Lyophilized bioluminescent bacterial reagent for the detection of toxicants

A reagent, useful in the detection of environmental insults comprising bacterial cells containing a stress promoter operably linked to a lux gene complex has been prepared by lyophilizing the cells in a specified medium. The reagent may be used immediately upon rehydration where a positive test for the presence of an environmental insult is given by an increase in light production from the cells.

FIELD OF INVENTION 
The present invention relates to a method for the detection of sublethal 
levels of environmental insults using a lyophilized bioluminescent 
bacteria as a test reagent. 
TECHNICAL BACKGROUND 
Increasing public concern and mounting government regulations have provided 
impetus for the development of environmental sensing systems capable of 
detecting contaminants in soil and ground water. Highly sensitive and 
specific detection systems incorporating analytical tools such as Gas 
Chromatography and Mass spectro-photometry have been known for several 
years; however, these systems require expensive equipment and skilled 
operators. Moreover, sample preparation and data analysis is often 
cumbersome and time consuming. 
Toxicity assays involving living organisms such as Daphnia, used in the 
standard U.S. water toxicity test, are simpler; however, these tests are 
non-specific and not particularly rapid. Somewhat more rapid are cell 
based toxicity assays that incorporate a bacterial cell as the sensitive 
element. These systems use bacterial cells as a reagent in a conventional 
automated analytical system. For example the RODTOX system (Central 
Kagaku., Tokyo, Japan) is a batch assay that measures bacterial oxygen 
consumption and was designed for use in sewage plants. Other bacteria 
based systems such as the GBI TOXALARM system (Genossenschaft Berliner 
Ingenieuirkollective, Berlin, Germany) can measure the presence of 
specific chemicals. The GBI TOXALARM is known to be able to detect the 
presence of as little as 0.1 ppm potassium cyanide in a sample. These 
detection systems are useful, but are hampered by cumbersome and complex 
detection systems. Recently, the phenomenon of bacterial bioluminescence 
has been regarded as providing a simpler and more sensitive mode of 
detection in environmental sensing systems. 
Bacterial bioluminescence is phenomenon in which the products of 5 
structural genes (luxA, luxB, luxC, luxD and luxE) work in concert to 
produce light. Naturally bioluminescent organisms have been used as the 
sensitive element in toxicity tests. The MICROTOX system, (Microbics 
Corp., Carlsbad, Calif.) is an example. The MICROTOX system measures the 
natural baseline luminescence of Photobacterium phosporeum and relates 
this to the hostility of the environment around the organism. Since the 
three couples, ATP level, NADPH level and FMNH.sub.2 level, between light 
production and the central metabolic events of energy generation are 
necessary for light production in Photobacterium phosporeum, any insult 
that interferes with the availability or interaction of these metabolites 
will cause a decrease in the activity of the bioluminescence(lux) system 
and therefore a related decrease in light production by the organism. 
A main attribute of bioluminescent systems is that the decrease in light 
production is rapid, occurring within minutes of exposure to an insult. 
Another key advantage of these systems is that light detection can be 
exquisitely sensitive (down to the level of single photons), and is 
readily adaptable to portable field units. Furthermore, the logistics of 
light detection precludes the necessity of having the detector contact a 
wet, biological sample, which is a key weakness in competing technologies 
(such as ion-selective electrodes), where detector fouling and corrosion 
are responsible for significant down time. 
Although the MICROTOX and similar systems are useful, their sensitivity is 
limited to detecting levels of insults that kill or cripple the cell 
metabolically. To be detected by these systems, the insult must have 
reached a level high enough to either interfere with the central 
metabolism of the cell or to inactivate the Lux proteins. 
Frequently it is necessary to be able to detect levels of insults at levels 
below those needed to affect cell metabolism. Such is the case in waste 
treatment facilities where lethal concentrations of pollutants can 
eradicate the useful microbial population, incurring significant cost and 
plant down time. A preferred sensing system would be one that would be 
able to detect the presence of insults at sublethal levels, before a 
useful microbial population could be harmed. Such an early warning could 
be used to trigger prompt remedial action to save the indigenous microbial 
population. 
Recently recombinant bacteria have been developed by fusing the lux 
structural genes to chemically responsive bacterial promoters and then 
placing such chimeras in appropriate hosts. These recombinant bacteria are 
sensor organisms that glow in response to specific stimuli. An example of 
this type of gene fusion is described by H. Molders (EP 456667). Here, 
indicator bacterial strains are provided (by vector mediated gene 
transfer) containing a met promoter, specifically inducible by Hg ions, 
fused to a bacterial luciferase (lux AB) gene complex which is responsible 
for bioluminescence. The test system of Molders relies on the induction of 
the mer promoter by the presence of mercury and the subsequent increase in 
light emission from the recombinant bacteria for the test results. 
Recently Applicants have disclosed a method for the detection of 
environmental insults involving a bacterial detector organism comprising a 
stress inducible promoter operabaly linked to a lux gene complex (WO 
96/16187). A variety of stress promoters were enabled including groEL, 
groES, dnaK, dnaJ, grpE, lon, lysU, rpoD, clpB, clpP, uspA, katG, uvrA, 
frdA, sodA, sodB, soi-28, narG, recA, xthA, his, lac, phoA, glnA, micF, 
and fabA. Each of the stress promoters are sensitive to different classes 
of environmental stresses, thus permitting a wide array of detection. 
