Things Were Not As They Appeared - Characterization of Chitinase and Reductase Genes in. Bacterial Isolates From the Salamander Microbiome

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1 Things Were Not As They Appeared - Characterization of Chitinase and Reductase Genes in Bacterial Isolates From the Salamander Microbiome Erin Carter Department of Biological Sciences, Fordham University, Bronx, NY

2 Abstract Evidence suggests that the microbiome of salamanders protects them from wildlife disease caused by cutaneous fungal growth. A previous study identified 39 species that constitute the microbiome of salamanders in the Greater New York area using 16s rrna primers that generated a ~250bp product. Of those species, somee exhibited antifungal ability as indicated by a zone of inhibition on a plate of fungal zoospores. The mechanism of Stenotrophomonas rhizophila s antifungal ability was recently characterized as due to production of antifungal metabolites and use of a chitinase to degrade fungal cell walls. The two genes of interest were thus a chitinase and aldo/keto reductase. Degenerate primers were designed based on S. rhizophila sequences aligned with other organisms gene sequences. PCR showed amplification of these genes in S. rhizophila, as expected, but also in, Serratia liquefaciens, and Serratia fonticola. The 16s rrna primers used as a control generated a ~1000bp product that was sufficient to recharacterize Bacillus, Pseudomonas, Serratia, and Acinetobacter as other species than originally thought. One of the S. rhizophila isolates was determined to be another Stenotrophomonas species, S. maltophila based on the chitinase and reductase sequences. This indicated that the region amplified was identical in both of these species. Introduction According to previous studies, nearly 200 amphibian species have gone extinct thus far, and 43% of the remaining species are in danger of going extinct (Pimm et al. 2014, IUCN 2015). One wildlife infectious disease, chytridiomycosis is caused by the growth of the parasitic chytrid fungus, Batrachochytrium dendrobatidis (Bd), on amphibian skin and results in suffocation and death by cardiac arrest (Carver et al. 2010). There is evidence that the microbiome of

3 salamanders protects them from this wildlife disease. Previous studies on amphibian infectious disease conducted by the Lewis lab at Fordham University s Calder Center have shown that the eastern redback salamander, Plethodon cinereus, which resists infection by the chytrid fungus, possesses cutaneous bacteria that inhibit growth of the fungus (Higashino & Lewis 2016). In this study, salamanders were swabbed and their microbiota plated on general purpose agar. Morphologically distinct colonies were isolated, DNA extracted and sequenced with 16s rrna primers that produced a roughly 250 base pair product. 39 species were identified. Many of the species identified exhibit antifungal activity against chytrid fungus. They show a zone of inhibition when streaked on top of zoospores plated on agar (Flechas et. al. 2012). The mechanism of bacterial antifungal ability is not well characterized in many species that exhibit antifungal ability. It was previously thought that competition for resources between fungal and bacterial species reduced fungal growth on salamander skin. However, Wolf et. al. (2002) recently identified that is antifungal due to the production of two metabolites, beta-phenylethanol and dodecanal. Beta-phenylethanol inhibits protein synthesis by binding to and inhibiting a key protein. The enzyme aldo/keto reductase is capable of forming beta-phenylethanol (Kai et al. 2009). Additionally, it is understood that chitinase proteins degrade fungal cell wall, which also inhibits growth. S. rhizophila possesses genes that encode both of these proteins (Roberts and Selitrennikoff 1988). We have identified other antifungal bacteria that are part of the salamander microbiome. The question remains whether or not these antifungal bacteria possess the aldo/keto reductase or chitinase gene despite the fact that they have never been well characterized in these species. While the possible presence of these genes in other organisms does not confirm that they possess the same mechanism of antifungal ability, their presence could inform further studies.

