Author: Nadyne A. Luis
Institution: Chaminade University of Honolulu
Date: December 2006
Sponges are a prolific source of biological compounds with diverse bioactivities. However, structural similarities between the metabolites of the sponge and its associated bacteria, those found within its tissue, indicate that these compounds are of bacterial origin. 16S rRNA gene sequence analyses have been widely used to identify and characterize both culturable and unculturable populations of marine bacteria. The purpose of this research was to isolate sponge-associated bacteria and screen them for antibacterial activity. In this study, a total of 178 potentially different bacteria were isolated from the Hawaiian sponge Suberites zeteki. Due to time restrictions, only the first forty of the bacterial isolates were subjected to further analyses. 16S rRNA gene analyses identified many isolates with a diversity of bacterial groups, including the genus Bacillus and Vibrio. Three of the seven representative bacterial isolates tested inhibited the growth of Bacillus subtilis, a microbe commonly used in bioactivity screening.
Sponges have long been known as a source of bioactive secondary products exhibiting antimicrobial, antitumor, antiviral or general cytotoxic properties with pharmaceutical and medical relevance (Schmitz). They diet on microorganisms, filtering seawater through the choanocyte chambers, transferring the microorganisms to the mesohyl and rendering the expelled seawater essentially sterile. It was later discovered that some of the sponge-produced metabolites possess structural similarities to metabolites of the sponge-associated bacteria, suggesting that the metabolites are of microbial origin (Unson et al). This discovery and the ease of cultivating bacteria as opposed to sponges shifted the focus of research to the cultivation of bacteria associated with sponges for bioactive compounds. It is suggested that the metabolites produced by microbial symbionts participate in the defense of the host sponge, serving as a chemical defense against predators (Unson et al).
However, many of the marine bacteria, including those associated with sponges, have not been isolated, identified or tested for bioactivity, particularly because of the difficulty associated with culturing many of these microbes. 16S rRNA gene sequence analysis has been used as a method for bacterial identification due to the highly conserved nature of the gene sequence, especially from the populations that have remained unculturable using traditional laboratory techniques. This gene sequence is approximately 1,500 nucleotide bases in length and codes for the portion of the ribosome that is responsible for the translation of mRNA to amino acids in protein synthesis, which should have only slight variations due to this specialized and essential function. These variations, although relatively slight, have evolved over millions of years, rendering them a suitable target sequence to estimate the distance of relation between organisms to the species level. Although the gene sequence can vary from one organism to another, there are regions of the sequence that will generally be the same between organisms. Thus universal or relatively specific primers can be used to initiate PCR amplification of all bacterial 16S rRNA sequences or those of specific taxa, respectively.
Biodiversity studies of sponge-associated bacteria have been conducted in Australia as a means of identifying and phylogenetically characterizing species of marine bacteria based on their 16S rRNA gene sequence, and determining symbiotic relationships between the bacteria and specific species of sponges. The research underscores the importance of bacterial diversity studies targeting marine sponges to give a more accurate estimate of the amount of bacteria associated with marine organisms, their environment, and the seawater. Current research does not reflect host-specificity relationships, which would potentially increase the magnitude of the microbial diversity previously estimated. The study also helped to determine host specificity of certain species of marine bacteria for specific species of sponge, simultaneously delineating those associated with seawater, but concluded that more research must be done on the patterns of sponge-bacteria interactions in varying locations before any assumptions can be made (Taylor et al). Other research conducted by Thiel & Imhoff, and Hentschel et al have isolated and identified previously unknown sponge-associated bacteria, and have discovered that some of these isolates demonstrated anti-microbial properties in an inhibition zone bioassay. They postulate that the metabolites of these bioactive microbes could be used in the development of new antibiotics.
This work screened sponge-associated bacterial isolates for antimicrobial activity in a bioassay using Bacillus subtilis, a bacterial strain commonly used for this purpose, and identified the isolates using 16S rRNA gene sequencing and phylogenetic analysis. Results from this work will help identify a new category of bioactive compounds with potential to replace current treatments that are becoming increasingly less effective against microbial resistance. Additionally, this research cataloged the bacteria associated with a species of sponge and will aid in studies on host specificity, simultaneously producing more accurate measurements of the marine microbial diversity.