One of the principle utilities of such detector organisms is in the 
monitoring of waste water treatment facilities as well as the testing of 
environmental samples at remote of isolated sites. For the purposes of 
field testing it is inconvenient to transport detector organisms to a site 
for testing all the while maintaining the cells in the appropriate growth 
condition to allow for maximum sensitivity in detection. A reagent, that 
could be handled with less stringency would be much more adaptable for 
remote field use. To that end a number of detection systems that require 
living cells have attempted to use lyophilized or freeze dried cells as 
reagents. 
Freeze dried or lyophilized cells have been used as reagents in a number of 
field applications and detection kits. For example McKinney et al., (DE 
2100476) demonstrate that freeze-dried microorganism compositions are 
useful as reagents for the remediation of oil. Cultures of Candida 
lipolytica are freeze-dried and mixed with vermiculite and exposed to an 
oil layer where the yeast grows rapidly. McKinney demonstrates that cells 
may be freeze dried and reconstituted and still retain enough viability to 
function biochemically after a sufficient period of time for acclimation 
and growth. However, McKinney does not address whether the cells are 
capable of all normal metabolic functions immediately after 
reconstitution, and does not teach that mechanisms governing transcription 
and translation are operational until after a period of acclimation and 
cell growth. 
Yates (Appl. Environ. Microbiol., 44, 1072, (1982) disclose a method for 
the detection of mycotoxins involving the use of the naturally 
bioluminescent Photobacterium phosphoreum. The concentrations of 
mycotoxins causing 50% light reduction (EC50) in Photobacterium 
phosphoreum were determined immediately and at 5 h after reconstitution of 
the bacteria from a dried state. Yates determines the presence of 
mycotoxins on the basis of a reduction in light from the photobacterium, 
and notes that higher concentrations are needed to produce a 50% reduction 
in light at 0 hr. post rehydration. 
Yates shows that a reduction in light production is possible from 
Photobacterium in response to the presence of mycotoxin immediately after 
freeze dried are reconstituted. However, since the metabolic requirements 
for light production in Photobacterium do not require synthesis of new 
proteins, Yates does not address whether translational and transcriptional 
elements are functional in cells 0 hr. post rehydration. 
Recently Corbisier et al., (J. Biolumin. Chemilumin., 9, 289, (1994)) have 
disclosed a genetically engineered Alcaligenes bacteria comprising several 
metal sensitive promoters fused with a lux gene complex from either V. 
fischeri or V. harveyi, useful as microbial bioluminescent sensors for the 
detection of metals. Corbisier has demonstrated that these cells may be 
lyophilized and reconstituted without adversely affecting the cellular 
bioluminescent apparatus. However, the method of Corbisier still allows 
for a significant period of time for cell acclimation before the cell 
sensor is used. 
The above methods demonstrating the use of freeze dried cells as biological 
reagents have shown that freeze dried cells may retain their metabolic 
activity after rehydration, however, appear to require a period of time of 
acclimation and growth for full metabolic abilities to return. Applicants 
have previously shown that bacterial cells transformed with plasmid 
containing a stress promoter operably linked to a lux gene complex were 
useful as detector organisms in a method for the detection of 
environmental stresses and toxicants. The mechanism postulated is that the 
presence of an environmental toxicant activates the stress promoter which 
in turn drives the lux gene complex to synthesize new proteins responsible 
for light production by the cell. The presence of the insult or toxicant 
is determined on the basis of an increase in light production (in contrast 
to the decrease seen in Yates supra) and requires new protein synthesis. 
Applicants have now made the surprising discovery that these stress 
detector cells may be freeze dried and upon rehydration are immediately 
useful in a method for the detection of environmental insults. The ability 
of these cells to be used immediately, after rehydration is surprising 
since, to date, no freeze dried cell has been taught that demonstrates the 
level of metabolic activity needed to synthesize new proteins so soon 
after rehydration. In all other instances cells must be subject to a 
period of acclimation, or initial growth before new protein synthesis is 
seen. For example, in the method of Yates supra, light production is seen 
to decrease in response to the presence of a mycotoxin, immediately after 
rehydration. However, the metabolic requirements needed for a test using a 
naturally bioluminescent cell (Photobacterium) which relies on a decrease 
of light production, is only for the presence of active Lux proteins, 
reducing potential (NADH) and ATP. In contrast, the requirements for a 
test using a detector cell that relies on genetic regulation for light 
production include the presence of active Lux proteins, ATP,CTP, TTP and 
GTP, RNA polymerase and all requirements for translation and 
transcription. Hence the requirements of the cells within Applicants' 
invention are far more stringent that of those taught in the art. 
SUMMARY OF THE INVENTION 
Disclosed is a method for detecting the presence of an environmental insult 
with a lyophilized biological reagent said reagent comprising a detector 
organism containing an expressible lux gene complex under the control of a 
stress inducible promoter sequence, the method comprising the steps of: 
(i) rehydrating the lyophilized biological reagent in a suitable amount of 
water wherein a baseline bioluminescence is produced; 
(ii) immediately contacting the rehydrated reagent with a sample suspected 
of containing an environmental insult to form a reagent mixture; 
(iii) incubating the mixture for at least 20 minutes and at a temperature 
of up to 30.degree. C. and; 
(iv) detecting a change in bioluminescence from the mixture. 
