62
Journal of Food Protection, Vol. 55, No. I, Pages 62-70 (January 1992)
Copyright©, International Association of Milk, Food and Environmental Sanitarians
Bacterial Bioluminescence:
Applications in Food Microbiology
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J. M. BAKER', M. W. GRIFFITHS', and D. L. COLLINS-THOMPSON 2
Departments of !Food Science and ^'Environmental Biology, University of Guelph, Guelph, Ontario NIG 2W1, Canada
(Received for publication June 10, 1991)
ABSTRACT
Many marine microorganisms (Vibrio, Photobacterium) are
capable of emitting light, that is, they are bioluminescent. The
light-yielding reaction is catalyzed by a luciferase, and it involves
the oxidation of reduced riboflavin phosphate and a long-chain
Journal of Food Protection 1992.55:62-70.
aldehyde in the presence of oxygen to produce a blue green light.
The genes responsible for the luciferase production, (lux A and
lux B), aldehyde synthesis (lux C, D, and E), and regulation of
luminescence (lux I and lux R) have all been identified, and recent
research has resulted in the discovery of three new genes (lux F,
G, and H). The ability to genetically engineer dark microorgan-
isms to become light emitting by introducing the lux genes into
them has opened up a wide range of applications of biolumines-
cence. Assays using bacterial bioluminescence for the detection
and enumeration of microorganisms are rapid, sensitive, accurate,
and can be made specific. It is these attributes that are making in
vivo bioluminescent assays so attractive to the food industry.
Bioluminescent bacteria (those capable of emitting
light) are classified into four major genera: Vibrio,
Photobacterium, Alteromonas, and Xenorhabdus. They are
classified as motile, Gram-negative rods, and function as
facultative anaerobes (25,26). The first three genera are
marine in origin and can be isolated from seawater or
certain luminous ocean species. They can exist free living
in the ocean, as symbionts with various marine fish in their
digestive tracts or specialized light organs, as saprophytes
on dead fish, and as parasites in Crustacea and insects (26).
The marine organisms coexisting with the luminescent
bacteria can use the luminescence for a variety of purposes
such as attraction of prey, communication between species,
and escape from predators. However, it is not known what
specific benefit these bacteria derive from this energy
consuming property, either free living or when associated
with higher organisms (77). Detailed biochemical and ge-
netic studies have been carried out on species of Vibrio and
Photobacterium. The results of these studies have led to an
advancement in the understanding of bacterial biolumines-
cence. Researchers are now beginning to discover a wide
range of applications and uses for bioluminescence. This
review will cover the luminescence reaction in bacteria at
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
a biochemical and genetic level. The applications of bacte-
rial bioluminescence will also be presented, with an empha-
sis on the applications in food microbiology.
BIOCHEMISTRY OF THE
BIOLUMINESCENT REACTION
The bioluminescent reaction, catalyzed by the enzyme
luciferase, involves the oxidation of a long-chain aldehyde
and reduced riboflavin phosphate (FMNH2) and results in
the emission of a blue green light.
FMNH2 + 0 2 + RCOH — te'fc™e—>
FMN + RCOOH + H20 -(-light (490 nm)
The primary source of energy for the light is supplied by
the conversion of the aldehyde to the corresponding fatty
acid. Long-chain aldehydes are essential for the lumines-
cence reaction and the aldehyde, tetradecanal, appears to be
the natural substrate for the luminescence reaction (25).
The reaction is highly specific for FMNFL, which is formed
by the reaction:
NAD(P) H + FMN —FMN °»^^^— > FMNH2
Thus, the luciferase reaction may be driven by coupling it
to any system that produces FMNH2. Electrons for the
reduction of flavin mononucleotide (FMN) are provided by
the reducing power derived from the electron transport
pathway (75). The mechanism of the reaction leading to
light emission is shown schematically in Fig. 1. FMNH2
plus the luciferase results in luciferase-bound FMNH2.
Luciferase-bound FMNH2 reacts with oxygen to form the
4a-peroxyflavin intermediate. The intermediate reacts with
a long-chain aldehyde to form an excited species - the
flavin-4a-hydroxide (72). This compound is highly stable
and decays slowly resulting in the light emission and the
oxidation of the substrates (26).
THE ENZYME LUCIFERASE
Luciferase is the enzyme catalyzing the bioluminescent
reaction and it is linked to the respiratory pathway. It is an
external flavin mono-oxygenase or mixed function
oxygenase. All luciferases are heterodimers (77 kDa) con-
sisting of two nonidentical subunits a (40 kDa) and [3 (37
 BACTERIAL LUMINESCENCE
hv
NAD(P)H + H A NADPH + H T
FMN RCOOH
0X1DO- FATTY ACID
LUCIFERASE
REDUCTASE REDUCTASE
| I
NAD(P) .J^ FMNH„ RCHO V V
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Figure 1. Schematic representation of the biochemical pathways involved
in bacterial bioluminescence. [From Engebrecht el al, 1983 (11)].
kDa) (25,26). The active site is located primarily on the a
subunit, but the p subunit is still essential for the light-
emitting reaction and may contribute to the structure of the
active centre. Neither the a or p subunit alone exhibits
luciferase activity, but both preparations regain activity
when combined with the second subunit - leading to the
conclusion that an individual subunit does not appear to
possess an active centre. Studies indicate that the aldehyde
Journal of Food Protection 1992.55:62-70.
binding site is, like the FMNH2 binding site, at or near the
interface of the luciferase a and p subunits (75).
