGTTTCTTCTCGGCCGGCAAT840 
GTGACTCATGCACATGGGGCGTGGCGGCACACGGACGCGTCGAATGACTTTCTGTCGGGG900 
AAGTACTACCAACACATCGCCCGTACTCTGGAGCGCGGCAAGTTCGATCTGTTGTTTCTG960 
CCTGACGGGTTGGCCGTCGAGGACAGCTACGGGGACAACCTGGACACCGGTGTCGGCCTG1020 
GGCGGGCAGGGTGCAGTCGCCTTGGAGCCGGCCAGTGTGGTCGCAACCATGGCCGCGGTG1080 
ACCGAGCACCTGGGTCTTGGGGCAACCATTTCGGCGACCTACT ATCCCCCGTATCACGTT1140 
GCTCGGGTGTTCGCGACGCTCGATCAGTTGTCAGGGGGTCGGGTGTCCTGGAACGTCGTC1200 
ACCTCGCTCAACGACGCTGAAGCGCGCAACTTCGGCATTAATCAGCATCTGGAACACGAC1260 
GCCCGCTATGACCGCGCCGA TGAGTTCTTGGAAGCGGTCAAGAAACTCTGGAACAGCTGG1320 
GACGAGGACGCCCTCGTGCTGGACAAGGCGGCCGGCGTGTTCGCCGATCCCGCGAAGGTG1380 
CACTACGTCGATCACCACGGGGAGTGGCTGAATGTGCGCGGACCTCTGCAGGTACCGCGT1440 
TCACCTCAGGGTGAGCCGGTGATCCTGCAGGCCGGCCTGTCGCCCCGGGGTCGGCGCTTC1500 
GCCGGGAAGTGGGCCGAGGCCGTCTTCAGTCTTGCACCCAACCTCGAGGTGATGCAGGCC1560 
ACCTACCAGGGCATCAAAGCCGAGGTCGACGCTGCGGGGCGCG ATCCCGATCAGACGAAA1620 
ATCTTCACCGCCGTGATGCCGGTACTCGGCGAAAGCCAGGCGGTGGCACAGGAACGACTG1680 
GAATATCTCAACAGTCTGGTCCATCCGGAAGTGGGACTGTCGACGCTATCCAGTCACACC1740 
GGCATCAACCTGGCGGCGTA CCCTCTCGACACTCCGATCAAGGACATCCTGCGGGATCTG1800 
CAGGATCGGAATGTCCCGACGCAACTGCACATGTTCGCCGCCGCAACGCACAGCGAAGAG1860 
CTCACGCTGGCGGAAATGGGTCGGCGCTATGGAACCAACGTGGGGTTCGTTCCTCAGTGG1920 
GCCGGTACCGGGGAGCAGATCGCTGACGAGCTGATCCGCCACTTCGAGGGCGGCGCCGCG1980 
GATGGTTTCATCATCTCTCCGGCCTTCCTGCCGGGCTCCTACGACGAGTTCGTCGACCAG2040 
GTGGTTCCGGTTCTGCAGGATCGCGGCTACTTCCGCACCGAGT ACCAGGGCAACACTCTG2100 
CGCGACCACTTGGGTCTGCGCGTACCACAACTGCAAGGACAACCTTCATGACAAGC2156 
MetThrSer 
1 
CGCGTCGACCCCGCAAACCCCGGTTCAGAACTCGATTCCGCCATCCGC2204 
ArgValAspProAlaAsnProGlySerGluLeuAspSerAlaIleArg 
510 15 
GACACACTGACCTACAGCAACTGCCCGGTACCCAACGCTCTGCTCACG2252 
AspThrLeuThrTyrSerAsnCysProValProAsnAlaLeuLeuThr 
202530 35 
GCATCGGAATCGGGCTTCCTCGACGCCGCCGGCATCGAACTCGACGTC2300 
AlaSerGluSerGlyPheLeuAspAlaAlaGlyIleGluLeuAspVal 
4045 50 
CTCAGCGGCCAGCAGGGCACGGTTCATTTCACCTACGACCAGCCTGCC2348 
LeuSerGlyGlnGlnGlyThrValHisPheThrTyrAspGlnProAla 
5560 65 
TACACCCGTTTTGGGGGTGAGATCCCGCCACTGCTCAGCGAGGGGTTG2396 
TyrThrArgPheGlyGlyGluIleProProLeuLeuSerGluGlyLeu 
7075 80 
CGGGCACCTGGGCGCACGCGTCTACTCGGCATCACCCCGCTCTTGGGG2444 
ArgAlaProGlyArgThrArgLeuLeuGlyIleThrProLeuLeuGly 
859095 
CGCCAGGGCTTCTTTGTCCGCGACGACAGCCCGATCACAGCGGCCGCC2492 
ArgGlnGlyPhePheValArgAspAspSerProIleThrAlaAlaAla 
1001051101 15 
GACCTTGCCGGACGTCGAATCGGCGTCTCGGCCTCGGCAATTCGCATC2540 