The method can be used with a sample containing a diverse microbial 
population and includes embodiments wherein the change in bioluminescence 
of step (iv) are increases or decreases in bioluminescence. A stress 
inducible promoter may be selected from the group consisting of groEL, 
groES, dnaK, dnaJ, grpE, lon, lysU, rpoD, clpB, clpP, uspA, katG, uvrA, 
frdA, sodA, sodB, soi-28, narG, recA, xthA, his, lac, phoA, glnA, micF, 
and fabA. 
Also disclosed is a lyophilized biological reagent comprising a transformed 
bacteria containing an expressible lux gene complex under the control of a 
stress inducible promoter sequence. 
The invention also concerns a kit containing the lyophilized biological 
reagents disclosed herein along with suitable solvents and/or buffers. A 
preferred embodiment for detecting the presence of an environmental insult 
comprises the following in packaged combination: 
(i) an aliquoted lyophilized biological reagent comprising: 
(a) a detector cell containing a DNA fragment comprising a stress promoter 
gene operably linked to the lux gene complex; 
(b) a suitable buffer; and 
(c) a cryoprotective reagent; 
(ii) a rehydrating reagent; and 
(iii) a suitable growth media. 
The kit can further include a means for measuring light output from the 
biological reagent.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein the following terms may be used for interpretation of the 
claims and specification. 
The terms "promoter" and "promoter region" refer to a sequence of DNA, 
usually upstream of (5' to) the protein coding sequence of a structural 
gene, which controls the expression of the coding region by providing the 
recognition for RNA polymerase and/or other factors required for 
transcription to start at the correct site. Promoter sequences are 
necessary but not always sufficient to drive the expression of the gene. 
A "fragment" constitutes a fraction of the DNA sequence of the particular 
region. 
"Nucleic acid" refers to a molecule which can be single stranded or double 
stranded, composed of monomers (nucleotides) containing a sugar, phosphate 
and either a purine or pyrimidine. In bacteria and in higher plants, 
"deoxyribonucleic acid" (DNA) refers to the genetic material while 
"ribonucleic acid" (RNA) is involved in the translation of the information 
from DNA into proteins. 
The term "transformation" refers to the acquisition of new genes in a cell 
after the incorporation of nucleic acid. 
The term, "operably linked" refers to the fusion of two fragments of DNA in 
a proper orientation and reading frame to be transcribed into functional 
RNA. 
The term "expression" refers to the transcription and translation to gene 
product from a gene coding for the sequence of the gene product. In the 
expression, a DNA chain coding for the sequence of gene product is first 
transcribed to a complimentary RNA which is often a messenger RNA and, 
then, the thus transcribed messenger RNA is translated into the 
above-mentioned gene product if the gene product is a protein. 
The term "bioluminescence" refers to the phenomenon of light emission from 
any living organism. 
The term "lux" refers to the lux complex of structural genes which include 
luxA, luxB, luxC, luxD and luxE and which are responsible for the 
phenomenon of bacterial bioluminescence. A lux gene complex might include 
all of the independent lux genes, acting in concert, or any subset of the 
lux complex. 
The term "stress" or "environmental stress" refers to the condition 
produced in a cell as the result of exposure to an environmental insult. 
The term "insult" or "environmental insult" refers to any substance or 
environmental change that results in an alteration of normal cellular 
metabolism in a bacterial cell or population of cells. Environmental 
insults may include, but are not limited to, chemicals, environmental 
pollutants, heavy metals, changes in temperature, changes in pH as well as 
agents producing oxidative damage, DNA damage, anaerobiosis, changes in 
nitrate availability or pathogenesis. 
The term "stress response" refers to the cellular response resulting in the 
induction of detectable levels of stress proteins. 
The term "stress protein" refers to any protein induced as a result of 
environmental stress or by the presence of an environmental insult. 
Typical stress proteins include, but are not limited to those encoded by 
the genes groEL, groES, dnaK, dnaJ, grpE, lon, lysU, rpoD, clpB, clpP, 
uspA, katG, uvrA, frdA, sodA, sodB, soi-28, narG, recA, xthA, his, lac, 
phoA, glnA, micF, and fabA. 
The term "stress gene" refers to any gene whose transcription is induced as 
a result of environmental stress or by the presence of an environmental 
insult. Typical E. coli stress genes include, but are not limited to 
groEL, groES, dnaK, dnaJ, grpE, lon, lysU, rpoD, clpB, clpP, uspA, katG, 
uvrA, frdA, sodA, sodB, soi-28, narG, recA, xthA, his, lac, phoA, glnA, 
micF, and fabA. 
The term "heat shock gene" refers to any gene for which its synthesis is 
positively controlled by the structural gene encoding the sigma-32 protein 
(rpoH). 
The term "stress inducible promoter" refers to any promoter capable of 
activating a stress gene and causing the expression of the stress gene 
product. 
The term "detector organism" refers to an organism which contains a gene 
fusion consisting of a stress inducible promoter fused to the lux gene 
complex and which is capable of expressing the lux gene products in 
response to an environmental insult. Typical detector organisms include 
but are not limited to bacteria. 
