Detection of Luciferase Gene Sequences in \'Visibly Non-Luminous\'
Bacteria from the Chesapeake Bay^^

Nagappa Ramaiah^1^, Jacques Ravel, Jongsik Chun^2^, William L. Straube,

Russell T. Hill and Rita R. Colwell\*

*Center of Marine Biotechnology, University of Maryland Biotechnology
Institute,*

*701, East Pratt Street, Baltimore, MD 21202*

Key words: Bioluminescent bacteria; *lux* gene sequences; Chesapeake
Bay; DNA probes

1\. ABSTRACT:

A 745 bp *lux*A fragment was amplified from *Vibrio harveyi* (UM 1503),
radiolabeled, and used as a probe to detect and quantify *lux*A
genotypes in culturable bacterial populations from the Chesapeake Bay.
DNA samples from 55 reference strains were also examined for this gene.
While the *lux*A -positive bacteria were at the most 6% of the
culturable heterotrophic bacterial community in the Bay, only those
reference strains known to be luminescent contained the *lux*A gene, as
indicated by PCR. Results in all cases were confirmed by PCR of DNA
extracts and Southern hybridization analyses using an internal probe for
*lux*A amplification products. We examined *lux*A encoding regions from
these environmental and many laboratory strains through an analysis of
their sequences. These sequence analyses (of the DNA samples positive
for this gene) from the visibly non-luminescent bacteria suggested least
or no difference in their homology with *lux*A sequences from the known
marine luminescent bacterial species. Our results indicate that *lux*A
-positive bacteria isolated from the Chesapeake Bay are not generally
luminescent on phenotype enumeration, implying that gene probe
techniques are required for examining *lux*A gene distribution in
environmental samples.

^1^Present Address: National Institute of Oceanography, Dona Paula, Goa
403 004, India

^2^Present Address: Korean Collection for Type Cultures, Korea Research
Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong Taejon
305-600, Republic of Korea

\*Correspondence to: R. R. Colwell, Center of Marine Biotechnology,
University of Maryland Biotechnology Institute,* *701, East Pratt
Street, Baltimore, MD 21202

^^Contribution No. \_\_\_ from the Center of Marine Biotechnology,
University of Maryland Biotechnology Institute and No. \_\_\_\_ from the
National Institute of Oceanography.

2\. INTRODUCTION:

Bioluminescence is a characteristic of a few genera of marine and
estuarine bacteria that are extensively studied with regard to their
taxonomy, ecology and phylogeny \[1-5\]. The well studied bioluminescent
bacterium, *Vibrio fischeri* \[6, 7\] is a symbiont colonizing light
organs in several marine fish and squid species. Strains of these
species are divided into visibly luminous and non-visibly luminous
classes based on differential light production on laboratory media. Only
the non-visibly luminous class is proficient at colonizing the light
organ of the squid, *Euprymna scalopes* under natural conditions where
both classes co-occur in the water column \[8\]. Visibly and non-visibly
luminescent strains of *V. cholerae* were reported by Palmer and Colwell
\[9\]. They observed that most non-visibly luminescent strains were
found to contain luciferase gene sequences. In general, however, counts
of bioluminescent bacteria in marine and estuarine waters have been
based on visual detection of luminescence and omitted dark variants.
Marine luminous bacterial numbers enumerated in this manner range from
nondetectable levels to over 20% of the culturable populations \[2, 4,
5, 10\]. The number of bacteria capable of expressing bioluminescence
under appropriate environmental conditions is likely to be considerably
higher than the number of visibly luminous bacteria. Cell density
dependent autoinduction of bioluminescence has been reported in
laboratory strains \[11\]; this and other induction methods may be
important under environmental conditions.

Genes encoding and subunits of bacterial luciferase have been cloned
\[12, 13\] and expressed \[14\]. Such studies made it possible to use
specific sequences from these genes as hybridization probes to identify
bacterial species that contain luciferase genes \[5, 15, 16\] using
molecular methods rather than depending on enumeration of visibly
luminous bacterial colonies. Wimpee et al. \[16\] developed species
specific *lux*A probes and examined their specificity by hybridization
studies with bacteria isolated from environmental samples. The present
study aimed at enumeration of those bacteria potentially capable of
expressing bioluminescence in environmental samples. Molecular detection
of gene sequences homologous to the *lux*A gene of *Vibrio harveyi* by
PCR and hybridization was used to detect and count visibly luminous as
well as dark variants from environmental samples and in a collection of
laboratory strains, particularly from the genus *Vibrio*.

3\. MATERIALS AND METHODS:

*3.1 Stock cultures and growth conditions*

Bacterial species studied are listed in Table 1. Stocks of these
cultures were subcultured on Luria agar and grown overnight at 30 ºC.