ALDEHYDE BIOSYNTHESIS
Aldehydes are essential in the bioluminescent reaction
and luminescent cells have a mechanism of synthesizing
aldehydes from fatty acids. A multienzyme fatty acid re-
ductase complex that produces aldehydes has been purified
and characterized from Photobacterium phosphoreum (25).
The net reaction:
R-COOH + ATP + NADPH
—-> R-CHO + AMP +PPi +NADP
is the reduction of fatty acid to aldehyde with the oxidation
of NADPH and the cleavage of ATP to AMP and PPi.
Maximum activity is obtained with tetradecanoic acid (25).
Two enzyme activities are necessary for the reaction to
occur. Acyl-protein synthetase is responsible for the ATP
dependent activation of the fatty acid, and a reductase
component catalyzes the NADPH-dependent reduction of
the activated fatty acid. Activation of the carboxyl groups
is catalyzed by the synthetase and results in an acyl-AMP
intermediate being formed. A NADPH linked reduction of
the acyl group to aldehyde is effected by the reductase
(25,26). Acyl-transferase is also present in the fatty acid
reductase complex and is involved in the generation of fatty
acids. Current data suggest that acyl-transferases are in-
volved in diverting fatty acids into the luminescence system
from the lipid biosynthesis pathway.
THE lux GENES
Escherichia colt cells have been transformed with
cloned genes coding for the luminescence functions and
expression has resulted in light emission. This has ad-
vanced understanding of the gene organization and regula-
tion of luminescence in the bacterial bioluminescence sys-
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
63
tern. There are seven genes and two operons required for
the complete Lux phenotype. Five of the seven genes are
" - " • , - ATP structural, coding for the luciferase and the fatty acid
reductase. The other two genes are regulatory, responsible
for producing an autoinducer and a receptor. The two
operons are transcribed in opposite directions. The right
L AMP+
ppi
+
NADP" 1
operon carries genes for the autoinducer, luciferase, and the
fatty acid reductase. The left operon carries the gene for the
receptor protein. Fig. 2 shows the organization of the
Vibrio fischeri lux genes.
Operon
Regulation Aldehyde Luciferase Aldehyde
-J I I l_
0 1 2 3 4 5 6 7 8 9
kbp
Figure 2. Organization of the Vibrio fischeri lux genes. [Modified from
Hastings el al, 1985 (13)].
Luciferase genes lux A and lux B
The a subunit is coded for by lux A and lux B codes
for the p subunit. Both genes are approximately 1 kbp in
length and are located immediately adjacent to one another
and are transcribed in the same direction. Aldehyde depen-
dent luminescence requires only a 2.5 kbp DNA fragment
with the lux A and B genes under a suitable promoter. Only
the luciferase genes are necessary for luminescence (25). It
is now known that lux A and lux B arose by gene duplica-
tion (6), and there is about 30% identity in amino acid
sequence between any luciferase a and p subunits regard-
less of bacterial species (4,14,38).
Fatty acid reductase genes lux C, D, and E
The genes coding for aldehyde synthesis, lux C, D, and
E, must also be transformed and expressed in E. coli to
obtain light emission without addition of aldehyde. The lux
C gene encodes for the reductase, the lux D gene encodes
for the acyl transferase, and the lux E gene encodes for the
acyl-protein synthetase (25).
Regulatory genes lux 7 and lux R
Two regulatory genes, lux I and lux R, involved in the
genetic control of luminescence, have been identified in V.
fischeri MJl (11,12). The genes lux F, lux G, and lux H
have recently been identified (36,37). Lux F codes for a
flavoprotein of about 24 kDa with no known function at
this time. Lux G and lux H are both 25 kDa proteins also
of unknown function. Meighen speculates on some possible
functions of these new proteins and also provides an up-
dated series of organizational maps (26). The in-depth
study of luminescent bacteria on a molecular level is
starting to result in exciting discoveries about these organ-
isms and the mechanisms responsible for their unique
characteristic.
 64 BAKER, GRIFFITHS AND COLLINS-THOMPSON
Regulation of luminescence
A significant portion of the cellular energy of biolumi-
nescent bacteria, (10% or more) can be utilized in produc-
ing light (11), and the expression of this phenotype is
tightly regulated by physiological factors.
Autoinduction. In 1977, Nealson (29) reported that the
synthesis of the luminous system of Photobacterium fischeri
(now V. fischeri) was stimulated, at the level of transcrip-
tion, by an autoinducer produced by the bacteria them-
selves. He also found that differences in light intensity were
accounted for by differences in the amounts of autoinducer
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produced. He noted a lag in luminescence and attributed it
to two factors. The first factor was a negative one, an
inhibitor that had to be removed. The second factor was a
positive one, the production by the bacteria of an autoinducer
stimulating the synthesis of the luminous system.
It was discovered later that the autoinducer for V.
fischeri is N-p-ketocaproylhomoserine lactone. Analogs of
this autoinducer could not stimulate luminescence in other
species (25) suggesting some degree of species specificity
in autoinduction.
