Shining the Light on the Structural Color of Tenacibaculum Ectocooler

Chi Nguyen Microbial Diversity, Marine Biology Laboratory 2017

Introduction

The use of color is ubiquitous through out all three domains of life and shown to play many important roles. Colors in nature are categorized into two groups, pigmented and structural color (Figure 1). Pigmented colors result from the selective absorption and reflection of colors at specific wavelengths. The most well characterized pigmented colors include chlorophyll and !-carotene that selectively reflect wavelengths in the green and orange spectrum, respectively (Figure 1). In contrast to pigmented color, structural colors do not result from selective absorption of colors but rather from non-specific and diffused diffraction of light at multiple wavelengths. This phenomenon occurs when light crosses between mediums of differing refractive indices (Figure 2), typically from air onto a surface comprised of regularly placed materials1. Changing in color by altering the viewing perspective, also called iridescence, is a defining characteristic of structural color and results from differential interactions of wavelengths (Figure 2). Colors in Nature

Pigmented Color Structural Color

Chlorophyll

Figure 1. Colors in nature. The physics of pigmented versus structural color and examples of where they occur in nature.

The usage of pigmented colors in animals for mate attraction and for evading predation has been well documented. It is thought that animals use structural colors for similar function. In contrast to animals, the function of bacteria iridescence is not well understood. Studies on the Himalayan tropical plant, Begonia pavonina, suggest that its blue-ish iridescence is a result of more selective reflection of blue light and may play a role in photo-protection against damaging UV light2. The photoprotective function of iridescence, if true, may also play important roles in bacterial iridescence such as Tenacibaculum Ectocoolest, discovered on surface marine water at Woods Hole by Rebecca Mickols. Since then, several other species of Tenacibaculum have been discovered my members of the 2017 Microbial Diversity students on sea surface of grass as well as in symbiotic relationship sea corals. Similarly,The Tenacibaclum Physics has also beenof isolatedIridescence from the surfaces of marine sea sponges near Japan3.

Figure 2. Wave function and iridescence. (Left) Light waves can interact constructively, destructively, or a combination of both. Iridescence occurs because the sum of lights wavelengths changes depending on viewing angle. (Right) An schematic of the iridescent layer of bird feathers.

While little known about the function of structural colors in bacteria, it has been shown that cell-cell organization plays key roles in iridescence. Studies in the bacterium Cellulophaga lytica illustrates that ordered cell-cell contact is key in iridescence and disruption of ordered cell-cell “lattice” results in loss of iridescence4. These results are consistent with studies in animals and plants using electron microscopy (EM) and small- angle X-ray scattering (SAXs) that illustrate highly ordered cell-cell arrays in iridescent birds, insects, and plants5,6 (Figure 3). At the molecular level, iridescence in these animals and plants are the result of highly ordered polysaccharides, such as chitin and cellulose, or proteins, including keratin and collagen5,6. Both of these iridescent biomaterials are often found in complex with structural proteins that may aid in their organization5,6. In contrast to the characterized iridescent biomaterials in animals and plants, little is known about what macromoleculesOrganization comprise iridescence of in bacteria.Iridescence As such, the goal of my mini project is to identify the macromolecule(s) that make up iridescence in bacteria using T. ectocooler as a model organism.

Figure 3. Examples of highly ordered cells in iridescent plant, animals, and the bacterium Cellulophaga lytica (last two panels).

Vinod Saranathan et al. Journal of Royal Society 2012. ResultsZhonge Gu andet at. Chemical Discussion Review Society 2012. Rosenfeld E. et al. Applied and Environmental Microbiology 2012.

Whole Cell Assay Structural colors in plants and animals are comprised of the polysaccharides chitin and cellulose and the proteins keratin and collagen. Neither collagen or keratin are produced in bacteria, therefore it is more likely that bacterial iridescence may be comprised of polysaccharides (Figure 4). Production of complex sugar in bacteria are biosynthesized by transmembrane enzymes7,8, therefore it is likely that the sugar is tethered to the bacterial membrane. Given this rationale, I designed two whole cell assays to test the polysaccharide composition of T. ectocooler. Whole Cell Assay: Rationale Figure 4. Predicted iridescent macromolecules and their localization in T. ectocooler. Keratin and collagen are not made in bacteria, therefore it is likely that T. ectocooler iridescence is made of Iridescent protein polysaccharides. Polysaccharide biosynthesis Structural protein occurs on bacterial membrane, therefore the Iridescent sugar sugar is likely attached. The iridescent layer is likely uniformed, therefore it is likely intracellular, most likely the periplasm.