AspLeuAlaGlyArgArgIleGlyValSerAlaSerAlaIleArgIle 
120125 130 
CTGCGCGGCCAGCTGGGCGACTACCTCGAGTTGGATCCCTGGCGGCAA2588 
LeuArgGlyGlnLeuGlyAspTyrLeuGluLeuAspProTrpArgGln 
135140145 
ACGCTGGTAGCGCTGGGCTCGTGGGAGGCGCGCGCCTTGTTGCACACC2636 
ThrLeuValAlaLeuGlySerTrpGluAlaArgAlaLeuLeuHisThr 
150155160 
CTT GAGCACGGTGAACTGGGTGTGGACGACGTCGAGCTGGTGCCGATC2684 
LeuGluHisGlyGluLeuGlyValAspAspValGluLeuValProIle 
165170175 
AGCAGTCCTGGT GTCGATGTTCCCGCTGAGCAGCTCGAAGAATCGGCG2732 
SerSerProGlyValAspValProAlaGluGlnLeuGluGluSerAla 
180185190195 
ACCGTCAA GGGTGCGGACCTCTTTCCCGATGTCGCCCGCGGTCAGGCC2780 
ThrValLysGlyAlaAspLeuPheProAspValAlaArgGlyGlnAla 
200205210 
GCGGTGT TGGCCAGCGGAGACGTTGACGCCCTGTACAGTTGGCTGCCC2828 
AlaValLeuAlaSerGlyAspValAspAlaLeuTyrSerTrpLeuPro 
215220225 
TGGGCCGGG GAGTTGCAAGCCACCGGGGCCCGCCCAGTGGTGGATCTC2876 
TrpAlaGlyGluLeuGlnAlaThrGlyAlaArgProValValAspLeu 
230235240 
GGCCTCGATGAGCGC AATGCCTACGCCAGTGTGTGGACGGTCAGCAGC2924 
GlyLeuAspGluArgAsnAlaTyrAlaSerValTrpThrValSerSer 
245250255 
GGGCTGGTTCGCCAGCGACCTGG CCTTGTTCAACGACTGGTCGACGCG2972 
GlyLeuValArgGlnArgProGlyLeuValGlnArgLeuValAspAla 
260265270275 
GCCGTCGACGCCGGGCTGT GGGCACGCGATCATTCCGACGCGGTGACC3020 
AlaValAspAlaGlyLeuTrpAlaArgAspHisSerAspAlaValThr 
280285290 
AGCCTGCACGCCGCGAAC CTGGGCGTATCGACCGGAGCAGTAGGCCAG3068 
SerLeuHisAlaAlaAsnLeuGlyValSerThrGlyAlaValGlyGln 
295300305 
GGCTTCGGCGCCGACTTCCAG CAGCGTCTGGTTCCACGCCTGGATCAC3116 
GlyPheGlyAlaAspPheGlnGlnArgLeuValProArgLeuAspHis 
310315320 
GACGCCCTCGCCCTCCTGGAGCGCAC ACAGCAATTCCTGCTCACCAAC3164 
AspAlaLeuAlaLeuLeuGluArgThrGlnGlnPheLeuLeuThrAsn 
325330335 
AACTTGCTGCAGGAACCCGTCGCCCTCGATCAGT GGGCGGCTCCGGAA3212 
AsnLeuLeuGlnGluProValAlaLeuAspGlnTrpAlaAlaProGlu 
340345350355 
TTTCTGAACAACAGCCTCAATCGCCACCGA TAGGAACATCCGCATGACAC3262 
PheLeuAsnAsnSerLeuAsnArgHisArg 
360365 
TGTCACCTGAAAAGCAGCACGTTCGACCACGCGACGCCGCCGACAACGATCCCGTCGCGG3322 
TTGCCCGT GGGCTAGCCGAAAAGTGGCGAGCCACCGCCGTCGAGCGTGATCGCGCCGGGG3382 
GTTCGGCAACAGCCGAGCGCGAAGACCTGCGCGCGAGCGCGCTGCTGTCGCTCCTCGTCC3442 
CGCGCGAATACGGCGGCTGGGGCGCAGACTGGCCCACCGCCATCGAGGTCGTC CGCGAAA3502 
TCGCGGCAGCCGATGGATCTTTGGGACACCTGTTCGGATACCACCTCACCAACGCCCCGA3562 
TGATCGAACTGATCGGCTCGCAGGAACAAGAAGAACACCTGTACACCCAGATCGCGCAGA3622 
ACAACTGGTGGACCGGAAATGCCTCCAGCG AGAACAACAGCCACGTGCTGGACTGGAAGG3682 
TCAGCGCCACCCCGACCGAAGACGGCGGCTACGTGCTCAATGGCACGAAGCACTTCTGCA3742 
GCGGCGCCAAGGGGTCGGACCTGCTGTTCGTGTTCGGCGTCGTCCAGGATGATTCTCCGC3802 