With the added knowledge of the genes responsible for
luminescence, there is now a proposed mechanism for
Journal of Food Protection 1992.55:62-70.
autoinduction in V. fischeri involving both positive and
negative feedback regulation. The autoinducer would be
produced initially at a low constitutive rate as a product of
the lux I gene on operon R. When enough autoinducer has
accumulated during cellular growth, it would interact with
a receptor molecule encoded by the lux R gene on operon
L and activate transcription of operon R (the lux structural
genes). This results in the production of more autoinducer
and an exponential increase in synthesis of the lux en-
zymes. High levels of the receptor-inducer complex would
then turn off expression of the lux R gene. This explains
why luminescence becomes constant or declines at high
cell density. This model would predict that synthesis of the
autoinducer, as well as expression of the structural genes,
would increase during growth (13,25). In situations where
the autoinducer cannot accumulate, the bacteria will appear
very dim as found in organisms living free in the ocean
(26).
A recent study (27) showed that the structural genes
(lux ABCDE) of V. fischeri and V. harveyi are highly
conserved indicating that the light-emitting systems are
very similar in the two bacteria. However, it was also found
that the lux regulatory systems appear to have diverged. In
V. harveyi there was no open reading frame of greater than
40 codons within 600 bp of the start of lux C, which is
where lux I is located in V. fischeri. This led to the
conclusion that either the basic mechanism for the induc-
tion of luminescence or the location of the regulatory genes
in relation to the structural genes differs in the two Vibrio
systems.
Other regulatory controls. Catabolite repression par-
tially controls the bioluminescence expression systems of
V. harveyi and V. fischeri. Addition of glucose decreases
the expression of both luciferase and fatty acid reductase.
These functions are partially restored on addition of cAMP
(26). Arginine stimulates luminescence in V. harveyi. The
control of expression is at the transcriptional level (25).
JOURNAL OF FOOD PROTECTION, VOL. 55. JANUARY 1992
Oxygen is an important and variable factor with respect to
the expression of the luminescent phenotype. In some
species such as V. harveyi and P. leiognathi, lux gene
expression is inhibited by growth at low oxygen. Others
such as P. phosphoreum and V. fischeri exhibit stimulation
of the luminous system when grown in low oxygen (10).
Thus, when using the lux genes from these organisms in
recombinant studies, the oxygen levels should be monitored
to ensure that changes in luminescence result from the
condition or situation under investigation, not fluctuating
oxygen levels. As the bacterial luminescent reaction re-
quires oxygen, using this method to study anaerobic bacte-
ria is not feasible. The presence of iron inhibits luciferase
synthesis, and thus light emission, in all species of lumi-
nous bacteria studied (10).
INSTRUMENTATION
Light measurements from luminescent reactions (39)
and commercial luminometers have been reviewed (32,41).
The sensitivity of commercial luminometers has also been
assessed (17).
Most luminometers rely on sensitive photomultiplier
tubes for detection and amplification of photon emission.
The time course of the light emitted by an ongoing lumi-
nescent reaction is the physical property measured. Two
components of light reach the detector. One is proportional
to the amount of the limiting reactant in the luminescent
reaction. The other is a fairly constant level of light due to
outside causes including reagent impurities and plastic
phosphorescence. This background "noise" is much lower
than the luminescent component. Some luminometers are
quite sensitive, being able to detect amounts of ATP as low
as 100 femtograms in ATP bioluminescent assays (17).
However, extreme sensitivity may also lead to high stan-
dard errors when dealing with small cell numbers.
In a luminometer, the amount of light produced is
directly related to the total volume in the cuvette. This is
because the instrument reads light equally from all parts of
the cuvette. Another key parameter in luminometric mea-
surement is the rate of reaction of the production of light.
This rate is directly affected by the concentration of lumi-
nescent material in the sample. Diluting a sample slows the
rate of the luminescent reaction, thus reducing the light
output in photons per second. Measuring the light output
(rate of reaction) results in a lower number for the dilute
sample. The measurement of light emission is also affected
by turbidity and color of the sample.
Instruments in which photon detection and amplifica-
tion is achieved by avalanche photodiodes are also com-
mercially available. These instruments have a sensitivity
approaching photomultiplier-based luminometers but can
be made much smaller and are more rugged (<S). Newer
methods of measuring luminescent reactions involving pho-
ton counting cameras and charge-coupled device (CCD)
imaging, make low light level detection possible (15,16).
The CCD is a solid-state silicon detector operating on the
basis that a single photon incident on a silicon junction may
be absorbed and create a single electron-hole pair. The
 BACTERIAL LUMINESCENCE
CCD has a photosensitive area of 1 cm2. This area contains
light-sensitive elements called pixels defined by an array of
closely spaced electrodes. Photons liberate electrons which
then migrate towards the electrodes within each pixel. The
electrons accumulate in the pixels. After a specified time
the charges stored in each pixel may be read out serially by
a process called charge coupling. The charge from each
pixel may be used to create an analogue TV picture.
In an experiment done to assess the potential applica-
tion of CCD imaging in studying luciferase gene expression
in mammalian cells, a vaccinia virus recombinant contain-
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ing firefly luciferase genes was added to monolayers of
CV-1 cells (African Green Monkey kidney cells), and light
detection was measured by the CCD imaging method. After
only 6 h of infection, distinct light-emitting cells could be
visualized demonstrating the extreme sensitivity of this
method for the early detection of single foci of infection.
Data presented indicate that this is a rapid, non invasive,
sensitive method of detection which will offer a wide range
of potential applications to the study of biological systems
(15,16).
GENETIC TECHNIQUES USED TO
Journal of Food Protection 1992.55:62-70.