*3.2. Extraction of DNA*

Genomic DNA was extracted following a standard method incorporating a
hexadecyl trimethyl ammonium bromide (CTAB) treatment step \[17\].
Briefly, overnight grown cultures on a shaking incubator at 37 ºC in 100
ml Luria broth were harvested by centrifugation at 4000 x g. Cell
pellets were resuspended in 9.5 ml tris-EDTA (TE) buffer, 0.5 ml 10%
sodium dodecyl sulfate (SDS), and 50 µl (20 µg.ml^-1^ stock) proteinase
K and incubated for 1h at 37 ºC. 1.8 ml of 5M NaCl and 1.5 ml of 10%
CTAB in 0.7 M NaCl was added and the mixture incubated at 65 ºC. DNA was
purified by a standard phenol-chloroform extraction \[17\], precipitated
and treated with RNAase (DNAase-free) (Boehringer-Manheim, Indianapolis,
Ind.).

*3.3 Synthesis of PCR primers*

PCR primers for amplification of *lux*A gene fragments based on those of
Wimpee et al. \[16\] were: the forward primer,
5\'-CTACTGGATCAAATGTCAAAAGGACG-3\' and the reverse primer,
5\'-TCAGAACCGTTTGCTTCAAAACC-3\'. An internal probe with the sequence,
5\'-ATAAAGGTCAATGGCGTGATTTTG-3\' was used as to confirm identity of
amplification products. Oligonucleotides were synthesized using an
Applied Biosystems 380A Automated DNA Synthesizer, purified,
lyophilized, resuspended in ultrapure water, and concentrations
determined by UV spectrophotometry.

*3.4 PCR amplification*

PCR amplification of the *lux*A region was performed as described by
Wimpee et al. \[16\]. PCR products were resolved by agarose gel
electrophoresis.

*3.5 Probe preparation and labeling *

The *lux*A internal oligonucleotide probe was end-labeled with \[
-^32^P\] dATP following the protocol in Ausubel et al. \[17\]. Labeled
probe was used to confirm the identity of *lux*A fragments by Southern
hybridization analysis.

The *lux*A probe for colony and dot-blot hybridizations was prepared by
radiolabeling PCR amplification products. The 745 bp *lux*A PCR fragment
was resolved by low melting temperature agarose (NuSeive, FMC) gel
electrophoresis, visualized by a short exposure to longwave UV, excised
from gel, and purified using a Wizard PCR Preps DNA Purification System
(Promega Corp., Madison, Wis.), following the protocol provided by the
manufacturer. PCR fragment was radiolabeled with \[-^32^P\] dCTP by nick
translation using a kit supplied by Boehringer- Manheim (Indianapolis,
Ind.).

*3.6 Isolation and enumeration of bacteria*

Water samples were collected in the Chesapeake Bay during March and
August 1995 and November 1997. Bacterial counts in samples collected
during August 1995 included determination of total direct counts (TDC)
by acridine orange staining and epifluorescence microscopy according to
the method of Hobbie et al. \[18\], nucleoid-containing cells (NUCC) as
described by Zwiefel and Hagstrom \[19\], direct viable counts (DVC) by
the method of Kogure et al. \[20\], and colony forming units (CFU) on
TSA (1/10 strength, 1% NaCl wt/vol final concentration \[21\]).

*3.7 Colony hybridization *

Water samples were collected at four depths from four sampling stations
located at latitudes 37 24', 38 18', 38 45' and 38 58' N on the mid-axis
of the Bay. Four to five replicates of appropriate dilution of these
samples were spread plated on 1/10 strength trypticase soy agar (Difco).
Plates were incubated at 25 ºC until colonies had formed, usually after
about 30 h. Colony-forming units (CFU) from these samples were
enumerated and all plates with 150-200 colonies were selected for colony
blotting. Colonies were lifted onto Magna nylon membranes (Micron
Separation Inc., Westboro, Mass.), for probing for *lux*A signatures.
For evaluating specificity of *lux*A probe generated by PCR, about 650
individual purified strains isolated from water samples collected from
the surface and near the bottom at two locations in the Bay during March
1995 were used. Using sterilized toothpicks, fifty each of these
cultures were spotted per TSA plate (1/10 strength with 1% NaCl) to
prepare nylon membrane-bound DNA for colony hybridization.

Membranes were prepared as follows: lysis (10% SDS for 3 min);
denaturation (0.5M NaOH and 1.5M NaCl for 5 min); neutralization (0.5 M
Tris HCl, pH 8.0 and 1.5M NaCl for 5 min); rinse with 2X SSPE; and
baking at 80 ºC under vacuum for 2 h. Membranes were placed at 65 ºC in
prehybridization solution (5X SSC, 10X Denhardt\'s solution and 500 µg
Salmon Sperm DNA per ml). After prehybridization for 2 h, hybridization
was carried out at 65 ºC overnight. Washing conditions were as follows:
two washes in 1x saline sodium citrate (SSC), 0.1% SDS for 15 min each
at room temperature, two washes with 1x SSC, 0.5% SDS for 20 min each at
37 ºC, two washes with 1x SSC, 0.5% SDS of 30 min each at 55 ºC; and 1
wash with 1x SSC, 0.5% SDS at 65 ºC for 1h. Membranes were exposed at
-70 ºC for 1-8 h using X-Omat AR film.