METHODS AND MATERIALS
Isolation of Sponge-Associated Bacteria
A single, large colony of Suberites zeteki was collected from Rainbow Marina, Oahu, Hawaii and transported back to the lab in a barrel containing seawater. The sponge tissues were washed several times with sterile seawater to remove superficial debris. The internal yellow tissue was then dissected out and homogenized in sterile deionized water (dH20) so as not to contaminate the sponge-associated bacteria. 8 uL of the homogenized sponge tissue was then aseptically spread onto sterile Gause I (20.0 g starch, 1.0 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4 7 H2O, 0.1 g FeSO4, 2 g agar, 1 L filtered sea water) or 1.5% (w/v) agar in Marine broth (MB) 2216 (Difco, Becton Dickinson, Sparks, MD) media plates to increase the probability of obtaining a viable culture. The plates were then incubated at 25C for 7 days and isolated colonies were transferred to sterile Gause I and 1.5% agar in MB media plates, respectively. Each plate was divided into six sections, each section inoculated with one isolate to conserve space (Figure 1). The plates were then incubated at 25C for at least 10 days.
Preliminary Screening for Bioactivity Using Bacillus subtilis
Based on differences in morphological characteristics, seven representative isolates of the first forty were chosen for bioactivity screening. These isolates were prepared for bioactivity screening by inoculating 5 mL of Luria-Bertani broth, Miller (LB) with colonies from the Gause I or 1.5% agar in MB plate media cultures. The media was then incubated at 25°C for several weeks to account for potentially slower growing bacteria.
B. subtilis endospores were prepared by inoculating a 1.5% (w/v) agar in LB slant and incubating for 7 days at 37°C. Following the incubation, the endospores were obtained by adding 15 mL of sterilized dH2O to the slant and mixing. To kill any bacterial cells that may have been dislodged from the slant media and preserve the resistant endospore, the solution was removed and placed in a 65°C water bath for 30 minutes. The solution was further purified by removal of the supernatant after centrifugation at 13,000 rpm for 1 minute. The spores were washed three times with sterilized dH2O by centrifugation to pellet, removing the supernatant, and resuspension of the spores in sterilized dH2O. The suspended spore solution was then purified again in a water bath for 30 minutes at 65°C, and stored at 4°C until ready to plate the media.
Media plates for the bioassay were aseptically prepared by first pouring the bottom layer composed of sterile 0.5% (w/v) agar in LB. This layer solidified before pouring 5 mL of the upper layer containing 0.5% (v/v) of B. subtilis endospores in sterilized and slightly cooled 0.5% (w/v) agar in LB. Two layers of media were poured to create a medium that conserved endospore solution and concentrated it near the area in which the plugs were introduced. The plugs were prepared by punching out circles, 1.2 cm in diameter, from sheets of Whatman paper, that were autoclaved and dried in an oven at 57C for 30 minutes. After the plates set, each plug was then dipped into 5 mL of isolate cultures that have been incubating for several weeks at 25C, and carefully laid on top of the sterile dual layer plated media, then incubated at 37°C overnight. Bioactivity is visualized when the area surrounding the plug clears, creating a zone of inhibition.