1.! Keratin and collagen are not made in bacteria ! likely a sugar 2.! Polysaccharide biosynthetic enzyme are located at the membrane ! likely attached 3.! Uniform lattice formation ! likely intracellular To test whether polysaccharides composition affect iridescence, I streaked T. ectocooler on plates containing lysozyme, cellulase, chitinase, and sucrase in combination with proteinase K and toluene (Figure 5). As compared to untreated cells and protienase K alone, cells grown on plates containing lysozyme, chitinase, and sucrase displayed a decrease in iridescence. The addition of toluene, which as been shown to permeablize cells, by itself or in combination with digestive enzymes lead to further disruption of iridescence in T. ectocooler (Figure 5). These Wholeresults links Cell structural Assay integrity and polysaccharide composition with T. ectocooler iridescence.

30°C

+ Proteinase K Observe iridescence +Polysaccharide Digestingdigesting enzymesEnzymes and Iridescence Polysaccharide Sugar monomer/oligomer Toluene Untreated Proteinase K Lysosyme Cellulase Chitinase Sucrase -

+

Figure 5. Colony iridescence assay in the presence of sugar digesting enzymes. (Top) Experimental setup for colony iridescence assay. (Bottom) The results of the colony iridescence assay. The addition of toluene, lysozyme,Toluene, chitinase, and lysozyme, sucrose led to decreaseschitinase, in colony and iridescence sucrase. ! decreases in iridescence To further investigate the polysacharride composition of T. ectocooler, I developed a second assay in which I grew this bacteria in liquid culture to stationary phase, incubated them with digestiveIs there enzymes, a change treated in the the cellspolysaccharide with toluene, and composition? stained for polysaccharide composition using the WGA and ConA lectin (Figure 6). The WGA-Alexa488 and ConA- Texas Red conjugates were used to stain for N-acetyl-glucosamine and mannose/glucose, respectively. It is likely, that digestion with these polysaccharide-digesting enzymes will lead to higher staining intensity. Cells left untreated or with proteinase K (data not shown) alone showed little to no staining with either WGA or ConA (Figure 6). Whole Cell Assay

+ proteinase K + Polysaccharide digesting enzymes

Staining Requires Disruption of Outer

PolysaccharideMembrane Sugar monomer/oligomer Lectin Overlay

Toluene Toluene WGA lectin: N-acetylglucosamine - ConA lectin: Mannose/glucose

N Acetylglucosamine

+ N Acetylglucosamine

+ Mannose/Glucsoe

WGA stains for peptidoglycan Figure 6. Whole cell assay in liquid culture. (Top)ConA Experimental has low background setup. (Bottom) Images of cells treated and untreated by toluene and stained with WGA and ConA imaged under bright field (Left column), with filters set at 490 nm absorbance and 525 nm emission for WGA and 596 nm absorbance and 620 nm emission for WGA (Middle column), and the overlay of the two images (Right column). The top row are untreated cells. The bottom two rows are cells treated with toluene and proteinease K.

The addition of toluene alone or in combination with proteinase K resulted in WGA staining but no ConA staining. The presence of WGA staining in permeablized cells and absence of polysaccharide-digesting enzymes suggest this lectin stains for the naturally occurring sugar components of T. ectocooler, most likely the N-acetyl-glucosamine of the cell wall. WGA may also bind to other polysaccharides that may play a role in iridescence but the high background prohibits further use of this lectin for additional investigation of T. ectocooler sugar composition in response treatment with sugar-digesting enzymes. In contrast, ConA displays low background and was used in to test the effect of the addition of cellulase, chitinase, and sucrose. In the presence of toluene, proteinase K, and sugar- digesting enzymes, T. ectocooler displayed significant staining for ConA (Figure 7). This suggests that digestion with these enzymes lead to changes in polysacharride composition, specifically depolymerization of complex sugars. In combination, the results of these two assays illustrates that the T. ectocooler structural integrity and polysaccharides influences iridescence. These studies provides the first insights into the molecular details of bacterial structural colors. Polysaccharide Digestion