The term "lyophilized biological reagent" refers to a detector organism 
which contains a gene fusion consisting of a stress inducible promoter 
fused to the lux gene complex and which is freeze-dried in a specific 
medium and is capable of expressing the lux gene products in response to 
an environmental insult, immediately upon rehydration. 
The term "lyophilize" or "lyophilization" or "freeze-dry" will refer to a 
process for the removal of water from frozen bacterial cultures by 
sublimation under reduced pressure. 
The term "rehydration" or "reconstitution" will refer to the process 
whereby a specified amount of liquid, usually sterile water or growth 
media is added to a sample of lyophilized biological reagent resulting in 
the rejuvenation of detector organisms to a point where metabolic activity 
may be detected. 
The term "Relative Light Unit" is abbreviated "RLU" and refers to a measure 
of light emission as measured by a luminometer, calibrated against an 
internal standard unique to the luminometer being used. 
The present invention provides a method for the detection of environmental 
insults, such as chemical toxicants, at levels that are sublethal to the 
detector organism. The method incorporates a lyophilized biological 
reagent, the active part of which is the detector organism. The detector 
organism comprises a stress promoter operably linked to a lux gene complex 
so that when the detector organism comes in contact with the environmental 
insult the stress promoter is activated resulting in the production of the 
Lux proteins and the production of light from the organism. Unique to the 
present method is the fact that the lyophilized reagent containing the 
detector organism may be used immediately after reconstitution for 
detection without any acclimation or growth stabilization. 
This invention is anticipated to have broad applicability. Potential uses 
include monitoring of air and water quality, agrochemical and 
pharmaceutical design, manufacturing and fermentation process control, 
process monitoring and toxicity screening. These applications may benefit 
many enterprises including the chemical, beverage, food and flavor, 
cosmetics, agricultural, environmental, regulatory and health care 
industries. The method and reagent of the present invention is 
particularly useful in the monitoring of any area or media for the 
presence of sublethal levels of environmental toxicants. For example it is 
contemplated that the present invention will be particularly useful in the 
monitoring of the influx at waste water treatment facilities which is key 
to preventing contaminants from destroying the active microbial population 
in such facilities. Further, the lyophilized biological reagent is 
particularly adaptable for field testing of soil and ground water in and 
around both commercial and domestic sites where pollutants may pose a 
hazard. 
Environmental insults capable of being detected by the detector organism of 
the present invention include a variety of organic and inorganic 
pollutants commonly found in industrial sites, waste streams and 
agricultural run-off. Such compounds include but are not limited to the 
polyaromatic hydrocarbons (PAH), the halogenated aromatics as well as a 
variety of heavy metals such as lead, cadmium, copper, zinc, and cobalt. 
Compounds demonstrated to be detected by the method of the present 
invention include atrazine, benzene, copper sulfate, 
2,4-dichlorophenoxyacetic acid, ethanol, methanol, 2-nitrophenol, 
4-nitrophenol, pentachloro-phenol, phenol, toluene, dimethylsulfoxide, 
lead nitrate, cadmium chloride, sodium chloride, acetate, propionate, 
hydrogen peroxide, puromycin, mercury chloride, 2,4-dichloroanaline, 
propanol, butanol, isopropanol, methylene chloride, Triton X100, 
acrylamide, methyl viologen, mitomycin C, menadione, ethidium bromide, 
serine hydroxamate and xylene. Other environmental stresses detected 
included low phosphate levels, poor nitrogen source, poor carbon source 
and irradiation with ultraviolet light. 
Reporter genes: 
The preferred reporter gene for the present invention is the lux gene 
complex, responsible for bacterial bioluminescence and isolated from the 
bacteria Vibrio fischeri. Bacterial bioluminescence is phenomenon in which 
the products of 5 structural genes (luxA, luxB, luxC, luxD and luxE) work 
in concert to produce light. The luxD product generates a C.sup.14 fatty 
acid from a precursor. The C.sup.14 fatty acid is activated in an ATP 
dependent reaction to an acyl-enzyme conjugate through the action of the 
luxE product which couples bacterial bioluminescence to the cellular 
energetic state. The acyl-enzyme (luxE product) serves as a transfer 
agent, donating the acyl group to the luxC product. The acyl-LuxC binary 
complex is then reduced in a reaction in which NADPH serves as an electron 
pair and proton donor reducing the acyl conjugate to the C.sup.14 
aldehyde. This reaction couples the reducing power of the cell to 
bacterial light emission. The light production reaction, catalyzed by 
luciferase (the product of luxA and luxB), generates light. The energy for 
light emission is provided by the aldehyde to fatty acid conversion and 
FMNH.sub.2 oxidation, providing another couple between light production 
and the cellular energy state. 
The source of the bacterial lux complex was the pUCD615 plasmid containing 
the lux gene complex, fully described by Rogowsky et al. (J. Bacteriol. 
169 (11) pp 5101-512, (1987)). 
Stress Promoters: The present invention provides a stress inducible 
promoter sensitive to the presence of an environmental insult. Stress 
inducible promoters from both prokaryotic and eukaryotic cells may be used 
however promoters from bacteria are preferred and promoters from E. coli 
are most preferred. Suitable stress inducible promoters may be selected 
from, but are not limited to the list of genes under the heading 
"responding genes" given in Table I, below: 
TABLE I 
______________________________________ 
REGULATORY REGULATORY RESPONDING 
STIMULUS GENE(S) CIRCUIT GENES* 
______________________________________ 
Protein rpoH Heat Shock grpE, dnaK, 
Damage.sup.a lon, rpoD, 
groESL, lysU, 
htpE, htpG, 
htpI, htpK, 
clpP, clpB, 
htpN, htpO, 
htpX, etc. 