*3.8 Sequencing of *lux*A amplicons*

A single fragment of the expected size of 750 bp was amplified from the
DNA of all luminescent control strains and from the DNA of a few visibly
non-luminescent environmental samples. In addition to confirming the
identity of PCR amplification products by hybridization to a ^32^P
labeled *lux*A derived oligonucleotide, seven of these *lux*A PCR
products were selected for sequencing studies. PCR products were
purified by agarose gel electrophoresis in low melting point agarose,
single bands were excised and recovered using a Wizard kit (Promega
Corp., Madison, Wis). PCR products were directly sequenced using dye
terminator cycle sequencing reactions run on an ABI 373 automated
sequencer (Applied BioSystems). The resulting sequences were aligned
using PILEUP program in the University of Wisconsin Genetics Computer
Group (Madison, Wis.) software package. After manual adjustments, the
alignment was subjected to the phylogenetic analysis. Phylogenetic trees
were inferred by using the Neighbor-joining \[22\], Fitch-Margoliash
\[23\] and maximum parsimony \[24\] methods. Evolutionary distance
matrices were generated according to Dayhoff \[25\] for amino acid and
according to Jukes and Cantor \[26\] for nucleotide sequence,
respectively. The PHYLIP package \[27\] was used for all analyses. The
resultant unrooted tree topologies were evaluated using bootstrap
analyses \[28\] of the neighbor-joining method based on 1000
resamplings.

4\. RESULTS:

*4.1 Detection of *lux*A in reference strains*:

Of the 55 reference strains examined in this study, only those species
recognized as luminous (*Vibrio fischeri* UM 1373; *Vibrio harveyi* ATCC
14216, UM 1503 and an environmental strain, UM BB7, and *V. cholerae*
ATCC 14547) had *lux*A gene detectable by the PCR primers we used (Plate
I). The identity of PCR products was confirmed by hybridization with
*lux*A- specific internal probe. Results were further confirmed by
probing dot-blots of DNA from reference strains with a *lux*A gene probe
amplified from *Vibrio harveyi *UM1503 *lux*A gene by PCR.

*4.2 Enumeration of *lux*A- positive colonies in Chesapeake Bay water
samples*:

Pure cultures of bacteria isolated from water samples collected from the
surface and near the bottom at two locations in Chesapeake Bay during
March 1995 (stations 724 and 858; Table 2 ) were used for testing the
specificity of the *lux*A gene probe amplified from *Vibrio harveyi
*UM1503 by PCR. Of the 650 isolates tested, only 18 (less than 4%) were
*lux*A- positive. These results were confirmed by PCR analysis of five
probe positive and five probe negative colonies. Probes were used for
enumerating *lux*A genotypes isolated from water samples collected at
different locations in Chesapeake Bay (Table 2). For each sample,
approximately 400 (784 in November 1997; Plate II) colonies were tested.
While there was no depthwise variation discernible at any location,
*lux*A - positive bacteria usually comprised less than 5% of the
culturable bacteria. No luminous colonies were visible upon examination
of colonies in the dark. To confirm these results, additionally five
each of probe- positive and probe-negative isolates were examined by PCR
and by dot-blotting techniques. Cell suspensions of probe positive
isolates were checked for luminescence in a luminometer.

*4.3 Bacterial counts*:

There was no depth or location-wise variation discernible (Table 2) in
either total direct counts (TDC), direct viable counts (DVC) or
nucleoid-containing cells (NUCC). TDC ranged from 1.338 x 10^6^ to 6.383
x 10^6^ ml^-1^ and NUCC, varying from 3.75 x 10^5^ to 8.08 x 10^5^
ml^-1^, comprised 5. 9 - 22% of TDC. DVC were usually less than NUCC and
ranged from 2.7 - 10.7% of TDC. Colony forming units (CFU) were quite
low ranging from 1120 to 3940 ml^-1^. These CFU accounted only for
0.02 - 0.08% of the TDC.