Mannose/Glucose Overlay Chitinase Cellulase Sucrase + - -

- + -

- - +

Figure 7. ConASelective staining of depolymerized staining polysacharrides. of depolymerized (Left column, middle,polysaccharide and right column) Images of cells treated with different sugar digesting enzymes under bright field light, imaged using a filter set at 596 nm absorbance and 620 nm emission, and the overlay of the two images.

Sequence Alignment and Mutation Identification During the Microbial Diversity course of 2016, Rebecca Mickols not only isolated T. ectocooler, she also generated an iridescent mutant using methyl methanesulfonate (MMS). Both the wild type and iridescent mutant were sent out for whole genome sequencing, providing an ideal platform for further investigation of iridescence at the genetic level. The goal for this portion of my project is to identify the mutated genes in the T. ectocooler iridescence mutant. To attain this goal, I used a bioinformatics approach using the following steps. The contigs and assembled WT T. ectocooler were obtained from Open Science Framework. I used Burrows Wheelers Alignment to align the mutant contigs against the WT T. ectocooler reference genome. I then applied SamTools to sort the contigs and to identify variants (mutation sites) using MPileUp9. The summary of this pipeline is shown in Figure 8.

T. Ectocooler Iridescence Mutant Assembly

Linking genes to protein function

T. Ecto WT

Figure 8. Schematic of the pipeline used to identify potential mutated sites in T. ectocooler.

Using this pipeline, I was able to identify 435 variants or potentially mutated sites. These Data Intensive Biology Summer Institute. BWA and Samtools Variant Calling. mutatedhttp://angus.readthedocs.io/en/2017/variant-calling.html sites were then compared with the WT reference and annotated T. ectocooler genome. This lead to identification of 44 mutated genes containing 162 potential mutations sorted into 44 separate contigs. I then pursued identification of genes that were either mutated more than 10 times or with depth value greater than 65. These genes are listed in Table 1.

