Characterization of Gliding and Iridescent Mutant in marine Tenacibaculum discolor

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1 Characterization of Gliding and Iridescent Mutant in marine Tenacibaculum discolor Lynn Kee Stetson University, Deland FL Microbial Diversity 2016 Abstract Iridescence is a phenomenon where varying colored hues arise from the interaction of light with physical surfaces. The function and mechanisms regulating iridescence of bacterial species is not well characterized. Recent studies have implicated that gliding motility and physical sub-structures and arrangements are associated with iridescence of bacteria. A marine Tenacibaculum discolor was isolated from Woods Hole, MA and in this report the gliding motility and iridescence were characterized using microscopy analyses. Introduction Iridescence arises from the way in which light interacts with physical surfaces to give rise to hues of varying colors. Iridescence of bacteria species has not been fully characterized. Recent report by Kientz et al. describe iridescence in a bacterial species Cellulophaga lytica, and characterize that within a biofilm, cells are arranged in a hexagonal photonic crystal arrangement to give rise to the iridescence observed (Kientz et al., 2016). This report describes the isolation of an iridescent species Tenacibaculum discolor from seawater off the beach near Trunk River in Woods Hole. The iridescence is green in color. Microscopy analyses were conducted to characterize the iridescence, cell morphology and gliding motility dynamics in species Tenacibaculum discolor. Tenacibaculum discolor is from the phylum Bacteriodetes. Gliding is a type of motility that does not involve flagella or motors over physical surfaces. Gliding motility is found within several phyla including Chlorobi, Proteobacteria, Chroloflexi, Bacteriodetes and Cyanobacteria. To investigate mechanisms of gliding motility and iridescence, a genetic screen was conducted to identify mutants of Tenacibaculum discolor. A candidate mutant was characterized with microscopy to analyze the iridescence and gliding phenotypic differences between the mutant and wild-type Tenacibaculum discolor. Materials and Methods Sample Collection and Enrichment Sea water was collected from the beach by Trunk River at Woods Hole on July 4, The sea water was diluted in a 1:10 dilution series and 100ul of each dilution was plated on seawater complete (SWC) plates. The SWC medium Kee

2 contained: 1 liter SW base, 5g tryptone, 1g yeast extract, 3ml 99% glycerol. The SWC plates contained additional 15g agar. Colonies growth on SWC plates were observed over the week. Enrichment and isolated were performed by streaking on SWC plates and inoculation in SWC medium. Culture Preservation Isolated colonies from pure culture were inoculated into 5ml SWC medium for freezer stock preparation and shaken at 30C at 200rpm. Freezer stocks of bacterial culture were prepared with 90% culture and 10% DMSO, and 75% culture and 25% glycerol. Freezer stocks of isolates at -80C include: 162CLK2G, 162CLK2D, 162CLK4G, 162CLK5G, 162CLK6G, 162CLK7G. Genomic DNA Extraction, 16S Sequencing and Genomic sequencing Genomic DNA for Polymerase Chain Reaction was extracted by picking cells from a colony into Alkaline Lysis PEG200 (ALP). PCR of 16srRNA gene was conducted using: 2x GoTaq mix (Promega), Primers: 8Fand 1391F. 10ul of PCR reaction was run on a 1% agarose gel, and PCR product was sent for 16srRNA gene sequencing using the primer. Genomic DNA for genomic sequencing was prepared from a pelleted 1ml culture using a Promega Maxwell Extraction procedure. DNA was submitted for MySeq Illumina sequencing and will be analyzed after the course. Chemical Mutagenesis When a 50ml culture reached OD600 of 0.3, five 5mL aliquots were pipetted into separate, sterile 15Ml conical tubes. Each culture tube was incubated with a final concentration of the chemical mutagenesis agent methyl methanesulfonate (MMS): 10mM, 20mM, 40mM and 80mM. Final tube contained no MMS as control. Tubes were returned to 30C and shaking for 30 minutes at 200rpm. After incubation with MMS, MMS was removed from sample by pelleting 1mL of each sample and washing with 1mL SWC medium. Washed samples were serially diluted 1:10 and 10ul of the 10-1 to 10-6 dilutions were plated on SWC plates to determine mutagenesis survival of cells compared to no MMS control. 40mM MMS concentration was picked for a second round of chemical mutagenesis. 100ul of 10-4, 10-5, and 10-6 dilutions were plated on 10 plates for each dilution concentration. After 48 hours, colonies were screened for phenotypic differences compared to no MMS control. Candidate mutants were picked with sterile tip and streaked out and inoculated into 5ml SWC medium. Phenotypic characterization and microscopy Iridescence and motility were assessed visually by plating out liquid culture in dilution series on SWC plates. Dilution series was conduced to allow for growth of single colonies for comparison to no MMS treated sample. Imaging of Kee

