Isolation and characterization of bioluminescent bacteria from marine

environments of Puerto Rico

J. Malave-Orengo1, Eva N. Rubio-Marrero

2 and C. Rios-Velazquez

1*

Microbial Biotechnology and Bioprospecting laboratory, 1Department of Biology and 1Industrial Biotechnology Program,

University of Puerto Rico at Mayaguez, Mayaguez, Puerto Rico.

*Corresponding author:crioslab@gmail.com

Bacteria are the most abundant bioluminescent organisms. These species of bacteria are known to emit a blue/green light

for communication purposes provoked by the enzyme luciferase, regulated by the lux gene operon. Recently, the bacterial

bioluminescence genes (lux genes) have been applied in molecular biology and in environmental biotechnology as genetic

reporters and contaminant biosensors, respectively. Being Puerto Rico a tropical island, with diverse marine ecosystems,

we sought to search for novel bioluminescence species in order to test their potential use as genetic tools and bio-

indicators. General microbiological techniques were used for initial isolation and screening of 125 pure isolated

bioluminescent bacteria. We accomplished the microbiological characterization and a preliminary molecular analysis.

Ongoing experiments are in progress to amplify the 16sDNA of all the samples, and the lux region in order to compare the

genes of the isolates. The 16S rDNA amplified genes will be sent to sequence. The DNA data will be analyzed in silico

using available data bases. Furthermore, the potential of the isolated candidates for detection of specific water

contaminants will also be evaluated.

Keywords: bioluminescence; luciferase; bacteria; lux operon

1. Introduction

1.1 Bioluminescence in nature

Bioluminescence is a form of light produced by a chemical reaction in living organisms. The function of

bioluminescence may vary from one organism to the other, such as for defense against predators, for predation or for

communication with their mates [1]. It is exhibited by a diverse group of organisms although their number is very less

compared to the total number of known species. It has been estimated that luminous organisms may have come from

about 30 different evolutionarily distinct origins [1].

The enzyme luciferase and its substrate luciferin are discovered to be the agents responsible for bioluminescence.

The biochemical and physiological mechanisms responsible for the bioluminescence are different among the luminous

organisms and even though components of the reaction differ in structure, they have similar roles. In higher organisms

such as fish and squids, evidence suggests that they emit their own light or have an endosymbiosis with luminous

organisms to perform a phenomenon known as counter illumination. This strategy is performed by the squid Euprymna

scolopes, which uses luminous bacteria contained in his gut to erase the shadow that is cast by the moon in the oceans

bottom that is detected by predator when the squid swims at night [2]. In the world of insects, the first studies with

luciferase were carried out to measure available ATP [3]. The luciferase used was monomeric and was isolated from

firefly by the pioneer in bioluminescence William McElroy. He made protein extracts from abdominal tissue of firefly

and noticed that ATP (luciferine) was required for the reaction to take place [4].

In microorganisms such as Dinoflagellates, the mechanism of regulation is unique, because it depends on the

circadian cycle, which orientates the organisms to sense what stage of the day is [5]. When a physical stimuli or stress

is perceived by the eukaryotic cell a sudden drop in pH occurs that detaches a protein called luciferase binding protein

(LBP), this way the Dinoflagellate protein is released to perform the reaction in charge of sending an alarm for

predators in distance that would devour the organisms grazing on the dinoflagellate [6]. In dinoflagellates the reaction

take places on specialized organelles called scintillons located in the cytoplasm.

1.2 Light emitting reaction

Bioluminescence is the emission of light by living organisms. It involves the oxidation of a reduced riboflavin

mononucleotide (FMNH2) and a long chain aldehyde. This even produces a blue-green (490 nm) light that gives

bacteria an important ecological role. This mechanism which requires O2 is catalyzed by an enzyme called luciferase

which different types are present on fungi, insects and invertebrates. Luciferase is a heterodimer of 77 KDa, coded by

two similar genes is activated by FMNH2 reacts with O2 producing a compound that will form a highly stable complex

with the aldehyde that decades slowly by emitting luminous energy due to the oxidation of the substrates [7-9].

The aldehyde required for the reaction is synthesized by an enzymatic complex coded by three different genes

present in all bioluminescent bacteria, luxCDE. This substrate is derived from the fatty acid pathway and can be

recycled after each reaction. The three proteins that form the enzymatic complex are a reductase, a synthetase and a

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transferase that were identified through radioactive labeling. Other proteins are involved in the reaction and are only

present in certain species of bacteria. For example, lumazine and yellow fluorescent protein (yfp) are two different

proteins present in Photobacterium .phosphoreum and in some strains Vibrio fisheri respectively. Both are called

accessory proteins because they are not crucial for the reaction to take place. The first one (lumazine) shifts the color of

light to shorter wavelengths, therefore increasing the energy of the emission. The second protein shifts the light

produced to such a degree that the color changes completely to yellow (540nm) [10].