Table 1. Contig Contig Position Mutation WT Ref. Depth Predicted gene name NODE_10 33582 T C 92 dTDP-glucose dehydratase NODE_111 6352 A C 79 C-terminal Domain NODE_111 6190 C T 78 C-terminal Domain NODE_111 6151 C T 33 C-terminal Domain NODE_111 6073 A G 56 C-terminal Domain NODE_111 6004 T C 86 C-terminal Domain NODE_111 5980 T G 89 C-terminal Domain NODE_111 5932 A G 140 C-terminal Domain NODE_111 5689 C T 171 C-terminal Domain NODE_111 5605 A G 175 C-terminal Domain NODE_111 5595 T C 173 C-terminal Domain NODE_111 5563 A T 170 C-terminal Domain NODE_111 5287 G A 140 C-terminal Domain NODE_111 5254 G T 90 C-terminal Domain NODE_111 6374 C T 90 C-terminal Domain NODE_111 5242 T G 103 C-terminal Domain NODE_111 5200 A G 142 C-terminal Domain NODE_111 5169 C T 178 C-terminal Domain NODE_111 5164 A T 179 C-terminal Domain NODE_111 5161 A G 184 C-terminal Domain NODE_111 5153 G T 207 C-terminal Domain NODE_111 5125 G A 235 C-terminal Domain NODE_111 5122 T A 237 C-terminal Domain NODE_111 4601 T C 71 C-terminal Domain NODE_111 5290 A G 47 C-terminal Domain NODE_111 5247 G A 100 C-terminal Domain NODE_1 105409 C G 70 Putative outer membrane receptor NODE_37 1274 C A 37 Adhesion/Structural Proteins NODE_37 1466 C T 38 Adhesion/Structural Proteins NODE_37 1430 T C 33 Adhesion/Structural Proteins NODE_37 1469 A,C G 38 Adhesion/Structural Proteins NODE_37 1487 C T 37 Adhesion/Structural Proteins NODE_37 1601 T G 33 Adhesion/Structural Proteins NODE_37 1457 T C 40 Adhesion/Structural Proteins NODE_37 396 A C 83 Adhesion/Structural Proteins NODE_37 1261 C T 31 Adhesion/Structural Proteins NODE_37 1259 G A 30 Adhesion/Structural Proteins NODE_37 1088 T C 38 Adhesion/Structural Proteins NODE_37 1055 G T 36 Adhesion/Structural Proteins NODE_37 908 G A 49 Adhesion/Structural Proteins NODE_37 869 C T 46 Adhesion/Structural Proteins NODE_37 839 T C 48 Adhesion/Structural Proteins NODE_37 788 A G 53 Adhesion/Structural Proteins NODE_37 749 C A 56 Adhesion/Structural Proteins NODE_37 617 T C 54 Adhesion/Structural Proteins NODE_37 506 C T 64 Adhesion/Structural Proteins NODE_37 497 C T 59 Adhesion/Structural Proteins NODE_37 495 A G 61 Adhesion/Structural Proteins NODE_37 416 T,C A 76 Adhesion/Structural Proteins NODE_37 413 C,T A 77 Adhesion/Structural Proteins NODE_37 1355 T G 33 Adhesion/Structural Proteins NODE_48 14798 G A 134 OmpA membrane Associated Protein NODE_48 15073 G A 78 OmpA membrane Associated Protein NODE_48 15060 C A 79 OmpA membrane Associated Protein NODE_48 15007 G A 94 OmpA membrane Associated Protein NODE_48 14986 G A 114 OmpA membrane Associated Protein NODE_48 14919 A T 136 OmpA membrane Associated Protein NODE_48 14874 G T 159 OmpA membrane Associated Protein NODE_48 14850 G A 154 OmpA membrane Associated Protein NODE_48 14782 G A 136 OmpA membrane Associated Protein NODE_48 14721 T G 102 OmpA membrane Associated Protein NODE_48 14709 A G 100 OmpA membrane Associated Protein NODE_48 14712 T A 100 OmpA membrane Associated Protein NODE_4 3039 T A 70 Acetyl-CoA acetyltransferase NODE_53 1373 A G 66 hypothetical protein NODE_53 1367 C T 64 hypothetical protein NODE_53 1408 G A 57 hypothetical protein NODE_53 1421 C A 58 hypothetical protein NODE_53 1450 A G 49 hypothetical protein NODE_53 1485 A G 37 hypothetical protein NODE_53 1497 G A 31 hypothetical protein NODE_53 1387 G A 65 hypothetical protein NODE_53 1415 G A 58 hypothetical protein NODE_53 1366 G A 64 hypothetical protein NODE_53 1437 A G 56 hypothetical protein NODE_53 1353 A G 64 hypothetical protein NODE_53 1323 G C 56 hypothetical protein NODE_53 1302 T C 46 hypothetical protein NODE_53 1361 A G 64 hypothetical protein NODE_53 1605 G A 28 hypothetical protein NODE_53 1497 G A 31 hypothetical protein NODE_53 1527 A G 31 hypothetical protein NODE_5 31064 A G 71 Chitinase NODE_5 31053 G C 66 Chitinase

Of the eight nodes and the corresponding mutated genes, I was able to pursue further bioinformatics characterization of Node 111, 37 and 48 using NCBI Blastx. Mutations in nodes 37 and 48 map to a putative adhesion/structural and an OmpA membrane associated proteins, respectively. Node 111 contains a total of 25 mutations mapping to a putative C- terminal domain implicated in gliding motility12 (Figure 9). Interestingly, the same contig also contain two addition non-mutated proteins, a putative porP protein, found as part of the Type IX secretion system,12 and a protein that contains a four repeats of an “agglutinin” β-barrel fold, typical of sugar binding proteins14 (Figure 9). Together these three proteins appear to from an operon and bear high resemblance to components of the bacterial Type IX secretion system (SS)10 (Figure 10).