Species Identification and Phylogenetic Analysis Using Partial 16S rRNA Gene Sequence
Bacterial isolates were obtained by aseptically inoculating 50 uL LB broth with colonies taken from plated media, either Gause I or Marine Agar. The cultures were then incubated at 95°C for 10 minutes to lyse the cells, and then incubated at 4°C for at least 10 minutes to prevent denaturation of the DNA. The sample PCR reaction mixture included 2.5 uL of Taq DNA Polymerase PCR Buffer (Invitrogen, Carlsbad, CA), 1.25 uL of 50mM MgCl2 (Invitrogen), 1 uL of 2.5mM dNTPs (Promega, Madison, WI), 0.25 uL of Taq DNA Polymerase (Invitrogen), 1 uL of each universal primer (Qiagen, Valencia, CA) 10 pmol U341F primer (sequence 5' to 3': CCTACGGGRSGCAGCAG) and 10 pmol U1406R primer (sequence 5' to 3': GACGGGCGGTGTGTRCA), 17 uL of dH20, and 1uL of the LB culture, resulting in a total reaction volume of 25 uL. Amplification by PCR was conducted on a thermal cycler with a heated lid utilizing a cycling program that included an initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation at 94°C for 50 seconds, annealing at 55°C for 50 seconds, and extension at 72°C for 1 minute. This was followed by additional extension at 72°C for 10 minutes and holding at 4°C until ready to proceed to the next step (Dallas-Yang et al).
Using gel electrophoresis, the amplified gene sequence was separated from the reaction mixture by adding 2 uL of 6X loading buffer (Invitrogen) to 15 uL of the amplicon, then run at 100V on a 0.8% (w/v) agarose in TAE (0.2M Tris acetate, 5 mM EDTA, pH 8.2) gel with 5 uL 1 Kb DNA Ladder (Invitrogen) as the marker, or alternatively, 5 uL of 1 Kb DNA Ladder (Promega). The gel was then incubated in an ethidium bromide (EtBr) solution for 10 minutes and, using an ultraviolet transilluminator to visualize the product, appropriate bands, approximately 100 mb in size, of the representative isolates were then excised from the amplicon gel and purified using QIAquick(R) Gel Extraction Kit (Qiagen).
The samples were then prepared for sequencing according to the instructions for the Beckman Coulter CEQ 2000XL DNA Analysis System using the CEQ Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter, Fullerton, CA). The DNA sequencing reaction was prepared according to the manufacturer's directions using 1.6pmol of either of the universal primers, U341F or U1406R. The sequencing reaction was run on a thermal cycler with a heated lid using the following cycling program: initial denaturation at 96°C for 1 minute, 30 cycles of denaturation at 96°C for 20 seconds, annealing at 50°C for 20 seconds, and extension at 60°C for 4 minutes. This was followed by holding at 4°C until ready to proceed to the next step. The sequencing product was precipitated in individual microcentrifuge tubes and loaded into the CEQ using the procedures outlined by the Beckman Coulter DNA sequencer instructions.
The partial 16S rRNA gene sequences obtained were then compared to those submitted to the National Center for Biotechnology Information (NCBI) GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) to determine if the isolates were of a known species of bacteria. The sequences were then entered into the Vector NTI 9 program to produce a phylogenetic tree.
A total of 178 potentially different bacteria were isolated from the sponge Suberites zeteki. The isolates exhibited different growth rates, some growing on marine agar or Gause I media within a matter of days while other took several weeks to grow. In addition, the isolates produced different color pigments, ranging from an opaque white to gray to yellow-orange and even slightly pink in color (Figure 1). Also, some of the isolates produced colonies that were subsequently coated in a solid, calcareous-like, white shell that made additional manipulation difficult without picking up the media as well (Figure 2). Due to time constraints, only the first 40 isolates were selected for further analyses.
Seven isolates were chosen as representatives of the first 40 based on differences in morphological characteristics, as visualized to the unaided eye. Isolates number 1, 2, 3, 6, 9, 11, and 31 were cultured and assayed against the growth of B. subtilis (Figure 3). Isolate number 11 exhibited a large zone of inhibition surrounding the plug, while the zone of inhibition surrounding the plug of isolates 1 and 6 were very small. The other isolates did not show any inhibitory activities against B. subtilis.
All 40 isolates were subject to PCR amplification for 16S rRNA fragments using universal primers U341F and U1406R, and their resulting amplicons were purified, then subject to sequencing analysis. Thirty-eight of those yielded a partial 16S rRNA gene sequence that was used for BLAST analysis and identified 14 isolates from the Vibrio genus and 26 from the Bacillus genus (Table 1). Isolates 30 and 38 did not produce a useable DNA sequence for BLAST identification.