*4.4 Sequence data analysis*:

Seven *lux*A sequences determined in this study were aligned with
published sequences. Both nucleotide and amino acid sequences for each
of these strains was deduced by aligning them to the amino acid
sequences of *Xenorhabdus luminescence*. The resultant multiple
alignment of nucleotides and amino acid sequences is shown in Figs. 1
and 2. Nucleotide and amino acid sequences similarity/differences are
listed in Tables 3 and 4. Fig 3 depicts the *lux*A phylogenetic trees
from sequences currently available. These phylogenetic trees were
inferred using both *lux*A nucleotide and amino acid sequence data (Fig.
3a, b). In addition, the corresponding phylogenetic tree based on 16S
rRNA was generated using published data for comparative analysis (Fig.
3c) as 16S rRNA sequences of most test strains are available in the
ribosomal database project and GenBank databases. The phylogenies were
consistently similar for both amino acid and nucleotide trees. The 16S
rRNA tree was remarkably consistent as the same tree topology was
recovered from all three tree-making algorithms and supported by high
bootstrap values through branching points (Fig. 3c). The phylogenies
based on *lux*A and 16S rRNA sequences are generally similar. Alignment
of *lux*A sequences from all seven strains we examined suggested a very
high degree of similarity/realtionship with other available sequences.
Sequences from two visibly non-luminescent strains, 81 and 103 from the
Chesapeake Bay were very similar to the sequence from *V. cholerae* ATCC
14547. The sequence from strain 329 had a very close match with that of
*lux*A gene sequences from *V. harveyi* UM 1503. The *lux*A from these
two showed about 98% similarity. The *lux*A from *V. fischeri* UM 1373
differed by 33% from all other sequences. There is a difference of one
amino acid between *V. cholerae* ATCC 14547 and strain, env 103 and no
difference between *V. cholerae* ATCC 14547 and strain env 81. Also, as
was the case with nucleotide sequences similarity, the *lux*A amino acid
sequence of strain env 329 and the environmental strain of *V. harveyi*
are closer to each other than to *V. harveyi* ATCC 14126.

Non visibly luminous strains positive for *lux*A were grown in full
strength TSB (prepared with 3% \[w/v\] Instant Ocean) containing the
light stimulating agents such as arginine (100 µg.ml^-1^), glycerol (30
µl.ml^-1^) and n-decyl aldehyde (2 µl.ml^-1^) individually and in
combination to check if it was possible to obtain light emission in
these strains. There was no visible luminescence in any of these
strains. Also, non-visible luminescence was not detected when overnight
cultures of all these strains were examined in a luminometer (Dyna Micro
2000).

5\. DISCUSSION:

There have been limited observations on the occurrence and distribution
of bioluminescent bacteria, particularly from the northern/ upper zones
of the Chesapeake Bay. During this study, none of more than 12,000
bacterial colonies (*i.e.*, all colonies that were formed on TSA plates)
examined for bioluminescence emitted visible light. Since our sampling
from the Bay was conducted during spring, summer and fall, it is likely
that there is low incidence of bacteria exhibiting visible
bioluminescence in the Chesapeake Bay throughout the year under
different conditions of temperature and salinity. Several studies have
noted a wide tolerance for salinity fluctuations among luminous
bacterial species \[29 -- 31\] indicating that salinity is unlikely to
be the parameter determining the lower incidence of luminous bacteria in
the Chesapeake Bay.

Molecular methods were used to determine whether genes encoding
luciferase, with homology to the *lux*A gene from *V. fischeri*, were
present in bacteria from the Chesapeake Bay. Positive results were
obtained by PCR analysis with *lux*A- specific primers and an internal
probe. A total of 136 (ca. 1.8%) of over 7500 colonies examined by probe
hybridization method were *lux*A- positive. These *lux*A -positive
bacteria in environmental samples were not bioluminescent by phenotypic
examination, or when bioluminescence was checked using a luminometer.
The incidence of luminescent bacteria found in the Chesapeake Bay water
samples was lower than that found by Kaneko and Colwell \[32\] in
samples from its lower reaches. However, many of the cell counts of
bacteria, including the CFU, observed during this study are not very
different from the previously reported ones \[32\]. The NUCC, examined
for the first time from the Bay, do suggest that up to 20% of the TDC in
many samples are 'dead' (metabolically inactive) bacteria.

None of the characterized strains from culture collections, known as
non-luminescent species, examined in this study by PCR was positive for
*lux*A. DNA of all known luminescent strains examined in this study
(*Vibrio fischeri*, *V. harveyi*, and *V. cholerae*) hybridized with our
*lux*A probe, confirming its specificity and the potential value of a
probe -based approach for enumeration of *lux*A -positive bacteria in
environmental samples.

Wimpee et al. \[16\] hypothesized that many bacteria possess *lux*A
sequences. Palmer and Colwell \[9\] detected *lux*A genes in many
visibly non-luminescent *Vibrio cholerae* strains by PCR. Lee and Ruby
\[8\] detected and enumerated non-luminescent *Vibrio fischeri* by using
a hybridization probe. In this study the absence of visible luminescence
in the environmental strains that were positive for *lux*A by both PCR
and hybridization was confirmed both by biochemical assays and by
luminometry. Sequence analyses confirmed that the strains containing
genes homologous to *lux*A were taxonomically identical either to
*Vibrio harveyi* (strain \# 329) or to *V. cholerae* (strains \# 81 and
103). The sequences of *lux*A indicated that no major changes had
occurred in the *lux*A genes among non-visibly luminous (or,
nonluminous) bacterial strains carrying this gene. This suggests that
lack of expression of *lux*A, rather than structural changes within the
*lux*A genes was resulting in the non-luminescent phenotypes. However,
the biochemical pathway of light emission is quite complex \[33, 34\]
and it is possible that alterations or deletions had occurred in other
genes in this pathway, leading to dark variants of these species as has
been previously noted in some laboratory \[35\] and environmental
strains \[31\].