Type IX SS have been shown to be critical for gliding motility in several bacterial species. Specifically, mutation of Type IX SS protein components results in loss of gliding motility in these bacteria11. Our work demonstrates that mutation of the CTD at this “operon” results in loss of iridescence, providing a direct link between gliding motility and T. ectocooler structural color. It is likely that the loss of gliding motility results in the loss the ability of T. ectocooler to self organize, leading to disruption of organized cell-cell contact, typical of iridescent cells. However, this model alone do not address whether T. ectocooler do no iridesce because they are disorganized or that they are disorganized and also have loss their cellular iridescent structures. CTD has been shown to be exported out of the cell using Type IX SS leading to the formation of complex extracellular matrices10. Further, CTDs are predicted to contain immunoglobulin fold15, proteins often associated with heavy glycosylation. In addition, the agglutinin protein, in the same contig as CTD, is a homolog to RemA16, another protein secreted by the Type IX translocon, also implicated in sugar binding (Figure 10). Contig 1 Blast Results

Agglutinin Type IX SS Gliding/motility C-terminal Sugar binding protein domain (CTD) Secreted by Type IX SS

Figure 9. Blastn results of node 111. The three annotated genes are shown as red bars and mapped to their position in the contig. X indicates the gene mutated in the T. ectocooler iridescence mutant. The three genes appear to be contiguous and may represent an operon.

The presence of two polysaccharide-binding proteins in an operon associated with a Type IX SS protein, one of which is mutated in our T. ectocooler iridescent mutant, provides a direct link between gliding motility and bacterial structural color. This model fits well with our whole cell assay that provides evidence that polysaccharides play important roles in T. ectocooler iridescence. Together these studies provide the first insights into the molecular composition of iridescence and links polysaccharide composition with cell iridescenceType and motility IV Secretion (Figure 11). System and Iridescence

Type IX SS translocon Adhesion protein CTD CTD CTD Sugar remodeling protein Polysaccharide RemA RemA RemA RemA Extracellular matrix

Type IX secretion system (SS) is required for motility

Agglutinin Type IX SS Gliding/motility C-terminal Sugar binding protein Component of domain (CTD) the translocon Secreted by Type IX SS ! extracellular matrix

Figure 10. The type IX secretion system and its homogous proteins from node 111.

Yongtao Zhu et al. 2012. Journal ASM 2012 !"!#$%&'(%#)%*+%&,-&.#Future Directions !"!#$%&'(%#)%*+%&,-&.# ! Given additional time and opportunites, I would like to pursue ! further studies on T. ectocooler iridescence using additional cultivation, whole!"#$%&'$%()(*+ cell assay, imaging, genetics, !"#$%&'$%()(*+ !,&%"#-.$.%/& and bioinformatics techniques. Specifically, it would be interesting! to cultivate WT and mutant T. ectocooler and quantitatively correlate gliding motility with,&%"#-.$.%/& iridescence. I would ! "#$%&! '()!also *##)+,-).! like to repeat /0%'$
! the whole *! 23456! cell assay #)*7/(! using 8$)-.).!a combination 9 of sugar !digesting "#$%&! enzymes '()! and *##)+,-).! /0%'$
! *! 23456! #)*7/(! 8$)-.).! 9 !#'7*$%!4:;<=>6?1!/0+@-)')!&)%0+)A!*#!'()!'0@!($'!B$'(!=;C!$.)%'$'8!staining with additional lectins to gain a better sense of the polysaccharide composition!#'7*$%!4:;<=>6?1!/0+@-)')!&)%0+)A!*#!'()!'0@!($'!B$'(!=;C!$.)%'$'8! *%.! D>C! EF)78! /0G)7H!between 6()7)! WT *7)!and 0'()7!mutant ($'#! T. ectocooler. B$'(! &7)*')7! Additionally, $.)%'$'8! ,F'!it would -0B)7! be EF)78!interesting*%.! D>C! to EF)78!quantitative /0G)7H! 6()7)! *7)! 0'()7! ($'#! B$'(! &7)*')7! $.)%'$'8! ,F'! -0B)7! EF)78! compare the cell-cell organization of iridescent versus non-iridescent T. ectocooler. To /0G)7*&)H! gain better sense of the affect of these mutations, I would like to specifically/0G)7*&)H! knock out or ! known down these genes identified through MMS mutation,! individually and in combination, and test for iridescence. To demonstrate that specific genes are necessary and sufficient, I would like to perform complementation experiments of knocked out or knock down genes. It would also be insightful to perform pan-genomic studies to determine conservation of genes found to be important for iridescence between Tenacibaculum and non-TenacibaculumSummary: strains. A Shining Example of a Multi- Prong Approach