Based on the bioassay results and the BLAST identification, isolate 11, Bacillus vietnamensis(GenBank accession number AB099708.1), exhibited the most bioactivity against B. subtilis. Isolates 1 and 6, Bacillus sp. V4.BE.28 (GenBank accession number AJ244685.1) and Bacillus sp. NK7 (GenBank accession number AY654898.1) respectively, produced a weak reaction to B. subtilis in the bioassay. The remaining bioassay isolates, 2 and 31, were identified as Vibrio harveyi strain ACMM131 (GenBank accession number AY264922.1) and Vibrio fortis (GenBank accession number AJ514917.1) respectively, and were not reactive to B. subtilis. Isolates 3 and 9 were identified as the same bacteria, Bacillus sp. PAMU 1.13 (GenBank accession number AB118223.1), and were also not reactive to B. subtilis.
The partial 16S rRNA gene sequences were also analyzed using Vector NTI 9 software to determine phylogenetic relationships (Figure 4). The phylogenetic tree seems to exhibit two distinct clades that seem to reflect morphological differences made during initial isolation of the bacteria. The first clade, of the Bacillus genus, is further divided into two distinct clusters. The first cluster showed relation between a B. marisflavi strain, B. vietnamensis, and various B. species. The second also shows relation between B. megaterium and various B. species different from the first Bacillus cluster. The second clade consisted of primarily Vibrio species of bacteria, including V. harveyi strains, V. carallilyticus, and V. sp. CJ11052.
The BLAST results show some repetition in the identity of the isolates, indicating that there are not 178 seemingly different morphological bacterial species associated with this sample sponge. The different species of bacteria were isolated based on visual differences in morphology to the unaided eye. This could have left slower growing bacteria unnoticed and not included in any of the analyses.
These results are premature and need to be analyzed with caution because the majority of the isolates did not grow quickly enough in LB broth to be bioassayed. This leads to the possibility that some of the isolated bacterial strains did not grow under our experimental conditions, and that many do not grow at the same rate. This creates the illusion that the isolates do no show antimicrobial activity against B. subtilis. Additionally, the isolates that did not yield a gene sequence probably were not sensitive to the universal primers used or there simply was not an adequate amount of useable DNA for the PCR amplification or sequencing, which is vital in both techniques. Additionally, although 178 isolates seems abundant, many more isolates were overlooked because of the generally unculturable nature of numerous marine bacteria. Thus we are not only overlooking the isolates that could not be cultured, but the universal primers available are probably not general enough for the identification of some of the isolates, and future work needs to be oriented in this area. Additionally, there was also a problem with some of the gene sequences producing BLAST results with multiple identification possibilities, including various strains of the same species. Furthermore, to create a more detailed phylogenetic tree, the remaining isolates will need to be identified.
Through 16S rRNA gene sequencing and bioactivity screening of sponge-associated bacteria, we were able to document the identity of different species of bacteria and their potential bioactivity. Subsequently, it could be discovered that some species exhibits antimicrobial activity against known species of resistant pathogens. Further research could isolate and confirm the bioactivity of these compounds, and potentially design a new class of antibiotics. In addition, majority of the species of marine bacteria have yet to be isolated and identified, and the bioactivity of their biological compounds have not been tested. Studies in the biodiversity of sponge-associated bacteria will help to not only document new bacterial species, but the study will also generate data on what species of bacteria is connected with a specific species of sponge. This data will be extremely helpful in determining host specificity among not only sponges but potentially all other organisms. Learning about the associations between bacteria and other marine organisms can help to create a more accurate estimate of marine microbial diversity because host specificity has affects on an extremely diverse array of biological phenomena.
Support for this work was provided through NSF grant 0243600 and the University of Hawaii Sea Grant College Program, through the assistance of Dr. Michael Cooney and the Marine Summer Undergraduate Research Fellowship Program. The authors would also like to acknowledge Dr. Quanzi Li, Sang Hwal Yoon and Dr. Teena Michael for their expertise and encouragement.
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