Quantitative determinations -including molecular probe based ones- of
ubiquitous marine luminous bacteria is well served when one links the
usefulness of quantitative data on this highly nutritionally versatile,
phylogenetically clustered group with that of the ecological and
metabolic processes of the general heterotrophic communities in the
marine ecosystems. Results of this study confirm the utility of
PCR-based probes for detection of *lux*A- positive bacteria in
environmental samples. These observations further demonstrate that these
bacteria are generally not luminescent by phenotypic examination
employing standard methods (*e.g*., screening for luminescence in plate
and broth cultures in a darkened place or in a luminometer) for
enumerating them. Specific environmental conditions appear to be
required for expression of the luminescent phenotype that may not be met
in laboratory culture.

ACKNOWLEDGMENTS:

We thank Anwar Huq, Eric Wommack, Christopher Grim and Maria Matte for
assistance and helpful suggestions and the crew of RV CAPE HENLOPEN for
assistance in collecting samples. N. R. is grateful to the Department of
Biotechnology (Ministry of Science and Technology), New Delhi, for an
award of Overseas Associateship and to Dr. D. Chandramohan at the NIO
for encouragement and support. N. R. also acknowledges funding from a
UNESCO- American Society for Microbiology Travel Award.

# REFERENCES

\[1\] Nealson, K. H. and Hastings, J. W.** ** (1979). Bacterial
bioluminescence: its control and ecological significance. Microbiol.
Rev. 43, 496‑ 518.

\[2\] Orndorff, S. and Colwell, R. R. (1980). Distribution and
identification of luminous bacteria from the Sargasso Sea. Appl.
Environ. Microbiol. 39, 983‑987.

\[3\] Ramaiah, N. and Chandramohan, D.** **(1988). Distribution of
luminous bacteria and bacterial luminescence in the equatorial region of
the Indian Ocean. Microbiologica 11, 243‑254.

\[4\] Ramaiah, N. and Chandramohan, D. (1992). Ecology and biology of
luminous bacteria in the Arabian Sea, in Oceanography of the Indian
Ocean, Desai, B. N. (ed), pp 11-23, Oxford‑IBH, New Delhi.

\[5\] Ruby, E.G. and Nealson, K.H.** ** (1978). Seasonal changes in
species composition of luminous bacteria in nearshore waters. Limnol.
Oceanogr. 23, 530‑533.

\[6\] Nealson, K. H. and Hastings, J. W. (1992). The luminous bacteria,
in The Prokaryotes, A. Balows, Truper, H. G. Dworkin, M. Harder W. and
Schleifer, K. H. (eds), pp 625-640, Springer-Verlag, New York.

\[7\] Ruby, E. G. and Asato, (1993). Growth and flagellation of Vibrio
fischeri during initiation of the sepiolid squid light organ symbiosis.
Arch. Microbiol. 159, 160-167.

\[8\] Lee, K. H. and Ruby, E. G. (1995). Symbiotic role of the viable
but nonculturable state of *Vibrio fischeri* in Hawaiian coastal water.
Appl. Environ. Microbiol. 61, 278-283.

\[9\] Palmer, L.M. and Colwell, R.R.** **(1991). Detection of luciferase
gene sequence in nonluminescent *Vibrio cholerae* by colony
hybridization and polymerase chain reaction. Appl. Environ. Microbiol.
57, 1286‑ 1293.

\[10\] Makemson, J. C. Fulayfil, N. and Basson, P. (1992). Association
of luminous bacteria with artificial and natural surfaces in Arabian
Gulf seawater. Appl. Environ. Microbiol. 58, 2341‑2343.

\[11\] Graf, J. Dunlap, P. V. and Ruby, E. G.** **(1994). Effect of
transposon‑induced motility mutations on colonization of the host light
organ by *Vibrio fischeri*. J. Bacteriol. 176, 6986‑6991.

\[12\] Cohn, D. Mileham, A. Simon, M. Nealson, K. H. Raush, S. Bonam, D.
and Baldwin, T. (1985). Nucleotide sequence of the* luxA* gene of
*Vibrio harveyi* and the complete amino acid sequence of the alpha
subunit of bacterial luciferase. J. Biol. Chem. 260, 6139‑6146.

\[13\] Engebrecht, J. Nealson, K. H. and Silverman, M.** **(1983).
Bacterial bioluminescence: isolation and genetic analysis of functions
from* Vibrio fischeri*. Cell 32, 773.