4 Methods Figure 1: The zone of inhibition produced by plating zoospores and streaking antifungal bacteria as well. In order to see whether or not these genes exist in other organisms, we needed to use universal primers. They are able to recognize similar, but not identical, sequences by containing a mixture of degenerate primers that contain multiple primer sequences based on nonhomologous bases across the conserved sequence. Performing a Clustal alignment of multiple organisms chitinase and reductase genes revealed conserved sequences used as targets for priming. Any gaps in consensus sequence were filled with degenerate bases. The chitinase primers were: F: GYGTGGAYATYGACTGGG R: TGCCCATGTCGTAGSTCATC The reductase primers were: F: TSGGCTGCATGGGCMTGAG R: YASGGCACGAASCCGATGCC The Y base indicates the need for either a C or a T. The S indicates the need for either a C or a G. The M indicates the need for either an A or a C. The chitinase primer pairs were predicted to generate a ~250bp product, while the aldo/keto reductase primer pairs were

5 predicted to generate a ~500bp product. 3 samples of DNA from each species was used for testing. PCR was performed with each primer pair on each DNA sample at an annealing temperature of 55 degrees C. 16s rrna primers were used to generate a ~1000bp control DNA to confirm the identity of the isolate that was originally determined using primers that amplified a much smaller piece of DNA. The products were run on a 1% agarose gel with ethidium bromide to visualize the bands. The PCR products that amplified were purified using Qiagen DNA PCR purification kit, and sent for sequencing. Sanger sequencing results were analyzed using translation site, ExPASy, and MUSCLE Clustal Alignment. The nucleotide sequences were translated into amino acid sequences. These sequences were aligned by MUSCLE Clustal Alignment which identified the amino acids that matched and those that didn t. Additionally, it analyzes which amino acid mismatches are biochemically similar. To confirm that these translated amino acid sequences were not random amplifications of DNA, the NCBI s BLAST search was used. Results The first PCR reaction performed tested the 16s rrna primers. Gel electrophoresis confirmed presence of bacterial DNA with bands of the same size at ~1000 base pairs. The 16s rrna primers PCR products, when put through a BLAST search, indicated that the identification of was correct. The same was also true for Bacillus weidmannii. However, was misidentified as Serratia myotis, Acinetobacter guillouiae was misidentified as Acinetobacter modestus, and was misidentified as Pseudomonas taetrolens. One of the presumed Pseudomonas isolates was

6 actually Serratia fonticola. This suggests that as much sequencing as possible is necessary to correctly identify bacterial species. Table 1: Re-Characterization of Previously Identified Bacterial Species Using Primers that Recognize 1000 Base Pair Product What I thought I had What I actually had # Based on NT sequence of 250 base pairs of the 16S rrna Based on NT sequence of 1000 base pairs of the 16S rrna Stenotrophomonas maltophila Serratia myotis Serratia myotis Serratia myotis Pseudomonas taetrolens Pseudomonas taetrolens Pseudomonas taetrolens Serratia fonticola * Acinetobacter modestus Acinetobacter modestus Acinetobacter modestus Table 1: The leftmost column is the isolate number that corresponds to individual DNA samples used. The inside left column shows what the initial identifications of each isolate were after amplifying with 16s rrna primers that produced a ~250bp product. The inside right column is the new identification of each isolate based on the 1000bp product. The group column indicates how the isolates were grouped in the PCR reaction and for alignment analysis. The next PCR reaction performed tested the degenerate primers in S. rhizophila. Amplification occurred as expected because S. rhizophila is known to have the two interest genes. The PCR that tested these same primers with the rest of the species isolates showed

7 amplification in a number of the other isolates. The chitinase gene was amplified in Bacillus weidmannii and Serratia fonticola. The reductase gene was amplified in,, Serratia fonticola and. There were regions of homology across the sequences for the reductase and chitinase genes. Some of the consecutive sequences that didn t match S. rhizophila matched each other, suggesting variation in regions that are conserved in different organisms. Figure 2: The PCR results using degenerate primers for the chitinase and reductase genes. Discussion The most compelling results yielded from this project was the finding that using 16s rrna DNA sequences might be insufficient to determine which bacterial species you have. It is likely that a larger piece of DNA must be amplified, or that multiple genes that are conserved in bacteria must be used to correctly identify each isolate. This was particularly evidenced by the failure of the 1000 base pair 16s rrna to differentiate between S. rhizophila and S. maltophila. The reductase sequence was identified as S. maltophila, indicating that the species was not S. rhizophila. When the sequence that was amplified in each species was aligned it was perfectly homologous. The overall 16s rrna gene is ~1600 base pairs, so even with over half the sequence amplified, it wasn t enough to distinguish between these two species. Another accomplishment of the project was the identification of novel chitinase and reductase genes in previously poorly characterized bacterial species. When gel electrophoresis