DNA Damage.sup.b 
lexA, recA SOS recA, uvrA, 
lexA, umuDC, 
uvrA, uvrB, 
uvrC, sulA, 
recN, uvrD, 
ruv, dinA, 
dinB, dinD, 
dinF, etc. 
Oxidative 
oxyR Hydrogen katG, ahp, etc. 
Damage.sup.c Peroxide 
Oxidative 
soxRS Superoxide micF, sodA, 
Damage.sup.d nfo, zwf, soi, 
etc. 
Membrane fadR Fatty Acid fabA 
Damage.sup.e Starvation 
Any.sup.f 
? Universal uspA 
Stress 
Stationary 
rpoS Resting State 
xthA, katE, 
Phase.sup.g appA, mcc, 
bolA, osmB, 
treA, otsAB, 
cyxAB, glgS, 
dps, csg, etc. 
Amino Acid 
relA, spoT Stringent his, ilvBN, 
Starvation.sup.h ilvGMED, 
thrABC, etc. 
Carbon cya, crp Catabolite lac, mal, gal, 
Starvation.sup.i Activation ara, tna, dsd, 
hut, etc. 
Phosphate 
phoB, phoM, P Utilization 
phoA, phoBR, 
Starvation.sup.j 
phoR, phoU phoE, phoS, 
aphA, himA, 
pepN, ugpAB, 
psiD, psiE, 
psiF, psiK, 
psiG, psiI, 
psiJ, psiN, 
psiR, psiH, 
phiL, phiO, 
etc. 
Nitrogen glnB, glnD, N Utilization 
glnA, hut, etc. 
Starvation.sup.k 
glnG, glnL. 
______________________________________ 
*Genes whose expression is increased by the corresponding stimulus and 
whose expression is controlled by the corresponding regulatory gene(s). 
.sup.a Neidhardt and van Bogelen in E. coli and Salmonella typhimurium; 
Cellular and Molecular Biology (Neidhardt, F. C., 
et al. Eds., pp. 1334-1345, American Society of Microbiology, 
Washington, DC (1987)) 
.sup.b Walker in E. coli and Salmonella typhimurium; Cellular and 
Molecular Biology (Neidhardt, F. C., et al. Eds., pp. 1346- 
1357, American Society of Microbiology, Washington, DC (1987)) 
.sup.c Christman et al. Cell 41: 753-762 (1985); Storz et al. 
Science 248: 189-194 (1990); Demple, Ann. Rev. Genet. 25: 
315-337 (1991) 
.sup.d Demple, Ann. Rev. Genet. 25: 31 337 (1991) 
.sup.e Magnuson et al. Microbiol. Rev 57: 522-542 (1993) 
.sup.f Nystrom and Neidhardt, J. Bacteriol, 175: 2949-2956 (1993); 
Nystrom and Neidhardt (Mol. Microbiol. 6: 3187-3198 (1992) 
.sup.g Kolter et al. Ann. Rev. Microbiol. 47: 855-874 (1993) 
.sup.h Cashel and Rudd in E. coli and Salmonella typhimurium; 
Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., 
pp. 1410-1438, American Society of Microbiology, Washington, 
DC (1987)); Winkler in E. coli and Salmonella typhimurium; 
Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., 
pp. 395-411, American Society of Microbiology, Washington, DC 
(1987)) 
.sup.i Neidhardt, Ingraham and Schaecter. Physiology of the 
Bacterial Cell: A Molecular Approach, Sinauer Associates, 
Sunderland, MA (1990), pp 351-388; Magasanik and Neidhardt in 
E. coli and Salmonella typhimurium; Cellular and Molecular 
Biology (Neidhardt, F. C., et al. Eds., pp. 1318-1325, American 
Society of Microbiology, Washington, DC (1987)) 
.sup.j Wanner in E. coli and Salmonella typhimurium; Cellular and 
Molecular Biology (Neidhardt, F. C., et al. Eds., E. coli and 
Salmonella typhimurium; Cellular and Molecular Biology 
(Neidhardt, F. C., et al. Eds., pp. 1326-1333, American Society 
of Microbiology, Washington, DC (1987)) 
.sup.k Rietzer and Magasanik in E. coli and Salmonella typhimurium; 
Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., 
pp. 1302-1320, American Society of Microbiology, Washington, 
DC (1987)); Neidhardt, Ingraham and Schaecter. Physiology of 
the Bacterial Cell: A Molecular Approach, Sinauer Associates, 
Sunderland, MA (1990), pp 351-388 
Table I indicates the relationship of responding gene(s) with a particular 
regulatory gene(s) and a regulatory circuit and the associated cellular 
stress response triggered by a particular stimulus. 