INVESTIGATE THE lux GENES
Several researchers (3,7,11,26) working on aspects of
the lux genes have used a variety of genetic techniques in
their studies. In order to study luminescence in nonlight-
emitting organisms, the genes responsible for luminescence
must be introduced into the dark organism. Transduction,
transformation, and conjugation are all suitable methods of
introducing DNA (40).
Transduction
Transduction involves the use of phages, which can
carry the luminescence genes. The phages do not possess
the intracellular biochemistry to express the genes but will
efficiently introduce these cloned genes into an appropriate
host. Bacteriophages usually have a narrow host range,
growing on some or all strains within a species. By care-
fully choosing a narrow host range phage, one can test for
the presence of specific bacteria. For example, phages
carrying lux genes whose host ranges cover all known
strains of E. coli may be prepared. On infection of the host
bacterium, the lux genes are transduced into E. coli which
has the necessary intracellular chemistry to express them.
Detection of this organism is, thus, indicated by the emis-
sion of light. This detection system is rapid and specific
(40). The difficulty with this methodology is in defining the
host range of the phage for different species. Too broad a
specificity could introduce the genes into species other than
those desired, leading to a false-positive response. Too
narrow a specificity may not transfer the lux genes into all
of the strains of the desired species giving a negative
response (26). Detection of 100-1000 E. coli cells can be
obtained within 1 h in milk or urine (40). Salmonella
species transduced by phages containing the lux A and lux
B genes could be detected within 1 hour of infection at cell
numbers as low as 10-100 (26).
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
65
Transformation and conjugation
Transformation involving plasmids to transfer the lu-
minescence genes into a host has also been successful (40).
These plasmids contain purified DNA comprising appropri-
ately modified bioluminescence genes. At high population
densities (107 to 108 cells per ml) cells of E. coli trans-
formed with specific plasmids exhibited light emission
within 2 to 5 h (40). However, the time of onset of
luminescence was inversely proportional to the cell concen-
tration when transformed with a constant amount of DNA.
Conjugation involves the transferring of DNA from one
bacterial cell to another by plasmids containing the transfer
genes. Transformation and conjugation are the methods
most often used in the transfer of the lux genes into various
gram-positive and gram-negative bacteria. Meighen has
summarized the organisms and methods used for the trans-
fer of lux genes (26). Ulitzer and Kuhn (40) found that
transformation or conjugation of lux DNA was approxi-
mately 1000-fold less sensitive for detection of bacteria
than was transduction.
Early experiments performed on the lux genes con-
sisted of isolating the genes in the marine organisms and
expressing them in E. coli in order to further study the
luminescent reaction.
Belas et al. (7) in 1982 isolated the lux A and lux B
genes from V. harveyi and transformed them on plasmids
pBB123 and pBB128 into E. coli in order to examine the
regulatory mechanisms of the genes. They found that light
production could be enhanced by coupling the lux genes to
strong promoter segments. The lux genes were in fact
downstream of two bacteriophage promoters. Due to this
increase in light emission, they felt that the limiting factor
for expression of luminescence in E. coli was the availabil-
ity of substrates - perhaps the reduced flavin. They also
realized the possibility of cloning the genes necessary for
the aldehyde synthesis in order to eliminate the need to
supply exogenous aldehyde.
Miyamoto et al. in a 1987 study (28) transformed the
genes for luciferase and the genes for fatty acid reductase
from V. harveyi into E. coli. Only 6.3 kbp of DNA was
transformed, which had insufficient coding capacity to
synthesize regulatory proteins similar to those found in the
V. fischeri system, thus showing that transcription of regu-
latory proteins is not required for expression of lumines-
cence. In past experiments, where V. harveyi DNA contain-
ing the luciferase genes had been cloned into E. coli, only
very low levels of light were observed. When cells that
expressed high levels of light were found, Miyamoto et al.
(28) investigated to see if expression of the luminescence
system arose from an alteration in the recombinant DNA or
in the E. coli cells themselves. They found that the E. coli
had mutated to allow expression of the V. harveyi system.
There was no difference in the recombinant DNA isolated
from the luminescent E. coli and the original recombinant
DNA. Thus, it was concluded that the host E. coli dramati-
cally affects the expression of the luminescence genes of
marine bacteria.
Expressing the bacterial luciferase genes in Bacillus
subtilis as well as E. coli was examined by Karp et al. (20).
Efficient expression of genes from gram-negative bacteria
 66 BAKER, GRIFFITHS AND COLLINS-THOMPSON
in gram-positive bacilli is not seen as due to more stringent
binding of ribosomes and/or to large heterogeneity in RNA
polymerase sigma factors recognizing different promoter
regions in these organisms. Solving these expression barri-
ers is partially accomplished by fusing foreign genes with
special vectors which have a strong Bacillus promoter and
Shine-Dalgarno sequence for ribosome binding.
Karp et al. (20) cloned genes encoding light production
from V. harveyi into a shuttle vector to examine expression
of lux genes in B. subtilis. They found that light emission
in B. subtilis was higher when the plasmid containing the
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lux genes had the correct binding site for B. subtilis. They
also stated that lux genes provided a better way to study
expression barriers in B. subtilis because other indicators
require cell disruption and enzyme assay steps. Further
work (9,79) showed that B. subtilis containing lux genes
was found to differ from E. coli in light emission charac-
teristics. Light emission in E. coli is rapid after aldehyde
addition and stays constant or rises as the cells continue to
grow, reflecting regeneration of substrates for the luciferase
reaction (19). In B. subtilis, after addition of the long-chain
aldehyde, there is immediate light emission - a peak -
which, depending on growth phase, slowly decays. The
Journal of Food Protection 1992.55:62-70.
decay may be due to the obligate aerobic nature of B.
subtilis. It appears that it does not have enough capacity to
regenerate FMN to its reduced form for the luciferase
reaction. However, other reasons may exist for the differ-
ences in light emission between E. coli and B. subtilis (9).