\[14\] King, J.M.H. DiGrazia, P.M. Applegate, B. Burglage, R.
Sanseverino, J. Dunbar, Larimer, P. F. and Sayler, G.S. (1990). Rapid,
sensitive bioluminescent reporter technology for naphathlene exposure
and biodegradation. Science 249, 778‑781.

\[15\] Nealson, K. H. Wimpee, B. and Wimpee, C.** **(1993).
Identification of *Vibrio splendidus* as a member of planktonic luminous
bacteria from the Persian Gulf and Kuwait region with *lux*A probes.
Appl. Environ. Microbiol. 59, 2684‑2689.

\[16\] Wimpee, C. F. Nadeau, T. L. and Nealson, K. H. (1991).
Development of species‑specific hybridization probes for marine luminous
bacteria by using in vitro DNA amplification. Appl. Environ. Microbiol.
57, 1319‑1324.

\[17\] Ausubel, F.M. Brent, R. Kingston, R.E. Moore, D.D. Seidman, J.G.
Smith, J.A. and Struhl K.** **(ed.) 1987. Current protocols in molecular
biology, Greene Publishing Associates and Wiley‑Interscience, New York.

\[18\] Hobbie, J.E. Daleu, R.J. and Jasper, S. (1977). Use of nucleopore
filters for counting bacteria by flourescence microscopy. Appl. Environ.
Microbiol. 33, 1225‑1228.

\[19\] Zweifel, U.L. and Hagstrom, A. (1995). Total counts of marine
bacteria include a large fraction of non‑nucleoid containing bacteria
(ghosts). Appl. Environ. Microbiol. 61, 2180‑2185.

\[20\] Kogure, K. Simidu, U. and Taga, N. (1979). A tentative direct
microscopic method for counting living marine bacteria. Can. J.
Microbiol. 25:415‑420.

\[21\] Takizawa, M. Colwell, R. R. Hill, R. T. (1992). Isolation and
diversity of actinomycetes in the Chesapeake Bay. Appl. Environ.
Microbiol. 59, 997-1002.

\[22\] Saitou, N. and Nei. M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,
406-425.

\[23\] Fitch, W. M. and Margoliash, E. (1967). Construction of
phylogenetic trees. Science 155, 279-284.

\[24\] Fitch, W. M. (1972). Toward defining the course of evolution:
minimum change for a specific tree topology. Syst. Zool. 20, 406-416.

\[25\] Dayhoff, M. O. (1978). Atlas of Protein Sequence and Structure,
vol. 5 suppl 3. National Biomedical Research Foundation, Washington D.C.

\[26\] Jukes, T. H. and Cantor, C. R. (1969). Evolution of protein
molecules in mammalian protein metabolism. Munro, H. N. (ed), pp 21-132,
Academic Press, New York.

\[27\] Felsenstein, J. (1993). PHYLIP (phylogenetic inference package)
version 3.5c. University of Washington, Seattle.

\[28\] Felsenstein, J. (1985). Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39, 783-791.

\[29\] Feldman, K. Buck, J. D. (1984). Distribution and characterization
of luminescent bacteria in a temperate estuary. Estuaries 7, 93-97.

\[30\] Sbrilli, G. Cruscanti, M. Bucci, M. Gagg, C. and Bacci, E.
(1997). Marine heterotrophic bacteria as indicators in quality
assessment of coastal waters. Environ. Toxicol. Chem. 16, 135-139.

\[31\] Ramaiah, N. and Chandramohan, D. (1994). Bacterial
bioluminescence in marine pollution assessment in Ocean Technology
perspectives. Sushuilkumar, K. Agadi, V. V. Das, V. K and Desai B. N.
(eds), pp 967-980, Publi Inform. Dir, CSIR, New Delhi.

\[32\]`	`Kaneko, T. and Colwell, R. R. (1978). The annual cycle of
*Vibrio parahaemolyticus* in Chesapeake bay. Microbiol. Ecol. 1,
135-155.

\[33\] Kurfurst, M. Ghisla, S. and Hastings, J. W. (1982).
Bioluminescence emission of bacterial lucifersae with FMN analogs in
particular with 1-diaza-FMN, in Flavins and flavoproteins. (Massey, V.
and Williams, C. eds), pp 353-358. Elsevier, Amsterdam.

\[34\] Meighen, E. A. (1991). Molecular biology of bacterial
bioluminescence. Microbiol Rev. 55, 123-142.

\[35\] Barak, M. and Ulitzur, S. (1980). Bacterial bioluminescence as an
early indication of marine fish spoilage. Eur. J. Appl. Microbiol.
Biotechnol. 10, 155-165.