CTD CTD CTD CTD matrix

Extracellular RemA RemA RemA RemA RemA RemA !"#$%&'$%()(*+012 !"#$%&'$%()(*+012 ! ! I$&F7)!JH! !B$-.K'8@)!L-)M'N!*%.!+F'*%'!L7$&('N!M70+!-$EF$.!/F-'F7)H!I$&F7)!JH! !B$-.K'8@)!L-)M'N!*%.!+F'*%'!L7$&('N!M70+!-$EF$.!/F-'F7)H! ! CTD! - CTD! iridescence + Toluene + Polysaccharide digesting enzymes

Figure 11. Models for iridescence. In wild type T. ecocooler, excretion of CTD by the Type IX secretion system!"#$%&'$%()(*+ leads to formation 012 of an extracellular matrix.!"#$%&'$%()(*+ This matrix maybe 012 important for the organization of!"#$%&'$%()(*+ 012 !"#$%&'$%()(*+ 012 CTD, RemA, and potentially other sugar-binding protein that are responsible for iridescence. When CTD is mutated there is loss of the extracellular matrix leading to loss of iridescence. This! phenomena is also ! I$&F7)! DH! O$)B#! 0M!observed in the WT T. ectocooler! B$-.K'8@)! structural *%.!integrity!"#$%&'$%()(*+012 or polysaccharides are! +F'*%'!digested.I$&F7)! DH! O$)B#! 0M! ! B$-.K'8@)! *%.! !"#$%&'$%()(*+012! +F'*%'! /0-0%$)#! *'! .$MM)7)%'!!"#$%&'$%()(*+012 *%&-)#H! 60@! 70BP! $%/F,*').! 0G)7%$&('! *'! 700+! ')+@)7*'F7)H!/0-0%$)#! *'! .$MM)7)%'!!"#$%&'$%()(*+012 *%&-)#H! 60@! 70BP! $%/F,*').! 0G)7%$&('! *'! 700+! ')+@)7*'F7)H! 20''0+!70BP!$%/F,*').!0G)7%$&('!*'!QR!STH!3)M'!/0-F+%P!Methods !+F'*%'H!20''0+!70BP!$%/F,*').!0G)7%$&('!*'!QR!STH!3)M'!/0-F+%P! !+F'*%'H!

!"/#$%&'(%#0&&'121-'&3#0&2456-6#U$&('!/0-F+%P! Cell cultivation!B$-.K'8@)H! !"/#$%&'(%#0&&'121-'&3#0&2456-6#U$&('!/0-F+%P! !B$-.K'8@)H! ! T. ectocooler were grown on seawater complete (SWC) culture media! in both liquid and plate format. Black ink plates were made from SWC media containg 1% Papermate Ink. SWC media were actoclaved with the addition of 1.5% agar, let cooled to ~65°C and ! mixed with sterile filtered Papermate Ink. Cells on plates and liquid !cultures were typically ! V)%0+)!*%%0'*'$0%!,8!U456!.$#@-*8#!*!&)%0+)!#$W)!0M!Q1QQD1JR=!,*#)#1!*!VT!incubated at 30°C in a standing incubator or shaken overnight. ! V)%0+)!*%%0'*'$0%!,8!U456!.$#@-*8#!*!&)%0+)!#$W)!0M!Q1QQD1JR=!,*#)#1!*!VT! /0%')%'!0M!QJHXC!*%.!;D;Y!/0.$%&!#)EF)%/)#H!4%%0'*'$0%!F#$%&!Z70[[*!L5))+*%%!!"#$%&'$%()(*+012+/0%')%'!0M!QJHXC!*%.!;D;Y!/0.$%&!#)EF)%/)#H!4%%0'*'$0%!F#$%&!Z70[[*!L5))+*%%!!"#$%&'$%()(*+012+ DRJQ!/0.$%&!#)EF)%/)#1!Q!/0.$%&!#)EF)%/)#1!