1.3 Regulation of the lux operon

The characterization of the lux operon was determined through the cloning of a 9 kb DNA fragment obtained from V.

fisheri that was transformed into E. coli. Successful expression was witnessed when E. coli would emit light in the

manner as V. fisheri. Mutational analysis showed five crucial genes required for the reaction (luxAB and luxCDE). The

genetic organization was determined to be arranged as an operon, a diverging with a left and right side containing two

different promoters that are transcribed in opposite directions [11-12]. The first gene, lux I at the start of the right side

of the operon produces a small peptide that works as an autoinductor that triggers the synthesis of the structural genes,

luxCDABE, in that same order (figure 1).

Fig 1. Bioluminescence in bacteria is produced by the expression of lux genes. The Vibrio fischeri lux operon is organized in two

different arrangements: from luxI to luxG are expressed downstream and opposite to luxR [13].

All genes in the right side are closely linked to each other, less than 50bp separates them. Next to luxE there is an

additional gene that is present only in marine species not crucial for the reaction but its function is still unknown. To

the left side of the operon a gene is present that codes for another regulatory protein, a repressor with dual function in

all bioluminescent strains. This repressor has one site that interacts with the DNA and a second site that binds to the

small peptide (luxI). When attached to the autoinducer, it forms a complex that promotes transcriptional activation in

the right border through positive regulation. On the other hand when the autoinducer concentration is not high enough,

the repressor continues to be produced. Therefore, inhibiting the transcription of structural genes by binding to operator

located in the control region. This 218bp intergenic region between the left and right border of the operon works as the

control region. It contains the DNA binding site of the repressor and a site for camp receptor protein (CRP) [11].

1.4 Biological systems used as biosensors

Biological processes are known to be diverse in nature and also highly specific. Macromolecules such as enzymes,

toxins and DNA sequences are known to recognize or interact with specific molecules with certain properties or

conformations. An application that has grown in use and versatility is known as a biosensor [14]. This technology is a

modified biological model that can detect through a signal production a specific analyte or molecule. It consists mainly

of two main elements: a promoter and a bioreporter. The promoter or receptor will interact or bind depending to the

case and will translate that interaction into a detectable signal carried out by the reporter. A DNA sequence that codes

for a protein that produces a signal that can be color or light. Some of the biological systems used to detect specific

analytes are bacterial metabolism response, bacterial catalytic pathways such as a trichloroethylene (TCE) sensors

developed by using the TCE degrading bacterium Pseudomonas aeruginosa JI104, and enzymatic reactions like the

nitrite reductase (NIR) from Alcaligenes faecalis S-6 used to measure nitrite concentration, arsenic and mercury at

picomolar levels, concentrations not perceived by certain equipments [15]. Other biosensors are so versatile that can

even produce a measurable signal for physical, chemical and genotoxic agents having genes involved such as recA,

uvrA and alkA [16].

These are only some examples of how a biological process such as bioluminescence has impacted the scientific

community. In this study, bioluminescent bacteria were isolated from marine environments of the tropical island of

Puerto Rico. The goals of this work are to characterize these isolates in order to report an updated diversity of

bioluminescent candidates from various sites around the island. In addition, after confirming the identity of the isolates

by a molecular characterization, we sought to test the new found strains for their biotechnological potential.

2. Methods

2.1 Processing and sample collection

To accomplish the objectives of this research, samples were collected from marine water sources located around the

whole island using sterile autoclaved dilution bottles. Water sampling was performed when rain is not present as a

C D A B E G R

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dilution factor. Sterile bottles (stored in ice) were used to contain enough water volume to use, approximately from

300-500 µL. Luminescent Agar (LA; NaCl 3%, Peptone 1% and Yeast Extract .5%) was chosen as media to perform

duplicate spread plates for each sample. After a 25º C incubation period of 24 hours, plates were examined on a dark

room to presence bioluminescence. Positive candidates were isolated by performing streak plates using toothpicks to

obtain pure colony for morphology examination.

2.2 Microbiological and physiological characterization

To complete the microbiological characterization Gram stain was achieved to determine the exact morphology of the

candidates and was confirmed through Scanning Electron Microscopy (SEM). The isolated bacteria were stored at

minus 80 on 50% glycerol preservation to avoid cell lysis. To confirm viability of bioluminescent bacteria, inoculation

of stored cultures on LA (3% NaCl) media was regularly done. In order to complete characterization of the isolates, a

physiological profile was developed using a total of 12 biochemical assays such as: Oxidase, Nitrate reduction,

Gelatinase, Simmons citrate and Voges-proskauer test. To test catabolic metabolism the following were used: starch,

chitin, glucose, glycerol, mannitol, sucrose and lactose assays [17].

