Characterization of Chitinase and Reductase Genes in Bacterial Isolates From Salamander Microbiome




Erin L. Carter

Introduction

There is evidence that the microbiome of 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, DNA was extracted from pure cultures 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 Stenotrophomonas rhizophila is antifungal due to the production of two metabolites, beta-phenylethanol and dodecanal. 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.

Materials and Methods

Primer Design:
In order to see whether or not these genes exist in other organisms, we needed to use universal primers that were able to recognize similar, but not identical, sequences. 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
(250 bp product)

The reductase primers were:
F: TSGGCTGCATGGGCMTGAG
R: YASGGCACGAASCCGATGCC
(500 bp product)

Y = C or T, S = C or G, M = A or C

PCR:
3 samples of DNA from each species was used for testing.
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.

Sequence Analysis
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. To confirm that these translated amino acid sequences were not random amplifications of DNA, the NCBI’s BLAST search was used.

Results

The 16s rRNA primers PCR products, when put through a BLAST search, indicated that the identification of Stenotrophomonas rhizophila was correct. The same was also true for Bacillus weidmannii. However, Serratia liquefaciens was misidentified as Serratia myotis, Acinetobacter guillouiae was misidentified as Acinetobacter modestus, and Pseudomonas koreensis was misidentified as Pseudomonas taetrolens. One of the presumed Pseudomonas isolates was actually Serratia fonticola. This suggests that as much sequencing as possible is necessary to correctly identify bacterial species.

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 chitinase gene was amplified in Bacillus weidmannii and Serratia fonticola. The reductase gene was amplified in Bacillus weidmannii, Serratia liquefaciens, Serratia fonticola and Acinetobacter guillouiae. There were regions of homology across the sequences for the reductase and chitinase genes, but some variable sequencs as well.

Discussion

It was interesting to find 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.

Another accomplishment of the project was the identification of novel chitinase and reductase genes in previously poorly characterized bacterial 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. 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. If they do not possess these genes they must have another mechanism for inhibition, therefore, giving another branch point for study into antifungal mechanisms.

References

Carver, S., B. D. Bell, and B. Waldman. 2010. Does chytridiomycosis disrupt amphibian
skin function? Copeia 2010:487–495.

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): e44832.

Higashino S and J.D. Lewis. 2016. 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): 1001-1012.

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. 2014. 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): 169-176.

Wolf, Arite, et al. "Stenotrophomonas rhizophila sp. nov., a novel plant-associated bacterium with antifungal properties." International journal of systematic and evolutionary microbiology 52.6 (2002): 1937-1944.

Figures


Figure 1-Figure 1: The zone of inhibition produced by plating zoospores and streaking antifungal bacteria as well.


Figure 2-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.


Figure 3-Figure 2: The PCR results using degenerate primers for the chitinase and reductase genes.


Figure 4-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.


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 Bacillus weidmannii, 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.

Full Paper

Acknowledgments

I would like to thank Elle Barnes for allowing me to work on this portion of her dissertation and for all of her guidance throughout my time on this project. I would also like to thank Tony Evans and Faaria Fasih-Ahmad for all their help and dedication to seeing my project come to fruition. Lastly, I would like to thank Dr. Berish Rubin for his guidance and support in completing the project. The three of them were instrumental in making it the best it could be.


This document was last modified 05/15/2018.
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