Whole cell assay on plates Enzyme plates were made by spreading of 75 µl sterile filtered enzymes at the concentration listed in Table 2. Toluene was spread on black ink agar plates immediately before usage since it is volatile. Once the cells are added, the plates were incubated overnight at 30°C. Plates containing toluene were inculcated at 30°C lightly sealed in GasPak. Iridescence is observed and colonies were imaged using a dissecting microscope with a light source place six inches away at ~45° from the vertical axis

Tips: These plates are wet when freshly poured. It is best to make them the day before. On the day of use, spread the enzyme, wait for the plates to dry and immediately add the cell.

Table 2 Enzymes Concentrations (mg/ml) Proteinase K 0.5 Lysozyme 1 Cellulase 40 Chitinase 0.5 Sucrase 40

*All enzymes are reconstituted using SWC media.

Whole cell assay in liquid culture Cells were shaken overnight in liquid culture at 30°C. Digestive enzymes were added to the final concentration listed in Table 3 and shaken overnight at 30°C. The liquid cultures were aliquoted into 1 ml fractions and incubated with 1.5 µl and shaken at 30°C for 1.5 hours. Lectins were added to the cell-enzyme-toluene mix and incubated in the dark for at least two hours. The cells were imaged for fluorescence using a Zeiss Axioplan A1 microscope.

Tips: Toluene is not very soluble in water. Shaking it with the cells increases permeability.

Table 3 Enzyme Final concentration (mg/ml) Proteinase K 0.015 Cellulose 5 Chitinase 0.25 Sucrase 5

* All enzymes are reconstituted in SWC media

Sequence Alignment and Variant Calling Sequence alignment between T. ectocooler mutant contigs and the WT reference genome was performed using BWA. Variant calling was performed using Samtools.

Acknowledgements

I would like to extend my greatest gratitude towards Lisa “Bioinformatics Queen” Cohen for her endless patience in helping me with the bioinformatics studies related to this project. Her training has instilled a newfound interest in incorporating bioinformatics in other my projects outside this Microbial Diversity course. I would like to thank Rebecca Mickol for supplying the Tenacibaculum strain used in this study. I am also thankful towards Dr. Scott Dawson for providing the sequencing data for both the WT and mutant Tenacibaculum Ectocooler. I am grateful for his thoughtful discussion and assistance for the duration of my training throughout the course. I am especially thankful towards Dr. Dianne Newman who provided the guidance in directing me towards the working on this project. Further, I am grateful for her time and effort in guiding me through navigating important career choices. I am also thankful towards Dr. Jared Leadbetter for pushing me outside my scientific comfort zone and his continual encouragement during this course in regards to my scientific interests and career decisions. I would like to thank Dr. Viola Krukenberg and Georgia Squyres for their patience and time in helping with imaging on the fluorescent microscopes. I am grateful towards Dr. Kurt Hanselman for his teaching of geochemistry. His enthusiasm is encouraging and motivates me to further study redox chemistry in our environment. I am also thankful towards our endlessly patient and kind course coordinator, Dr. Gabriella Kovacikova, for being the glue that held the culture of the course together. I am thankful towards Dr. Titus Brown and Dr. Tracy Teal for their training in bioinformatics, skills that will be useful towards my own research. I like to also thank Dr. Kyle Costa and Gemma Takahashi for being such wonderful and helpful TAs. Their advice and training in cell cultivation has been invaluable and will be applied to my work. I am also grateful to have made great friends during the course, in particular Nadia Herrera and Lei Zhou. Their humor and thoughtful discussions helped me grow tremendously as a person and scientist this summer. Finally I would like to thank the rest of 2017 Microbial Diversity students, TAs, instructors, lectures, and staff whom have contributed the richness and tremendous success of this summer course.

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