Two systems were developed by Baldwin et al. (5) to
allow quantitative determination of the efficacy of pro-
moter or terminator elements based on light emission. The
terminator screening vector placed the lux A and lux B
genes downstream from the promoter with the multiple
cloning site between the promoter and the lux genes.
Fragments inserted into the multiple cloning site containing
termination signals caused a reduction in light intensity in
cells carrying this plasmid. The promoter screening vector
is similar, but no promoter is present so that cells carrying
the plasmids are dark without an inserted promoter.
Two papers were published in the early 1980s (11,12)
dealing with the identification of the genes and their prod-
ucts responsible for bacterial luminescence. Various ge-
netic techniques were used in order to define and character-
ize the genetic information. Determination of the genetic
organization and definition of the lux functions was per-
formed by transposon mutagenesis. Transposons mutate by
insertional inactivation of the target gene causing loss of
function. Nonluminous and dim mutants were isolated and
transposon insertions were located by restriction mapping.
The regions encoding aldehyde, luciferase, and regulatory
functions lux genes A to E and lux I and lux R were
identified. These functions were assigned to the genes by
examining the phenotypes of mutants.
Very recent work (2) with lux genes has involved the
fusion of the lux A and lux B genes into a lux AB gene on
the plasmid pHG 165-3M. This fusion is necessary for
facilitated insertion of the genes into eukaryotic cells, since
expression in eukaryotes requires separate promoters before
each gene. This fusion is expected to lead to an increase in
applications for the luciferase genes in eukaryotic systems.
JOURNAL OF FOOD PROTECT/ON, VOL. 55. JANUARY 1992
It was found that the fusion of the genes resulted in a good
level of luciferase production in transformed cells (2). The
fused gene has successfully been expressed in both yeast
cells and Drosophila melanogaster cells (2). The amount of
light emitted by yeast cells (Saccharomyces cerevisiae) was
quite low (1% of the activity in cells without the fusion),
and this could be due to the fact that the supply of FMNH2
is extremely limited in yeast. The researchers felt that the
amount of light emitted from Drosophila cells should be
sufficient for many kinds of experiments with the organ-
ism. Generating in vivo luminescence is more difficult in
eukaryotes because there is limited availability of FMNH2,
although it appears that aldehydes can cross the cell mem-
brane (26).
The scope of work done on the lux genes from biolu-
minescent bacteria, from identifying the genes and their
products to inserting them into other organisms, all stems
from a desire to use the emission of light in a wide range
of applications. The assay is sensitive, quick, relatively
inexpensive, does not destroy cell integrity, and can be
made specific by using host specific vectors. For the above
reasons, coupled with the successes achieved in expressing
the genes in foreign hosts, a wide array of applications has
been, and still is being developed.
APPLICATIONS OF lux GENE TECHNOLOGY
Many different disciplines are investigating the use of
lux gene technology. Biomass assays are being developed
to monitor water quality and for clinical diagnosis of
bacteruria (31).
Work has been done to produce E. coli cells that emit
stable light thus allowing them to be used as biosensors
(22,23). Evidence has been produced that suggests this
new bioluminescent based sensing method could be devel-
oped for direct analysis of biodegradative microbial activity
in complex environmental matrices such as soil (21).
Ulitzer and Kuhn (40) have used lux genes to deter-
mine the antibiotic susceptibility of bacteria. E. coli cells
possessing the lux genes exhibited decreased light emission
in the presence of certain antibiotics. Suppression of lumi-
nescence depended upon the cell's susceptibility to and the
concentration of the antibiotic. This is of great importance
in diagnostic medical microbiology. A commercially avail-
able toxicity test, Microtox, based on bacterial biolumines-
cence has the potential of replacing animal testing.
Lux genes have been inserted into the broad host range
vector pUCD4 resulting in the recombinant plasmid
pUCD607. The recombinant plasmid was mobilized into
plant pathogenic bacteria. This allowed bacterial invasion
in host plant tissues to be quantified by light emission even
before the onset of visual symptoms (30). This may have
potential for monitoring storage life of fruits and veg-
etables. These are just a few examples of the many appli-
cations now being found for bacterial bioluminescence.
APPLICATIONS OF lux GENE TECHNOLOGY
IN FOOD MICROBIOLOGY
In a review of the potentials of in vivo biolumines-
cence in microbiology, Stewart states three components he
 BACTERIAL LUMINESCENCE
feels are necessary for novel developments in lux gene
technology (34). The first is the potential of a high level of
light output from individual cells. The second is the rapidly
expanding ability to transfer the biochemistry of light
production by the cloning of lux gene vectors into normally
dark microorganisms. The third component involves the
use of instruments originally designed for in vitro ATP
assays to allow the detection of light from only a few
hundred bacteria per ml. All of these are important in
implementing bacterial bioluminescent assays into the realm
of microbiological testing. Whereas a number of ATP
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luminescent techniques are presently being employed in
various aspects of food microbiology, the application of
bacterial bioluminescence is still on the research or proto-
type level. Many experiments have been, and are being
done, to determine the feasibility and usefulness of the
proposed assays. Some of the applications of use to the
food industry include the detection of specific bacterial
pathogens and indicator microorganisms, monitoring hy-
giene on-line, determining the effectiveness of spore de-
struction, monitoring starter culture integrity, biocide and
virucide challenges, and studying recovery of sublethally
injured cells.