Vectors 
The invention also provides a transformation vector containing a stress 
inducible promoter-lux gene fusion, capable of transforming a bacterial 
host cell for the expression of the Lux proteins. A variety of 
transformation vectors may be used, however, those capable of transforming 
E. coli are preferred. pGrpELux.3, and pGrpELux.5 are two specific 
examples of suitable transformation vectors whose construction is given in 
detail in the following text. These vectors represent only a sample of the 
total number of vectors created for the purpose of introducing stress 
promoter-lux reporter fusions into host cells. However, it will be readily 
apparent to one of skill in the art of molecular biology that the methods 
and materials used in their construction are representative of all other 
suitable vectors. 
pGrpELux.3 and pGrpELux.5 are vectors containing the grpE promoter. 
pGrpELux.3 and pGrpELux.5 were created by the method of direct cloning. 
Transformation vectors such as these are common and construction of a 
suitable vector may be accomplished by means well known in the art. The 
preferred source of the lux genes is a pre-existing plasmid, containing a 
promoterless lux gene complex. Similarly, preferred sources of the stress 
inducible promoter DNA for the construction of the transformation vector 
are either also a pre-existing plasmid, where the stress inducible 
promoter DNA is flanked by convenient restriction sites, suitable for 
isolation by restriction enzyme digestion, or the product of a PCR 
reaction. 
The pGrpELux.3 and pGrpELux.5, vectors are constructed from the E. coli 
stress gene grpE, and the lux gene complex. pGrpE4 is an E. coli vector 
derived from pUC18 (Pharmacia, Cat. No. 27-4949-01). pGrpE4 contains the 
grpE gene, including its promoter, bounded at the 5' end by an EcoRI site 
and at the 3' end by a BbuI site. Additionally, the grpE promoter is 
bounded at the 3' end by a PvuII site and an HaeIII site just downstream 
of the EcoRI site (FIG. 2). Digestion with EcoRI and BbuI restriction 
enzymes yields a 1.1 kb fragment which corresponds to the grpE gene. 
Further digestion with PvuII produces two fragments, one of which contains 
the grpE promoter. The 3' PvuII site on the grpE promoter fragment is 
converted to an EcoRI site via ligation to phosphorylated EcoRI linkers. 
Further digestion by HaeIII yields a grpE promoter fragment conveniently 
bounded by a 5' HaeIII site and a 3' PvuII site (FIG. 2). 
The pUCD615 plasmid containing the lux gene complex is fully described by 
Rogowsky et al. (J. Bacteriol, 169 (11) pp 5101-512, (1987)). Plasmid 
pUCD615 is a 17.6 kb plasmid which contains the genes for kanamycin and 
ampicillin resistance and contains the promoterless lux gene operon (FIG. 
2). pUCD615 is first digested with restriction enzymes EcoRI and SmaI, 
opening the plasmid, followed by ligation with the DNA fragments from the 
HaeIII digestion of pgrpE IV. 
Typically, the products of the ligation reactions are screened by first 
transforming a suitable host and screening for bioluminescence. A variety 
of hosts may be used where hosts having high transformation frequencies 
are preferred. XL1Blue (Stratagene, LaJolla, Calif.) and DH5-.alpha. 
(GIBCO-BRL, Gaithersburg, Md.) are two such hosts. Preferred methods of 
bioluminescence screening involve exposing gridded cultures of 
transformants to a suitable X-ray film, followed by visual analysis of the 
developed films for evidence of exposure. Reisolation of the plasmid from 
the transformed host and restriction digests followed by gel 
electrophoresis is used to confirm the existence of the correct plasmid. 
The plasmids pGrpELux.3 and pGrpELux.5, isolated from two different 
transformed colonies, are indistinguishable on the basis of restriction 
enzyme analysis. Under some experimental conditions cells transformed with 
pGrpELux.5 exhibited higher baseline bioluminescence than those 
transformed with pGrpELux.3 and hence pGrpELux.5 is preferred for the 
detection of many environmental insults. 
Transformed Hosts--Detector Organisms: 
The present invention further provides a transformed host cell capable of 
increased luminescence in the presence of an environmental insult. Many 
suitable hosts are available where E. coli is preferred and the E. coli 
strain RFM443 is most preferred. RFM443 is derived from W3102 which is 
fully described by B. Bachmann, in E. coli and Salmonella typhimurium; 
Cellular and Molecular Biology (Niedhardt et al. Eds., pp 1190-1220, 
American Society of Microbiology, Washington, D.C. (1987)). Transformation 
of RFM443 by pGrpELux.3 gives the new strain TV1060 which has been 
deposited with the ATCC under the terms of the Budapest Treaty. 
Transformation of RFM443 by pGrpELux.5 gives the new strain TV1061. The 
baseline of bioluminescence from strain TV1061 is greater than that from 
strain TV1060. E. coli TV1060 has been assigned ATCC No. 69142, and TV1061 
has been assigned ATCC No. 69315. 
It is well known that hydrophobic compounds are effectively excluded by the 
cell envelope from entry into gram negative bacteria, such as E. coli. 
Recently several E. coli strains containing a mutation for tolerance to 
colicins (tolC-) have been found to have the unexpected additional 
property of increased permeability of host cell envelopes to various 
organic molecules. (Schnaitman et. al. J. Bacteriol., 172 (9), pp 
5511-5513, (1990)). Optionally, it is within the scope of the present 
invention to provide a transformed bacterial host containing the tolC- 
mutation as a suitable detector organism. 