3 colonies were taking on Zeiss macroscope AXIO Zoom.V16 and epifluorescence scope Imager.A2. Kee

4 Results & Discussion Enrichment and Isolation Iridescent bacteria were isolated on SWC plates from the enrichment of bioluminescent bacteria. Iridescent bacteria were streaked out until pure culture was isolated. The iridescent bacteria have green iridescence and edges that are not even and spread out with finger-like projections. Initially the growth of the streak is yellow in color, but over time changes to light green color with iridescence depending on how the light hits the bacteria (Figure 1). A. B. C. Figure 1: Streaked plate of iridescent bacteria A. Growth and iridescent of bacteria on a streaked plate B. Growth and iridescent of two iridescent bacterial strains isolated from the same location. Right is the bacterial strain characterized in this paper, and on the right is the bacterial strain characterized by another member of the class. C. Edge morphology of streak imaged on the macroscope. Kee

5 Species Identification Identification of the species of bacteria was conducted by 16srRNA DNA sequencing from isolated colony. BLAST search on NCBI identified the species at Tenacibaculum discolor. The only available paper published on Tenacibaculum discolor described the isolation of this species form diseased sole and seawater for turbot (Piñeiro-Vidal, M et al., 2008). Piñeiro-Vidal et al. describe their isolates as gram-negative, rod-shaped, gliding bacterial strains that form yellow colonies with uneven edge. Streaked plates did not consistently give single colonies of uniform size. Therefore, single colonies were obtained by serial 1:10 dilution of colonies or liquid culture. The initial attempt at serial dilution allowed isolation of a single colony on a plate at 10-3 dilution (Figure 1). A 10-7 dilution from liquid culture was optimal for obtaining about 10 single colonies on a plate from liquid culture. A. B. C. D. Kee

6 E. Figure 2. Growth of single colony A. Initial colony growth is white-yellow in color. B. Colony turns light green with green iridescence. C. Bigger colony with edges of green iridiescence. D. Image of colony morphology with light shining from underneath (left) compared to no light from underneath and light from the top (right). E. Edge of colony. Kee

7 Bacteria cell morphology and shape was analyzed by phase microscopy. The cells were rod-shaped. Initial microscopy analysis of motility and cell shape were compared from liquid culture and from streaked plates. Cells from streaked plates had similar length, but cells from liquid culture were variable in cell length (Figure 3). Culture did not grow well in 5YE medium when compared to SWC medium (Figure 3C). A. B. C. Figure 3: Comparison of cell morphology in colony and liquid culture A. Cell length is variable in liquid culture. B. Cell length is uniformly similar from colony. C. Culture grown in 5YE (Left) medium and SWC (Right) Medium. Kee

8 Observations of gliding motility of Tenacibaculum discolor on agar To analyze gliding motility of bacteria, cells from colony were streaked onto a thin SWC agar pad made on a microscope slide, and left to grown by incubation overnight at room temperature. The next day, the edge of the streak was imaged under the phase microscopy (Figure 4). Individual cell movement could be seen moving from one area to join other cells from a different island or protrusion of cells. Additional cells move bi-directionally. A. B. C. Figure 4: Gliding motility of Tenacibaculum discolor A. Agar pad with streak of bacteria growth before imaging. B. Morphology of cells at the edge. Individual cell layers were observed, and gliding motility was captured in time-lapse imaging. Time stamp in red. C. A different time lapse of morphology of cells at edge of streak. Kee