Journal of Food Protection 1992.55:62-70.
Detection of specific bacterial pathogens and indicator
organisms
Presently in food microbiology laboratories, the mea-
surement of ATP by firefly luciferase is used to detect and
enumerate cells and has been used to assess shelf life and
the microbial quality of many kinds of foods (24,33). The
ATP assay is a rapid technique, but it lacks specificity for
identification of bacteria present. This specificity can be
achieved with lux genes from luminescent bacteria. As
discussed previously, lux genes can be introduced into
bacteriophages which will then absorb to specific bacteria
and transfer the light-emitting genes to those bacteria. By
knowing what type of bacteria one wishes to detect, it is
only a matter of obtaining the host specific phages for that
particular organism, performing the genetic manipulations,
combining the organisms with the phages, and measuring
the light emission, to obtain a quantitative assay of the
organism. Research has shown that the bioluminescent
assay is rapid (usually less than 1 h), sensitive [luminometers
can detect as few as 500 bacteria (35)], simple, specific,
and demonstrates a good correlation (linear relationship)
between cell numbers and bioluminescence.
The dark terrestrial organisms that need to be moni-
tored in food microbiology (pathogens, starter cultures,
hygiene indicators) lack the ability to produce luciferase or
fatty acid reductase, but they can supply FMNH2. There-
fore, all that is needed is the transfer of the genes for
luciferase and fatty acid reductase (35). In some cases only
the genes for luciferase are transferred and the aldehyde is
supplied exogenously as it can cross the cellular membrane.
By this means, one can confer bioluminescence on a par-
ticular set of bacteria growing in a complex mixture of
microbial organisms. Phage P22 is a narrow host range
phage infecting only Salmonella typhimurium. Stewart and
co-workers have constructed P22 containing lux genes. On
infection with this phage, as few as 100 cells of S.
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
67
typhimurium could be detected in 50 min. by monitoring
light expression (35). Lux containing bacteriophage should
be able to target other pathogens such as Campylobacter
spp. and Listeria monocytogenes. There may still be a need
for recovery and enrichment procedures prior to phage
detection, because of regulatory requirements, for example
zero tolerance for L. monocytogenes. However, due to the
sensitivity (100 per ml) and the specificity of the host and
phage, the enrichment time could be short providing simple
same day testing for pathogenic bacteria (34).
High numbers of nonpathogenic microorganisms present
within a food can indicate an increased probability of
pathogen contamination. Recombinant lux+ phages can de-
tect these indicator bacteria without recovery or enrichment
as long as they are in a food matrix at levels greater than
1000 per g. The assay takes only 30-50 min, thus allowing
for an index of food safety in less than 1 h (34).
Enteric bacteria are used in the food industry as indi-
cators of poor sanitary conditions. Lux genes have been
inserted into phages that infect a broad range of enteric
bacteria providing a reagent for an on-line hygiene test with
a detection limit of 1000 enterics per g (35). This test
would take less than an hour to perform, would not require
capital equipment, and could be operated by factory per-
sonnel. This would allow an accurate assessment of the
numbers of enterics in raw materials, the product during
manufacture, swabs from the floor and equipment, and the
end product. The test has shown promise in prototype form
(18,35). It would be particularly useful in the monitoring of
critical control points as part of the Hazard Analysis Criti-
cal Control Points (HACCP) system, whereby defined criti-
cal control points in a manufacturing process are to be
rapidly monitored by physical, chemical and microbiologi-
cal indicators. Presently, microbiological monitoring results
require from 8-24 h. As many hazards are microbiological,
it would be very advantageous to rapidly sample the micro-
bial flora. If these bacteriophages can be engineered to
provide a reagent capable of detecting changing levels of
organisms in an hour then perhaps on-line microbial testing
will become part of the HACCP system (35).
Sporeforming organisms and bioluminescence
Bacterial luciferase will function in a gram-positive
organism although it is originally from gram-negative or-
ganisms. The lux genes must be coupled to appropriate
gram-positive promoters and must be engineered to obtain
stable insertion via gram-positive replicons (regions con-
taining DNA necessary for the initiation of DNA replica-
tion) (35). Bacillus spp. are capable of producing heat
stable dormant endospores. The spores obtained from phe-
notypically bioluminescent vegetative cells are dark. This is
expected because the spores show no detectable metabo-
lism, ATP or electron transport, thus having no energy to
drive the light reaction. When the spore germinates, the
onset of electron transport and the initiation of metabolism
are early events. For lux containing spores, germination is
accompanied by the emergence of bioluminescence. This
provides a sensitive, real-time monitor of the germination
and outgrowth process (35).
 68 BAKER, GRIFFITHS AND COLLINS-THOMPSON
Spores that have been killed or injured, and are thus
unable to germinate, produce no light. Therefore, those
chemical and physical processes designed to eliminate
bacterial spores could be monitored within 1 h in a
luminometer assay. Researchers feel that a rapid biological
indicator for thermal processing or sterilizer efficacy moni-
toring could be available in the near future (35).