##### Table 1. Bacterial species examined for *lux*A gene by PCR and DNA dot-blot analyses

*Aeromonas hydrophila* ATCC 1546

0.  *A. hydrophila* ATCC 7966

*Escherichia coli* ATCC 34309

RC75 *E.coli* O157

*Klebsiella pneumoniae* ATCC 13883T

*Plesiomonas shigelloides* UM 6451

*Pseudomonas aeruginosa* ATCC 27852

*Vibrio aestuarensis* ATCC 3504

*V. alginolyticus* ATCC 17749

*V. anguillarum* ATCC 19105

*V. anguillarum* UM 2445

*V. (Beneckea) campbelli *ATCC 25920

*V. cholerae* UM 400

*V. cholerae* UM 928

*V. cholerae* O1 El Tor ATCC 39315

*V. cholerae* O1 0100

*V. cholerae* O139 UM NT330

*V. cholerae* ATCC 14035T

*V. cholerae* O1 ATCC 11623

*V. cholerae* O139 Al 1877

RC8 *V. cholerae* O1 088

RC25 *V. cholerae* O1 6367

RC42 *V. cholerae* non O1 ATCC 14547

RC44 *V. cholerae* ATCC 25574

RC45 *V. cholerae* Y334

RC47 *V. cholerae* non O1 ATCC 25872

RC48 *V. cholerae* O31Y1 NRT 36S

RC60 *V. cholerae* IGR TMA21

RC72 *V. cholerae*

*V. damsella* UM 4236

*V. diazotrophicus* UM 4232

*V. fischeri* UM 1373

*V. furnessi* UM 4367

*V. harveyi* ATCC 14216

*V. harveyi* UM 1503

*V. harveyi* UM BB7

*V. metsschnikovi* UM 4368

*V. mimicus* ATCC 33653T

*V. mimicus* UM 6812

*V. mimicus* ATCC 33653

*V. mimicus* UM RB653L

RC54 *V. mimicus* UM 4194

contd

RC55 *V. mimicus* UM4198

RC56 *V. mimicus* UM4205

RC57 *V. mimicus* UM4208

RC59 *V. mimicus* UM4053

*V. natriegens *UM 4239

*V. neries* UM 4231

*V. parahaemolyticus* ATCC 15338

*V. parahaemolyticus* ATCC 17802

*V. tubiashi* ATCC 19109

*V. vulnificus* UM C7184

*V. vulnificus* UM E4125

*V. vulnificus* UM 2543

Strains designated with UM or RC are the culture collections at the
Center of Marine Biotechnology

Table 2. Microbiological parameters of water samples collected during
different months in the Chesapeake Bay.

Station Location Sampling TDC DVC NUCC CFU *lux*A probe

depth (m) positive\*

## March 1995

724 37º 24\' N 1 NA NA NA 3.86 4/150

76º 05\' W 19 NA NA NA 4.82 5/200

858 38º 58\' N 1 NA NA NA 2.88 5/150

76º 23\' W 19 NA NA NA 5.71 4/150

### August 1995

724 37º 24\' N 1 2.700 0.311 0.375 2.52 0/378

76º 05\' W 6 6.383 0.237 0.640 3.56 15/392

10 2.442 0.157 0.890 4.16 6/416

19 1.837 0.229 0.396 2.86 12/429

818 38º 18\' N 1 3.758 0.425 0.413 1.12 15/392

76º 18\' W 9 5.375 0.218 0.793 2.44 0/366

18 2.492 0.146 0.780 2.78 5/417

30 1.856 0.177 0.539 1.98 4/396

845 38º 45\' N 1 3.000 0.372 0.506 2.24 8/415

76º 25\' W 4 5.625 0.275 0.808 3.44 0/395

9 2.150 0.208 0.520 1.62 0/405

13 1.338 0.295 0.540 1.54 0/385

858 38º 58\' N 1 2.725 0.383 0.495 3.94 0/394

76º 23\' W 5 5.375 0.233 0.743 3.76 4/376

10 1.806 0.278 0.728 1.68 16/386

19 1.545 0.193 0.568 3.04 24/425

## November 1997

845 38º 45\' N 1 NA NA NA 2.68 9/784

TDC- total direct counts; DVC- direct viable counts and NUCC- nucleoid
containing cells multiplied by 10^6^ ml and, CFU- colony forming units
multiplied by 10^3^ ml; \* number of colonies positive for *lux*A of the
total number examined by DNA-DNA hybridization employing colony-lifts
procedure ; NA- not analysed.

Fig. 1. Alignment of *lux*A nucleotide sequences. Sequences obtained
from GenBank database are: *Kryptophaneron alfredi* symbiont; GenBank
accession number, *Xenorhabdus luminescence* ATCC 29999; GenBank
accession number, *Photobacterium leiognathi* ATCC 25521T; GenBank
accession number , *Photobacterium phosphoreum* NCMB844; GenBank
accession number). Sequences obtained in this study are *Vibrio harveyi*
ATCC 14216T; *Vibrio cholerae* ATCC 14547 ; *Vibrio fischeri* UM1373,
*Vibrio harveyi *luminescent environmental isolate, Chesapeake isolate
\# 81 (visibly non-luminescent, VNL) Chesapeake isolate \# 103 (VNL) and
Chesapeake isolate \# 329 (VNL). The numbering is based on the sequence
of *Xenorhabdus luminescence*.