9 Chemical Mutagenesis To identify novel genes that regulate gliding motility and iridescence, chemical mutagenesis was conducted by sub-culturing a growing population of Tenacibaculum discolor with treatment of the chemical carcinogen and alkylating agent MMS. Treatment of MMS results in a decrease in the number of cells, suggesting that MMS treatment causes an increase in cell death compared to control due to mutagenesis. Plated MMS treated cells were assessed for phenotypic differences in colony size and iridescence. Four candidate colonies were picked and streaked into a new SWC plate and inoculated in SWC medium and left to grow overnight. To characterize growth and iridescence, dilution series of mutant 4 and wild-type cells were plated and allowed to grow. 22 hours post-plating, phenotypic colony differences were observed under the macroscope (Figure 5A, B). Representative images were taken comparing wild-type cells and candidate mutant 4. Wild-type cells had uneven finger-like projections that projected outwards from the colony that appeared to be of different cell thickness (Figure 5A, B). Colony mutant 4 had smoother edges and under color camera a pink hue was visible, in addition to differences in texture. Five-hour time lapses were taken of the two colony types growing (data not shown). To assess that the phenotype observed was not due to differences in agar thickness, both wild-type and mutant 4 cells were plated on the same SWC agar plate, and colonies from both cell types that were next to each other were analyzed. Comparison demonstrated that on the same agar plate, wild-type and mutant 4 had different colony morphology and shape 22 hours post-plating. Although it is interesting to note that when mutant 4 and wild-type were on the same plate, the mutant 4 colony on the right did have small projections that were similar to that of wild-type (Figure 5C). The edge morphology was further analyzed at higher magnification (Figure 5 D, E). Mutant 4 did not have the thinner, pointy thicker projections that were visible in wild-type colony edge. The thicker edges in Mutant 4 appeared more blunt, with thinner, more flat cell layers radiating out. Figure 5: Comparison of colony morphology of wild-type and mutant 4 at 22 hours postplating. A. Whole colony morphology of wild-type. B. Whole colony morphology of mutant 4. C. Whole colony morohology wild-type and mutant 4 on the same agar plate. D. Edge of colony morphology of wild-type on thin agar plate. E. Edge of colony morphology of mutant 4 on thin agar plate. Kee

10 A. B. C. D. E. Kee

11 Colony morphology was further analyzed at 48 hours post-plating. Both wild-type and mutant 4 had projections at the edges but mutant 4 edges had pink hue and not a yellow-green hue, possibly to due cell thickness differences at the edges (Figure 6 A and B). To further investigate this, the edges were analyzed at higher magnification on the macroscope. Two hour time lapses were taken at the edge of the scope at 6.3x magnification (Figure 6) Time lapses show that for wild-type, there are thicker pointy projections radiating from the colony and thin variable cell layered root-like growth and gliding at the very edge (Figure 6C). Time lapse of mutant 4 show that there are more blunt edged thicker projections, and then from these projections there are more flat cell layers that glide. In some areas above a flat cell layer, there were flat-tiered layered gliding. To visualize individual cell gliding motility of individual colonies, dilution series of cells were plated onto thin agar plates. Agar plates were left to grow until colonies formed, then imaged on the Zeiss Imager. A2 epifluorescent scope. Time lapses of gliding of wild-type showed individual cells moving separately from other cells and then together in a pack in a cell layer. Gliding motility of mutant 4 showed that there were no cells that glided individually on the agar surface. Instead cells glided together that individual cells could not be visualized (Figure 6E). Flat multi-tiered cell layers on the very edge seen in Figure 6D were not observed in the mutant 4 colony grown on agar plates (Figure 6F). Whether the thickness of agar affects the very edges of the colony of mutant 4 needs to be further characterized. Interestingly, the mutant 4 colony appears to have red iridescence on the edges of the green iridescence (Figure 6D). Figure 6. Comparison of colony morphology of wild-type and mutant 4 at 48 hours postplating. A. Whole colony morphology of wild-type B. Whole colony morphology of mutant 4 C. Imaging of wild-type colonies on thin agar plate D. Imaging of mutant colonies on thin agar plate E. Edge of colony morphology of wild-type on thin agar plate F. Edge of colony morphology of mutant 4 on thin agar plate Kee