A similar approach for studying germination of spores
of Clostridium spp. would be advantageous. However,
problems may be encountered due to the anaerobic nature
of the organism.
Downloaded from jfoodprotection.org by WDAS Country Access Consortium on 01/06/20. For personal use only.
Lactic acid bacteria and starter cultures
A plasmid based replicon pSB 154 has been engineered
to introduce bioluminescence into a wide range of lactic
acid bacteria including Lactococcus lactis, Lactobacillus
casei, and Lactobacillus plantarum. Only the lux A and lux
B genes are expressed, so aldehyde must be supplied for
bioluminescence (35). Levels of light emission from these
organisms are less than those from the enteric bacteria
recombinants, but it is possible to monitor in real-time
concentrations of 106 bacteria per ml. Further research is
expected to result in improvements in quantum output, but
Journal of Food Protection 1992.55:62-70.
useful reagents have been developed. Many of these assays
have a relation to the dairy industry. The presence of
bacteriophages or antibiotics can cause starter culture fail-
ure in cheese or yogurt manufacture. These agents impair
growth of the starter and allow contaminating microorgan-
isms to proliferate. Dye reduction tests are used currently to
evaluate antimicrobial activity in milk, but these require
several hours of incubation before a result is obtained.
Bioluminescent lactic streptococci are suited for use as
indicators of the presence of lytic phages or antibiotics.
This light-emitting component of the starter culture can be
monitored for a loss of luminescence that indicates the
presence of an inhibitory substance (26,34,35). Using a
bioluminescent derivative of Lactobacillus casei, the tech-
nique allowed detection of penicillin G down to levels of
0.03 |Xg/ml (0.05 units/ml) in 30 min and bacteriophages at
concentrations as low as 105/ml in 100 min (1).
Certain species of lactic acid bacteria are currently
being investigated as probiotics in the control of intestinal
flora in poultry and pigs. Bioluminescent bacteria could
play a role in assessing the colonization and attachment to
the gut epithelium of organisms such as Lactobacillus
ingested live at substantial levels (35).
Effectiveness of biocides and virucides
Biocides are used for microbiological control in manu-
facturing, environmental, engineering, service, and other
industries as preservatives or disinfectants (J8). Material
such as food waste, organic soil, dust and other organisms
can neutralize biocide activity without reducing apparent
chemical activity (35). For this reason, monitoring of bio-
cide levels is essential to provide adequate protection.
Current testing techniques (dipslides, dye reduction tests,
microbiological challenge tests) require in excess of 18 h
and are thus not suitable for on-site evaluation. Biolumines-
cent microorganisms offer the potential for rapid biocide
testing. The test takes only 10-15 min and gives a good
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
correlation with the viable count evaluation (18,34). Since
dead cells produce no light, the biocidal effect is measured
as a decrease in the light output of the bioluminescent
culture. These results may lead to rapid on-site evaluation
of biocide efficacy in process water control and water-
holding tanks where bacteria are known to proliferate
(34,35). This method has been used to test the effectiveness
of certain biocides on L. monocytogenes with good results
after 15 min exposure to the biocides (3). The genes for
luminescence have been introduced into L. monocytogenes
cells by transforming them with a recombinant plasmid.
The plasmid (pSB292) is a broad host range gram-positive
vector which replicates in L. monocytogenes and thus
provides a means for incorporation of the lux genes into the
Listeria cells. It was demonstrated that bioluminescence
closely correlates to cell growth of L. moncytogenes until
the approach of stationary phase when bioluminescence
begins to decay (3).
Assessing antiviral activity usually requires cell culture
work or electron microscopy facilities (18). A rapid biolu-
minescent test has been developed by inserting lux A and
lux B genes into Lambda phage. After exposure to hypo-
chlorite (virucidal agent), the lux* phages were unable to
infect a nonbioluminescent E. coli and so did not elicit a
bioluminescent response. Untreated lux* phages did pro-
duce a bioluminescent phenotype in E. coli even after the
E. coli had been treated with hypochlorite. This demon-
strates the potential of using lux* phages to screen for
virucidal activity.
Recovery of sublethally injured cells
Sublethal injury refers to the state where a microorgan-
ism is neither actively growing or biochemically dead.
Microorganisms in this state may arise in a food after
heating, chilling, freezing, moisture reduction, irradiation,
or exposure to preservatives. Sublethally injured cells can
retain their pathogenic traits which makes enumeration of
these cells during microbial analysis very important. They
usually need a period of resuscitation in a nonselective
medium for intracellular repair (18). For Salmonella, an
overnight resuscitation in an appropriate preenrichment
medium is needed before any attempt to determine its
presence is made (35). This compromises efforts to reduce
the time for bacterial detection and enumeration based on
new rapid technologies. Protocols used currently to monitor
recovery are based on observing colonies obtained under
different resuscitation conditions and overnight incubation
(18). Researchers now believe that in vivo bioluminescence
may provide a real-time tool for probing the recovery of
microorganisms from sublethal injury. This injury affects
the quantitative efficiency of in vivo bioluminescence. It is
possible with sensitive luminometers to watch the recovery
of sublethally injured cells to optimum bioluminescence
without having to observe a single cell division. Work done
indicates that three hours into the recovery period the
viable count data and bioluminescence reflect near perfect
correlation (18). Different protocols for recovery can be
assessed with great sensitivity both to optimize chemical
conditions and to reduce the time required (35): soon it
should be possible to investigate the effect of media,
 BACTERIAL LUMINESCENCE
temperature, and other environmental parameters on recov-
ery in vivo (18). Recovery from sublethal stress is an
emerging concern in food microbiology. Any method to
advance its understanding should be pursued with vigor.