Fig. 2. Alignment of deduced *lux*A amino acid sequences. Sequences
obtained from GenBank database are: *Kryptophaneron alfredi* symbiont;
GenBank accession number, *Xenorhabdus luminescence* ATCC 29999; GenBank
accession number, *Photobacterium leiognathi* ATCC 25521T; GenBank
accession number , *Photobacterium phosphoreum* NCMB844; GenBank
accession number). Sequences obtained in this study are *Vibrio harveyi*
ATCC 14216T; *Vibrio cholerae* ATCC 14547 ; *Vibrio fischeri* UM1373,
*Vibrio harveyi *luminescent environmental isolate, Chesapeake isolate
\# 81 (visibly non-luminescent, VNL) Chesapeake isolate \# 103 (VNL) and
Chesapeake isolate \# 329 (VNL). The numbering is based on the sequence
of *Xenorhabdus luminescence*.

*RUSSELL PLEASE NOTE*:

**YOU MAY HAVE TO PLEASE LOOK UP GenBank Accession Numbers for all
strains mentioned above. Or, DR. JONGSIK CHUN MAY HAVE THEM WITH HIM; I
WILL REQUEST HIM TO PROVIDE YOU WITH THESE NUMBERS.**

Fig. 3. Unrooted neighbor-joining trees based on *lux*A nucleotide (A),
*lux*A amino acid (B) and 16S rRNA (C) sequences. The trees were based
on 651 (A), 217 (B) and .. (C) unambiguously aligned positions,
respectively. Asterisks indicate the branches that were also recovered
in the Fitch and Margoliash, and maximum parsimony methods (see text for
details). The numbers at the node exhibit the levels of bootstrap
supports based on

neighbor-joining analyses of 1,000 resampled data sets. The scale bar
represents 0.1 nucleotide substitution per position.

*RUSSELL PLEASE NOTE*:

**DR. JONGSIK CHUN WILL PROVIDE ALL THREE REQUIRED TREES FOR THIS
FIGURE**.

Plate I. *Lux*A PCR products from different bacterial strains. Amplicons
in lane 1 to 8 serially are from *Vibrio parahaemolyticus* ATCC 15338
(negative control); *Vibrio fischeri* UM1373; *Vibrio harveyi* ATCC
14216T; *Vibrio harveyi *luminescent environmental isolate; *Vibrio
cholerae* ATCC 14547 ;Chesapeake isolate \# 81 (visibly non-luminescent,
VNL); Chesapeake isolate \# 103 (VNL); Chesapeake isolate \# 329 (VNL);.
Lane 9 not loaded. and lane 10 has the marker DNA of 1Kb ladder

Plate II. DNA-DNA (dot-blot) hybridization showing *lux*A positive
colonies in a water sample from the Chesapeake Bay. Colony-lifts were
hybridized with a 745bp *lux*A probe. Dark spots are of colonies
positive for *lux*A. Positive controls are circled.

Table 3. Percent similarity of luxA nucleotide sequences from: 1
*Xenorhabdus luminescence *ATCC 29999*; 2, Vibrio harveyi* ATCC 14216T;
3,* Vibrio harveyi *UMBB7 luminescent environmental isolate; 4,
Chesapeake isolate \# 329 (visibly non-luminescent, VNL); 5, *Vibrio
fischeri* UM1373; 6, *Vibrio cholerae* ATCC 14547; 7, , Chesapeake
isolate \# 81 (VNL); 8, Chesapeake isolate \# 103 (VNL); 9,
*Photobacterium leiognathi* ATCC 25521T; 10 *Photobacterium phosphoreum*
NCMB844 and 11, *Kryptophaneron alfredi* symbiont.

Table 4. Percent similarity of luxA amino acid sequences from: 1,
*Xenorhabdus luminescence *ATCC 29999*; 2, Vibrio harveyi* ATCC 14216T;
3,* Vibrio harveyi *UMBB7 luminescent environmental isolate; 4,
Chesapeake isolate \# 329 (visibly non-luminescent, VNL); 5, *Vibrio
fischeri* UM1373; 6, *Vibrio cholerae* ATCC 14547; 7, , Chesapeake
isolate \# 81 (VNL); 8, Chesapeake isolate \# 103 (VNL); 9,
*Photobacterium leiognathi* ATCC 25521T; 10 *Photobacterium phosphoreum*
NCMB844 and 11, *Kryptophaneron alfredi* symbiont.

*RUSSELL PLEASE NOTE*:

**DR. JONGSIK CHUN WILL PROVIDE THE CONTENTS OF BOTH THESE TABLES**.