12 A. B. C. D. E. F. Kee

13 Differences in gliding motility and dynamics during early colony formation was investigated. 12 hours post-plating, colonies were analyzed under the macroscope and imaged for a 9-hour time lapse. Differences in gliding dynamics are visible (Figure 8 and 9). In wild-type the colony edges are more uneven with protrusions of different sizes. As the colony is building up in mass, layers are built from many small initial masses of cells of different sizes that pop up in the middle of the colony, followed by the joining of the masses together to form of a single cell layer and continuation of this pattern (Figure 8). In comparison to wild-type colony, the colony is build in concentric ring pattern, where each layer is build on top of the other a layer at a time (Figure 9). The protrusions at the edges of mutant 4 also appear more uniform in size. Figure 8: Time lapse of early colony formation of wild-type. Time lapse was taken every 5 minutes for 9 hours. Shown above is an image from every hour. Kee

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15 Figure 9: Time lapse of early colony formation of Mutant 4. Time lapse was taken every 5 minutes for 9 hours. Shown above is an image from every hour. Kee

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18 In addition to gliding dynamics, the iridescence and color of mutant 4 compared to wild-type was analyzed after the colony had spread out 5 days post-plating (Figure 10). Wild-type had more green iridescence around the edges and within colonies. Mutant 4 had some green iridescence around the edges, but there were yellow-red iridescence in the center. Figure 10. Comparison of iridescence in wild-type compared to mutant 4 Left. Plate of wild-type colonies Right. Plate of mutant 4 colonies. Kee

19 Future experiments to be conducted involve first characterizing the genetic mutations in Mutant 4 strain. Determining the genes containing mutations and the encoded protein will provide insight into the potential mechanisms into which the mutations cause the gliding and iridescent phenotypic differences compared to wild-type. A possible mechanism is that mutations in gene(s) regulating the production or secretion of polysaccharides or surfactants could be involved in gliding differences. Polysaccharides and surfactants are thought to regulate the gliding motility of bacteria. Mutant 4 appears to still glide but the individual cells do not appear to glide alone but with other cells, given rise the appearance of more even protrusions and uniform cell layers. In addition to gliding phenotypic differences, Mutant 4 differences in iridescence. The mechanism in which bacteria iridescence is still not fully characterized. Modeling conducted by Kientz et al propose that a hexagonal sub-structure of the biofilm of the bacterial species Cellulophaga lytica regulate the iridescence. Whether Tenacibaculum discolor has hexagonal sub-structures is not known. However, macroscope analyses of the original colony isolated from Tenacibaculum discolor after the colony has covered about half of the surface of the SWC plate (from Figure 1) at high magnification, areas of the colony have a wave-like sub-structure pattern with indentations (Figure 11). Whether this patterning is associated with the iridescence pattern still needs to be investigated in future studies. Figure 11. Colony surface morphology Wave-like and indentation patterns observed on the surface of colony at higher magnification. Kee

20 The isolate of Tenacibaculum discolor was sent for genomic sequencing. Future genomic analyses will be conducted to assemble and analyze the genome and annotate genes. References: Kientz, B., et al. A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms. Nature (2012) Pin eiro-vidal, M., et al. International Journal of Systematic and Evolutionary Microbiology (2008), 58, Acknowledgements: I am very grateful for the funding sources that allowed me to attend this course, provided by Simons MD Scholarship and Helsmsley Charitable Trust - Microbial Diversity. I am thankful for the support and resources provided by the Microbial Diversity Course Directors Jared Leadbetter and Dianne Newman, the faculty Kurt Hanselmann, Scott Dawson, George O Toole, and Teaching Assistants, especially Kyle Costa. Kee