1. ABSTRACT:

A 745 bp luxA fragment was amplified from Vibrio harveyi (UM 1503), radiolabeled, and used as a probe to detect and quantify luxA genotypes in culturable bacterial populations from the Chesapeake Bay. DNA samples from 55 reference strains were also examined for this gene. While the luxA -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 luxA 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 luxA amplification products. We examined luxA 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 luxA sequences from the known marine luminescent bacterial species. Our results indicate that luxA -positive bacteria isolated from the Chesapeake Bay are not generally luminescent on phenotype enumeration, implying that gene probe techniques are required for examining luxA gene distribution in environmental samples.

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

2Present 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 a and b 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 luxA 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 luxA 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 luxA 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 luxA 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 luxA internal oligonucleotide probe was end-labeled with [ g -³²P] dATP following the protocol in Ausubel et al. [17]. Labeled probe was used to confirm the identity of luxA fragments by Southern hybridization analysis.

The luxA probe for colony and dot-blot hybridizations was prepared by radiolabeling PCR amplification products. The 745 bp luxA 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 [a -³²P] 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 luxA signatures. For evaluating specificity of luxA 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 luxA 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 ³²P labeled luxA derived oligonucleotide, seven of these luxA 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 luxA 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 luxA gene detectable by the PCR primers we used (Plate I). The identity of PCR products was confirmed by hybridization with luxA- specific internal probe. Results were further confirmed by probing dot-blots of DNA from reference strains with a luxA gene probe amplified from Vibrio harveyi UM1503 luxA gene by PCR.

4.2 Enumeration of luxA- 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 luxA gene probe amplified from Vibrio harveyi UM1503 by PCR. Of the 650 isolates tested, only 18 (less than 4%) were luxA- positive. These results were confirmed by PCR analysis of five probe positive and five probe negative colonies. Probes were used for enumerating luxA 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, luxA - 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 10⁶ to 6.383 x 10⁶ ml^-1 and NUCC, varying from 3.75 x 10⁵ to 8.08 x 10⁵ 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 luxA 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 luxA phylogenetic trees from sequences currently available. These phylogenetic trees were inferred using both luxA 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 luxA and 16S rRNA sequences are generally similar. Alignment of luxA 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 luxA gene sequences from V. harveyi UM 1503. The luxA from these two showed about 98% similarity. The luxA 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 luxA 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 luxA 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 luxA gene from V. fischeri, were present in bacteria from the Chesapeake Bay. Positive results were obtained by PCR analysis with luxA- specific primers and an internal probe. A total of 136 (ca. 1.8%) of over 7500 colonies examined by probe hybridization method were luxA- positive. These luxA -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 luxA. DNA of all known luminescent strains examined in this study (Vibrio fischeri, V. harveyi, and V. cholerae) hybridized with our luxA probe, confirming its specificity and the potential value of a probe -based approach for enumeration of luxA -positive bacteria in environmental samples.

Wimpee et al. [16] hypothesized that many bacteria possess luxA sequences. Palmer and Colwell [9] detected luxA 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 luxA by both PCR and hybridization was confirmed both by biochemical assays and by luminometry. Sequence analyses confirmed that the strains containing genes homologous to luxA were taxonomically identical either to Vibrio harveyi (strain # 329) or to V. cholerae (strains # 81 and 103). The sequences of luxA indicated that no major changes had occurred in the luxA genes among non-visibly luminous (or, nonluminous) bacterial strains carrying this gene. This suggests that lack of expression of luxA, rather than structural changes within the luxA 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 luxA- 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.

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Table 1. Bacterial species examined for luxA gene by PCR and DNA dot-blot analyses

Aeromonas hydrophila ATCC 1546

1. 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 luxA probe

depth (m) positive*

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

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

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 10⁶ ml and, CFU- colony forming units multiplied by 10³ ml; * number of colonies positive for luxA of the total number examined by DNA-DNA hybridization employing colony-lifts procedure ; NA- not analysed.

Fig. 1. Alignment of luxA 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 luxA 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 luxA nucleotide (A), luxA 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. LuxA 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 luxA positive colonies in a water sample from the Chesapeake Bay. Colony-lifts were hybridized with a 745bp luxA probe. Dark spots are of colonies positive for luxA. 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.

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