Reagent Preparation--Cell lyophilization: 
Methods of preserving cells are varied and well known in the art 
(Maintenance of Microorganisms Kirsop, B. E., and Snell J. J. S., Eds, 
(1984), Academic Press, New York). The method chosen will depend on such 
factors as cell viability, genetic mutations, frequency of culture use and 
others. For cultures whose primary utility is use in field tests an kits, 
drying, freeze drying (lyophilization) or freezing are the most suitable. 
Although it is contemplated that any of these methods are compatible with 
the present invention the method most preferred is lyophilization. 
Lyophilization of cultures is a process that involves the removal of water 
from frozen cultures by sublimation under reduced pressure. 
When freeze drying living organisms several elements must be taken into 
account to allow for both the maximum viability and maximum storage time 
for the cells. At the time of harvesting cultures should be healthy and 
actively growing in either the logarithmic or early stationary phase and 
at a density of about 10.sup.8 /ml. A basic requirement in the medium for 
the preservation of the cells is a cryoprotective agent. A variety of 
cryoprotective reagents are known including skim milk, sucrose, dextran, 
horse serum, and inositol. For the purpose of the present invention 
sucrose is preferred at a concentration of about 12%. 
The choice of media and cryoprotective agents is an empirical process and a 
choice is made on the basis of highest cell viability and storage 
parameters. In the present application four different combinations of 
media and cryoprotective reagents were analyzed for their effect on cell 
viability, onset of induction of bioluminescence, and stability of 
baseline luminescence. The four lyophilization media are listed below: 
A. LB media with glucose (1%) 
B. Minimal Media with casamino acids (2%) and glucose (1%) 
C. Minimal Media with casamino acids (2%), glucose (1%), and sucrose (12%) 
D. Minimal media with casamino acids (2%), glucose (1%), and skim milk 
(12%). 
Of the above media it was found that lyophilization media (C) gave the best 
cell viability in combination with rapid onset of bioluminescent 
inducibility and stability of baseline luminescence. 
In the present method cells were grown to about an absorbance of 2 at O.D. 
600 (Log-phase growth) in LBG broth containing kanamycin and portions of 
the culture were subcultured into lypohilization media (D) above and grown 
until again reaching log phase densities. At this point cells were 
harvested by centrifugation, resuspended in lyophilzation media and frozen 
at -70.degree. C. in a lyophilization vial. Vials were placed on the 
lyophilizer and lyophilized for at least 3 hours at .ltoreq.20 millitorrs 
and -100.degree. C. Vials were sealed and stored at refrigerated or 
freezer temperatures until rehydrated. 
In order to rehydrate the lyophilized cells for use in the test method, 
lyophilized reagent was resuspended in a volume of sterile water equal to 
the volume of the samples prior to lyophilization. Cells were then 
immediately exposed to a sample suspected of containing an environmental 
insult and monitored for change in bioluminescence. Bioluminescence is 
measure on a luminometer of a type similar to that made by Dynatech 
Laboratories Inc. (Chantilly, Va.) 
The following examples are meant to illustrate the invention but should not 
be construed as limiting it in any way. From the above discussion and 
these Examples, one skilled in the art can ascertain the essential 
characteristics of this invention, and without departing from the spirit 
and scope thereof, can make various changes and modifications of the 
invention to adapt it to various usages and conditions. 
EXAMPLES 
General Methods 
E. coli TV1061 contains a plasmid with the E. coli grpE heat shock promoter 
fused to the Vibrio fischeri luxCDABE reporter genes and are fully 
described in the DETAILED DESCRIPTION section, above. Materials and 
Methods suitable for the maintenance and growth and lyophylization of 
bacterial clutures may be found in Manual of Methods for General 
Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, 
Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, 
eds), pp. 210-213. American Society for Microbiology, Washington, D.C. All 
reagents and materials used for the growth, maintenance and lyophilization 
of bacterial cells were obtained from Diffco Laboratories or Sigma 
Chemical Company unless otherwise specified. 
Example 1 
Preparation of Lyophilized Biological Reagent 
Example 1 describes the preparation of the biological reagent by the 
process of lyophilization in specially formulated media. 
E. coli TV1061 cells were grown in LBG broth containing the following 
components in g/L: tryptone, 10; yeast extract, 5; sodium chloride, 10; 
glucose, 10 and Kanamycin at a final concentration of 2.5 g/L. Cultures 
were allowed to grow to mid-log phase at O.D.600 of 2. 
This culture was then used to inoculate the production medium consisting of 
the following ingredients (g/L): ammonium sulfate, 0.3; magnesium sulfate, 
0.45; sodium citrate dihydrate 0.047; ferrous sulfate seven hydrate, 
0.025; thiamine-HCl 0.06; potassium phosphate dibasic, 1.95; sodium 
phosphate monobasic, 0.9; biotin 0.005; casamino acids, 20.0; trace 
element solution, 1 mL stock; uracil 0.1; glucose, 20.o; calcium chloride 
dihydrate, 0.026. 
Trace element solution was composed of the following (g/L): zinc sulfate 
seven hydrate, 8; copper sulfate five hydrate, 3; manganese sulfate 
monohydrate, 2.5; boric acid, 0.15; ammonium molybdate four hydrate, 0.1; 
cobalt chloride six hydrate, 0.06. 