AGCAGGGT GCGATCATTGCTGCCGCTATCCCGACATCGCGGGCTGGCGTTACGCCCAACG3862 
ACGACTGGGCCGCCATCGGCATGCGGCAGACCGACAGCGGTTCCACGGACTTCCACAACG3922 
TCAAGGTCGAGCCTGACGAAGTGCTGGGCGCGCCCAACGCCTTCGTTCTCGCC TTCATAC3982 
AATCCGAGCGCGGCAGCCTCTTCGCGCCCATAGCGCAATTGATCTTCGCCAACGTCTATC4042 
TGGGGATCGCGCACGGCGCACTCGATGCCGCCAGGGAGTACACCCGTACCCAGGCGAGGC4102 
CCTGGACACCGGCCGGTATTCAACAGGCAA CCGAGGATCCCTACACCATCCGCTCCTACG4162 
GTGAGTTCACCATCGCATTGCAGGGAGCTGACGCCGCCGCCCGTGAAGCGGCCCACCTGC4222 
TGCAGACGGTGTGGGACAAGGGCGACGCGCTCACCCCCGAGGACCGCGGCGAACTGATGG4282 
TGAAGGTC TCGGGAGTCAAAGCGTTGGCCACCAACGCCGCCCTCAACATCAGCAGCGGCG4342 
TCTTCGAGGTGATCGGCGCGCGCGGAACACATCCCAGGTACGGTTTCGACCGCTTCTGGC4402 
GCAACGTGCGCACCCACTCCCTGCACGACCCGGTGTCCTACAAGATCGCCGAC GTCGGCA4462 
AGCACACCTTGAACGGTCAATACCCGATTCCCGGCTTCACCTCCTGAGGATCTGAGGCGC4522 
TGATCGAGGCCGAGGCCACCGCGCGGCCGAGTCGCGAATCGCCCGCCGATACTCAGCTTC4582 
TCCATACGTACGGGTGCACACAAGGAGATA TTGTCAAGACCTGTGGATGAGGGTGTTTCA4642 
GGCGACCTCCGTTTCGCTTGATTCGTCGGGCTCAGCGGGTGAGATGTCGATGGGTCGTTC4702 
GAGCAGCTTGCCTTTGTGGAACACCGCGCCGGCACGGACCAGCGCGACCAGATGGGGGGC4762 
GTTGACCG CCGCCAGCGGGCTTGTGCGGCGTCGATCAGCTTGTAGGCCATGGCAATCCCG4822 
CTGCGACGTGACCCAGGGCCCTTGGTGACCTTGGTTCGCAACCGCACGGTCGCAAACGTC4882 
GATTCGATCGGATTCGTAGTGCGCAAGTGGATCCAGTGCTCGGCCGGGTACCG GTAGAAC4942 
TCCAGGAGCACGTCGGCGTCGTCGACGATCTTGGCGACCGCCTTGGGGTACTTCGCGCCG5002 
TAATCTACCTCGAAGGCCTTGATCGCGACCTGGGCCTTGTCGATGTCCTCGGCGTTGTAG5062 
ATTTCCCGCATCGCCGCGGTCGCACCTGGA TGAGCCGACTTGGGCAGCGCAGCAAGCACA5122 
TTGGCCTGCTTGTGAAACCAGCAGCGCTGTTCACGGGTATCCGGAAACACCTCCCGCAGT5182 
GCCTTCCAGAACCCCAGCGCCCCATCACCGACGGCCAGCACCGGGGCGGTCATCCCGCGG5242 
CGTCGGCA TGAGCGCAGCAGATCAGCCCACGACTCTGTGGACTCCCGGAACCCATCGGTG5302 
AGCGCGACGAGCTCCTTGCGGCCGTCGGCGCGGACGCCGATCATCACGAGCAAGCACAGC5362 
TTCTCCTGCTCCAGGCGGACATTGAGATGGATGCCGTCGACCCATAGGTACAC GAAATCG5422 
GTGCCCGAGAGATCCCGGTCGGCGAAGGCCTTCGCCTCGTCCTGCCATTGCGCGGTCAGC5482 
CGGGTGATCGTCGAGGCCGACAGCCCGGCACCAGTGCCGAGGAACTGCTCCAA5535 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 365 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetThrSerArgValAspProAlaAsnProGlySerGluLeuAspSer 
151015 
AlaIleArgAspThrLeuThrTyrSerAsnCysProValProAsnAla 
202530 
LeuLeuThrAlaSerGluSerGlyPheLeuAspAlaAlaGlyIleGlu 
354045 
LeuAspValLeuSerGlyGlnGlnGlyThrValHisPheThrTyrAsp 
505560 
GlnProAlaTyrThrArgPheGlyGlyGlu IleProProLeuLeuSer 
65707580 
GluGlyLeuArgAlaProGlyArgThrArgLeuLeuGlyIleThrPro 