Stewart (34) deals with how in vivo bioluminescence
can be a reporter of microbial stress by acting as a reporter
of gene expression. The luciferase expression is a very
versatile reporter gene. It does not require any special
genotype in the host strain in order to obtain expression
data and the response is obtained in real time without cell
disruption. This is important when exploring cellular re-
sponse to environmental stimuli. Another application inves-
Downloaded from jfoodprotection.org by WDAS Country Access Consortium on 01/06/20. For personal use only.
tigates the capacity of cells to supply high energy bio-
chemical intermediates (i.e., FMNH2) during or after some
type of cellular stress. Stress responses that affect the
production of intracellular FMNH2 can be monitored in lux
recombinant bacteria as changes in light output per cell
(34). For example, using bioluminescent recombinant strains
of S. typhimurium, workers have shown that 20% of the
population could survive a freeze/thaw cycle with a bio-
chemical system sufficiently intact to permit immediate and
sustained bioluminescence (34). By classical plate tech-
niques it was found that only 2.5% of the original inoculum
remained viable. This has important implications for the
Journal of Food Protection 1992.55:62-70.
food industry as these results suggest that appreciable
numbers of toxigenic cells (e.g., S. aureus) that are not
considered viable by conventional enumeration techniques
may continue to produce toxigenic compounds after pro-
cessing.
The monitoring of bioluminescent cells exposed to
different stresses may find application in the field of pre-
dictive microbiology. Trying to predict how an organism is
going to behave in a certain environment is not an easy
task, though many are pursuing it. By inflicting a variety of
stresses on different components of the cell's metabolism
and then monitoring light expression, one can see what
conditions are harmful or beneficial to the organisms.
ADVANTAGES AND DISADVANTAGES
OF THE BACTERIAL LUNINESCENT SYSTEM
IN FOOD MICROBIOLOGY
The advantages of assays performed with bacterial
luminescent genes have been repeated throughout this re-
view. The assays are rapid — fast enough for on-line
assays in situations where on-line monitoring was impos-
sible previously, and also fast enough for assays that
require immediate action, such as the detection of antimi-
crobials in milk. The rapidity may also in some cases
reduce costs as well. For example in testing the efficacy of
virucides, there would be no need for expensive tissue
culture supplies.
The method is simple enough to operate or use so that
plant workers could perform the test. It is a sensitive
method - very low numbers of cells can be detected with
luminometers and continued research may lower those
numbers even further. In most cases it is advantageous that
the in vivo bioluminescence assay is noninvasive. No cell
disruption is required to measure the light emission. It has
been shown to be an accurate method exhibiting good
JOURNAL OF FOOD PROTECTION, VOL. 55, JANUARY 1992
69
correlation with cell counts - both when increasing and
decreasing in numbers. When using narrow range phages to
introduce the lux genes, the assay can be specific to detect
a particular microorganism. A final advantage is that the
technology of bacterial bioluminescence is leading to a
great deal of innovation in many areas. New ideas are
emerging that were not thought possible before, such as
actually watching the recovery of an injured cell.
As with any new technique, the researchers developing
the technology are slow to mention too many disadvan-
tages. As mentioned in Meighen's review (26), there could
be a problem with phage or plasmid host ranges being
either too specific or too broad, resulting in false negatives
or false positives, respectively. More research into phages
and plasmids and their host specificity should rectify this
problem. Bioluminescent cells have been detected in milk,
but one can envisage a setback with solid foods, in that any
particulate matter may decrease the efficiency of gene
transfer within the food homogenate. Sample pretreatment
such as filtration may be a solution to this problem. How-
ever, further research needs to be done in this area.
A disadvantage for workers in the food microbiology
industry is that the bacterial bioluminescent assays are still
at the experimental and prototype stages.
SUMMARY
A great deal of research has been done in the last 10
to 15 years to investigate the mechanisms whereby marine
bacteria bioluminesce. From an initial understanding of
their biochemistry and the substrates required for light
emission, to investigation of their molecular biology and
defining what genes code for what enzymes, to genetically
engineering otherwise dark microorganisms to become light
emitting, these organisms have become very well defined.
Along with the basic scientific investigation of these organ-
isms, many applications of their luminescence are being
realized, thus leading to further research on these unique
microorganisms. The many advantages of assays using
bacterial luciferase should increase their use, especially in
food microbiology, once they become commercially avail-
able. These assays will not eliminate classical microbiology
methods, but they will further the influence of microbial
assays into areas such as the HACCP system where rapid
results are necessary. Advancements in genetically engi-
neering microorganisms to become bioluminescent coupled
with a desire for rapid microbiological assays by the food
industry will make in vivo bioluminescence a common
practice in the future. Bioluminescence coupled with low
level light detection systems such as photon counting and
CCD imaging (techniques capable of visualizing biolumi-
nescent single cells on food surfaces) will provide the food
microbiologist with a very powerful tool for studying food
as an ecosystem and will offer a unique opportunity to
study microbial behavior and interactions in food.
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