Production culture was grown at 26.degree. C., pH 7.0, Dissolved oxygen 
(DO2) 50%. Dissolved oxygen was controlled by increasing agitation and 
aeration during growth. (rpm 300-1200; aeration 100-300 L/H). When 
cultures reached an OD 600 of 1.8 (logarithmic growth), they were 
harvested by centrifugation (Sorvall Superspeed, 9000 rpm for 20 minutes, 
4 C). Medium was decanted and cells were kept on wet ice. Cell pellets 
were resuspended in half the volume of the starting culture with fresh 
production medium and an equal half volume of 24% sterile sucrose. Cells 
were resuspended and dispensed into sterile lyophilization vials. Vials 
were frozen at -70.degree. C. Cultures were kept frozen until the 
lyophilization process was complete. Vials were placed on the lyophilizer 
(FD-14-84, FTS Systems, Stone Ridge, N.Y.) using a manifold system and a 
presterilized filter(Pall Emflon II 0.2 micron absolute) to prevent 
contamination of culture and lyophilizer. Vials were lyophilized for at 
least 3 hours at .ltoreq.20 millitorrs and -100.degree. C. Vials were 
sealed and stored at refrigerated or freezer temperatures until 
rehydrated. 
Example 2 
Use of Lyophilization Reagent for the Detection of Environmental Stress 
Example 2 demonstrates the use of the lyophilized biological reagent for 
the detection of environmental stress. 
The detector organism, E. coli TV1061 containing the E. coil grpE heat 
shock promoter fused to the Vibrio fischeri luxCDABE reporter genes is 
grown, harvested and lyophilized as described in Example 1 to prepare the 
reagent. The reagent was resuspended in a volume of sterile water equal to 
the volume of the samples prior to lyophilization. Reconstituted cells 
were tested for their ability to respond to stress induction at three 
different times post-rehydration. Cells were either used immediately or 
were incubated for 30 or 60 minutes prior to use. Viable cells were 
measured by plating serially diluted rehydrated cells on LB plates. 
Assessment of the ability of cells to respond to stress was made by 
measuring the kinetic changes in light output following the addition of 20 
.mu.l rehydrated cells to 80 .mu.l LB medium with or without 2.5% (v/v) 
ethanol (final ethanol concentration was 0% or 2%, respectively). 
Bioluminescence from these treated cells in white microtiter plates 
(Microlite.TM., Dynatech Laboratories Inc.) was quantitated in a Dynatech 
ML3000 microtiter plate luminometer with temperature controlled at 
26.degree. C. The units of measurements are relative light units (RLU). 
As can be seen by the data in FIG. 1, cells receiving no ethanol maintained 
a constant baseline luminescence whereas cells in the presence of 2% 
ethanol demonstrated a 100 fold increase in light output. It is important 
to note that the cells used at 0 and 30 minutes post-rehydration exhibited 
similar light production kinetics demonstrating that no acclimation phase 
is needed for the instant reagent to be effective in this assay. 
Example 3 
Determination of Lyophilization Media 
Example 3 describes the selection of the most appropriate lyophilization 
media for the bioluminescent detector cell. 
E. coli TV1061 cells were grown in LBG broth and inoculated in the 
production medium as described in Example 1. After growth and harvesting 
from the production media, cell pellets were resuspended in half the 
volume of the starting culture in four different media for lyophilization. 
A. LB media with glucose (1%) B. Minimal Media with casamino acids (2%) 
and glucose (1%) C. Minimal Media with casamino acids (2%), glucose (1%), 
and sucrose (12%) D. Minimal media with casamino acids (2%), glucose (1%), 
and skim milk (12%). 
Cells were lyophilized as described in Example 1 and stored for testing. 
Upon rehydration cells lyophilized in each medium were analyzed for 
viability, stability of baseline luminescence during the rehydration 
process and baseline stability during the induction process. 
Cell viability was determined by plating the cells after rehydration and 
determining the number of viable cells on the basis of colony forming 
units (CFU). 
Stability of baseline bioluminescence during rehydration was determined by 
continuously monitoring the bioluminescence of rehydrated cells over a 30 
minute time period. Stability of baseline luminescence during induction 
was determined by monitoring the bioluminescence of control cells (not 
exposed to an environmental insult) throughout the time of the test, which 
was always 120 minutes. 
Lag time was determined by measuring the amount of time from induction to 
the first increase in light output. Average Lag time for healthy, 
non-lyophilized cells was 20 min. 
The results of the analysis are given in Table I below. 
TABLE I 
__________________________________________________________________________ 
Viable 
Initial Stable During 
Stable During 
Medium O.D. 600 
Cells/ml 
RLU Lag Rehydration 
Induction 
__________________________________________________________________________ 
A (LBG) 
1.8 2.1 .times. 107 
0.0002 
90 min 
YES NO 
B (MMG) 
1.8 1.9 .times. 107 
0.0001 
20 min. 
NO NO 
C (MMGS) 
1.8 1.0 .times. 109 
0.066 
20 min 
YES YES 
D (MMGSM) 
1.8 7.6 .times. 107 
0.0025 
20 min 
YES NO 
__________________________________________________________________________ 
As can be seen by the information in Table I, the only media that 
demonstrated good stability of light output during both the rehyrdation 
phase and the induction phase was medium (C). All instances where the 
baseline was not stable demonstrated a steady increase in light output, 
presumably due to the increasing health and metabolic activity of the 
cells. Media (C) gave the surprising result of providing cells capable of 
immediate high level metabolic activity without requiring the almost 
obligatory acclimation period.