2.3 Preliminary molecular analysis

In order to confirm the identity of the isolates based on the physiological and microbiological data, molecular analysis

was started by performing genomic DNA extraction [18]. Viability and presence of this genomic DNA was confirmed

by 1% agarose gel electrophoresis. As a second step, Polymerase Chain Reaction (PCR) was used to amplify the 16S

ribosomal unit with universal primers for bacteria (27F, 5-AGAGTTTGATCMTGGCTCAG and R 1492, 5-

TACGGYTACCTTGTTACGACT-3) [19]. Amplification parameters for bacterial 16S rDNA were as follow: an initial

denaturation of 94°C for 3 min., 30 cycles of (94°C 30 sec., 52.7°C 30 sec., and 72°C 1.30 min). Comparison of PCR

products from each sample will be done by performing Restriction Fragment Length Polymorphism (RFLP). The

purpose of this technique is to determine how related are the candidates to one another for future sequencing of the 16S

rDNA. A 2.5-3.0% gel electrophoresis will be used to confirm the result of RFLP. Each enzyme used in this technique

will be determined by developing a restriction enzyme profile using in silico tools.

3. Results

In order to survey bioluminescent bacteria from the island of Puerto Rico as a natural reservoir, a collection of 125

isolates from different marine sites have been collected (Figure 2). Sampled sites include the north and southwestern of

Puerto Rico with strains isolated from open areas and estuaries.

Fig. 2 Bioluminescent strains were isolated from different water sources including beaches and estuaries located both on the Atlantic

Ocean and Caribbean Sea (Red star = open areas such as beaches; blue star=estuary (http://www.puertorico.com/images/map-new.gif.).

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Some strains exhibit different bioluminescent intensities when observed in a darkroom. All isolates were Gram

negative when observed in bright field microscopy. In addition, the scanning electron microscopy confirmed their

morphologies from isolates from the Municipalities of Arecibo and Ponce (Figure 3).

In the physiological characterization samples differ in their inability to metabolize carbohydrates such as starch,

gelatin, citrate and chitin. Active presence of the enzymes gelatinase, amylase and the use of glycerol were not observed

for the strain B. In contrast, when strain D was expose to chitin and glycerol significant growth was observed in

minimal media. All strains gave a positive reaction to the oxidase test, and also required NaCl for growth. In contrast,

none of the isolates reported were able to ferment lactose or grow at a temperature of 4ºC.

In order to molecularly characterize the isolates presence of total genomic DNA was confirm through an 1% agarose

gel electrophoresis. This DNA was used to amplify the 16S rDNA which yielded a product of a 1.5 Kb (Figure 4).

Fig. 3 Some bioluminescent strains differ both in light emitting intensities and morphologies. Sample B exhibit a basic morphology

of lengthen bacilli, in contrast sample D shows a shorter bacilli morphology. All dark room photos were taken using the same time

exposure and in the absence of any light sources. Wavelength approximately 495-500nm.

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Fig. 4 Preliminary molecular characterization. A shows extracted high molecular weight DNA bands (1-6)close to 23Kb, lambda

Hind III use as ladder (M). B shows amplified products of expected 1.5Kb (7-12) using bacterial universal primers, 1kb use

as ladder (K).

4. Discussion

Luminous bacteria are the most ubiquitous of all bioluminescent organisms. In Puerto Rico many examples of this

phenomenon have been reported, in contrast to bacteria, the last survey was in 1980 [20]. The purpose of this study is

to report a collection of bioluminescent candidates from some coastal territory of the island. A preliminary

characterization including microbiological and molecular approaches has been initiated. The microbiological data

suggests differences in morphology and metabolic capacities of the isolates. This includes their capacity of producing

cytochrome oxidase, and catabolyze glycosidic bonds in complex carbohydrates by expressing amylase, gelatinase and

chitinase among others. Taxonomically, strains capable of producing gelatinase and amylase are candidates close to the

candidate Vibrio harveyi. In contrast, candidates that resulted negative to these assays suggest their identity to be close

related to other members of the Vibrionaceae family such as Vibrio fisheri. None of the strains resulted to be

psychrophilic or showed any growth on temperatures close to 4ºC which suggests a proximity to strains from the genus

Photobacterium.

Current in silico analysis of the 16S rDNA sequencing of the isolates was performed using BLAST (Basic Local

Alignment Search Tool), which suggests strain B to be Photobacterium leognathi. This corresponds with the

physiological data collected (data not shown). In contrast, the resulted 16S rDNA fragment sequenced for strain D

suggests to be closely related to Vibrio harveyi which was capable of catabolizing chitin and glycerol. Some candidates

were tested for their biotechnological potential by exposing them to p-nitrophenol, which proved to be resistant at a

final concentration of 50 ug/mL (data not shown). Bioluminescent bacteria have always been used as new sources of

gene reporters in molecular biology, moreover, their ability to use chitin, glycerol and other complex molecules indicate

their future use in the field of Biotechnology.

Acknowledgements We would like to thank The Amgen Bio-Minds Program (Biotechnology Mentorship Initiative to Develop

Scientists Program) for the contribution of making possible this manuscript be summited and published.

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