8590 95 
LeuLeuGlyArgGlnGlyPhePheValArgAspAspSerProIleThr 
100105110 
AlaAlaAlaAspLeuAlaGlyArgArgIleGlyValSerAlaSer Ala 
115120125 
IleArgIleLeuArgGlyGlnLeuGlyAspTyrLeuGluLeuAspPro 
130135140 
TrpArgGlnThrLeuValA laLeuGlySerTrpGluAlaArgAlaLeu 
145150155160 
LeuHisThrLeuGluHisGlyGluLeuGlyValAspAspValGluLeu 
165 170175 
ValProIleSerSerProGlyValAspValProAlaGluGlnLeuGlu 
180185190 
GluSerAlaThrValLysGlyAlaAspLeuPhe ProAspValAlaArg 
195200205 
GlyGlnAlaAlaValLeuAlaSerGlyAspValAspAlaLeuTyrSer 
210215220 
TrpLeuPr oTrpAlaGlyGluLeuGlnAlaThrGlyAlaArgProVal 
225230235240 
ValAspLeuGlyLeuAspGluArgAsnAlaTyrAlaSerValTrpThr 
245250255 
ValSerSerGlyLeuValArgGlnArgProGlyLeuValGlnArgLeu 
260265270 
ValAspAlaAlaValAspAlaG lyLeuTrpAlaArgAspHisSerAsp 
275280285 
AlaValThrSerLeuHisAlaAlaAsnLeuGlyValSerThrGlyAla 
2902953 00 
ValGlyGlnGlyPheGlyAlaAspPheGlnGlnArgLeuValProArg 
305310315320 
LeuAspHisAspAlaLeuAlaLeuLeuGluArgThrGlnGlnPheLeu 
325330335 
LeuThrAsnAsnLeuLeuGlnGluProValAlaLeuAspGlnTrpAla 
340345350 
AlaProGluPh eLeuAsnAsnSerLeuAsnArgHisArg 
355360365 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GTTTTCCCAGTCACGAC17 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AACAGCTATGACCATG16 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii ) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GCGAATTCCGCACCGAGTACC21 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
( ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ATCCATATGCGCACTACGAATCC23 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GGAATTCTAGACATATGAGGAACAGACCATGACTCAACAACGACAAATGC50 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CGAATTCTAGAATCAGGGGTCGACGCGGCTTGTCATG37 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GGAATTCAGATCTCATATGAGGAAACAGACCATGACAAGCCGCGTCGACC50 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CGAATTCAGATCTAATTCCTATCGGTGGCGATTGAGGC38 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GGAATTCTTAACATATGAGGAAACAGACCATGACACTGTCACCTGA46 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
( D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CGAATTCTTAATCAGCGCCTCAGATCCTCAG31 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 116 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CATATGCATTTAAAGGACGCATACGCGATGACTCAACAACGACAATGCATCTGGCCGGGT60 
ATACGTAAATTTCCTGCGTATGCGCTACTGAGTTGTTGCTGTTACGTAGACCGGCC116