8 confirmed that DNA was amplified in these isolates, performing a BLAST search on the translated protein sequences confirmed that they were relatively unidentified in these species. Each of the sequences were matched as S. rhizophila proteins. The protein products of the genes were superfamily proteins - GH18 chitinase and an aldo/keto reductase family. This was expected as the genes had not been previously characterized in the organisms the 16s rrna identified each isolate as. There are clear conserved sequences throughout the protein sequences shown, as well as upstream and downstream of this fragment. Mismatched amino acids show chemical similarities to the original sequences. The identification of these genes in other species of bacteria that have antifungal ability leads to possible further research into the mechanism of antifungal ability in these species. The mechanism of antifungal ability is potentially the same in these isolates as in S. rhizophila. It also brings into question the mechanism for antifungal ability in those organisms that do not possess these genes. If they do not possess these genes they must have another mechanism for inhibition, therefore, giving another branch point for study into antifungal mechanisms.

9 What I actually had Reductase translated AA sequence # Stenotrophomonas maltophila ALALGVTLLDTADMYGPHTNEVLVGKAIADRRDQVFLATKFGIRQEPSDSAA ALALGVTLLDTADMYGPHTNEVLVGKAIADRRDQVFLATKFGIRQEPSDSAA ALESGVTLLDTADMYGPHTNEVLVGKAIADRRDHVFLATKFGIRLEPSDTAA Serratia fonticola ALALGVTLLDTADMYGPHTNEVLVGKAIADRRDQVFLATKFGIRQEPSDSAA AVERGVTFFDSAEAYGPFRNEELLGEAFAACRDKVVIATKFGFKEGQVDAG AVERGVTFFDTAEGYGPYTNEELVGEALQPLRDKVVIGTKFGFDINESGETV AVEQGVTLFDTAEAYGPFKNEELLGQALAPHREKVVIATKFGFKDGHADA # What I actually had Chitinase translated AA sequence Stenotrophomonas maltophila Serratia fonticola DRRNMTLLVQEFRRQLDAVGTPRGQHLLLTAALPAGRVQTDGAYDPARSY DRRNMTLLVQEFRRQLDVVGTPRGQHLLLTAALPAGRVQTDGAYDPARSY DRRNMTLLVQEFRRQLDALDGGRGPHRLLTAALPAGRVQTDGPYDPALSY DRRNMTLLVREFRRQLDALDNKDGQHRLVTAALPAGRVQTDGPYDPARSY

10 Figure 3: Top: The amino acid alignment for a region of the chitinase gene. Blue letters indicate the mismatch of an amino acid based on the reference sequence of S. rhizophila. Bottom: the amino acid alignment for a region of the reductase gene. Blue letters indicate the mismatch of an amino acid based on the reference sequence of S. rhizophila. References Carver, S., B. D. Bell, and B. Waldman Does chytridiomycosis disrupt amphibian skin function? Copeia 2010: Flechas, Sandra V., et al. "Surviving chytridiomycosis: differential anti-batrachochytrium dendrobatidis activity in bacterial isolates from three lowland species of Atelopus." PLoS One 7.9 (2012): e Higashino S and J.D. Lewis Species richness of cutaneous bacteria varies with urbanization: Implications of habitat conditions on defense mechanisms of Plethodon cinereus. Unpublished. Kai, Marco, et al. "Bacterial volatiles and their action potential." Applied microbiology and biotechnology 81.6 (2009): Pimm, S. L., C. N. Jenkins, R. Abell, T. M. Brooks, J. L. Gittleman, L. N. Joppa, P. H. Raven, C. M. Roberts, and J. O. Sexton The biodiversity of species and their rates of extinction, distribution, and protection. Science 344. Roberts, Walden K., and Claude P. Selitrennikoff. "Plant and bacterial chitinases differ in antifungal activity." Microbiology134.1 (1988): Wolf, Arite, et al. " sp. nov., a novel plant-associated bacterium with antifungal properties." International journal of systematic and evolutionary microbiology 52.6 (2002):