Pseudo-Nitzschia Multiseries in Culture

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A model system for diatom-flavobacterium interactions H. M. van Tol, S. A. Amin, E. V. Armbrust School of Oceanography, University of Washington, Seattle

A metabolic requirement for complex carbohydrates and reduced nitrogen is thought The algicidal element is soluble and may be produced constitutively to drive the rapid marine Flavobacteriaceae response to increases in labile organic Washed bacteria Sterile filtered growth media matter during phytoplankton blooms. We find that the Flavobacterium Croceibacter t = 16 h Croceibacter Centrifuge t = 89 h 100 t = 184 h atlanticus appears to be closely associated with the cell surface of the diatom culture 40

Pseudo-nitzschia multiseries in culture. In co-culture experiments, C. atlanticus has Remove 30 50 supernatant a detrimental effect on P. multiseries, apparently inducing cell lysis. The type strain Wash pellet 20 0 was originally isolated from 250-m Sargasso seawater; we isolated two more strains 10 from P. multiseries cultures collected off the Pacific and Atlantic coasts. The avail- % inhibition 0 % inhibition −50 −10 ability of three C. atlancticus genomes, the P. multiseries genome, and several repre- −100 −20 sentatives in culture makes this an excellent system for modeling interactions be- Add dilution series to axenic diatoms in 48 −30 −150 well plates tween a bloom-forming diatom species and a closely associated flavobacterium. 1:100 1:10 1:1 10:1 100:1 1X 10X 100X 1000X 10000X These types of associations directly link primary productivity with the microbial Initial Pseudo−nitzschia : Croceibacter ratio Dilution loop, contributing to the lability of photosynthetically-derived organic matter. Figure 3 Dose response curves for HV2 cells washed in fresh media and for filter sterilized Measure relative fluorescence over time growth media Diatom cell surface hosts a distinct community of epiphytic bacteria We hypothesize that Croceibacter alternates between parasitic and predatory modes in nature Maximum- Roseovarius halotolerans Figure 1 Sulfitobacter mediterraneus ● Croceibacter can exist solely on the exudates produced by P. multiseries Sulfitobacter dubius likelihood 16S phy- ria e Sulfitobacter brevis ● Algicidal activity requires a metabolic investment from Croceibacter. Yet, ct a SA57 logeny of bacteria SA44 β b Sulfitobacter sp. PIC-74 - o SA33 p r the lysate may be consumed by other bacteria. te o isolated from four o te r SA47

73 HV1 Hyphomonas sp. DG895 Hyphomonas sp. DG1705 o ● P. multiseries death removes a steady supply of organic matter and the p SA36 SA46 b Pseudo-nitzschia - Sulfitobacter guttiformis SA38 α SA16 SA54 a 99 c SA40 dead host cell will start to sink away from the surface. SA11 90 t Thalassospira lucentensis multiseries strains SA6 e SA42 r Croceibacter cells 89 SA37 i 62 a SA53 SA25 collected at different SA23 Figure 4 Micrographs of Pseudo-nitzschia multiseries and Croceibacter SA52 SA4 SA51 Limnobacter sp. DG1290 geographic locations 99 atlanticus generated by light and fluorescence microscopy. For fluores- SA59 P. multiseries nucleus SA35 SA12 and maintained in SA30 SA7 cent visualization, P.multiseries and Croceibacter co-cultures were col- 83 SA56 SA5 culture for varying Pseudoalteromonas sp. c7 lected on a filter and stained with SYBR. When excited with blue light, SA43 Alcanivorax sp. JC109 lengths of time. Iso- SA49 5 μm 83 chlorophyll fluoresces red and stained DNA fluoresces green. 51 SA13 SA48 Saccharospirillum impatiens late names of the SA41 SA55 same colour were SA31 99 SA58 55 collected from the SA50 a Marinobacter sp. MCCB214 i We sequenced the genomes of the two diatom-associated strains and compared them with the SA26 r Marinobacter algicola e same P. multiseries t SA8 SA1 SA17 c

HV2 type strain

SA60 SA10 HV3 a SA14 strain (Amin, et al., SA29 SA24 b SA9 o Summary of Croceibacter atlanticus genome and isolate information te in preparation) Table 1 o Muricauda sp. H15 r p Croceibacter atlanticus γ- Name HTCC2559* SA60 Psemu1.scaffold1 HV2

Maribacter sp. HME8336 0.04 Isolated? Y Y N Y Location 250-m water, Sargasso Sea P. multiseries CLNN17, Bay of Fundy P. multiseries CLNN47, Bay of Fundy P. multiseries GGA2, Puget Sound Nitrosopumilus maritimus Status Complete Draft Complete Not sequenced C Sequencing method Sanger SOLiD Sanger - FB Number of bases 2,952,962 2,878,364 3,010,226 - Chromosomes 1 1 1 - %GC 33.90 33.66 33.78 - Croceibacter atlanticus was isolated from two geographi- Protein coding genes 2,678 2,637 2,769 - cally distant Pseudo-nitzschia multiseries cultures RNA coding genes 49 31 49 - The type strain was isolated by Cho & Giovannoni using high throughput culturing * This is the type strain for Croceibacter atlanticus. It was isolated and characterized in Cho & Giovannoni (2003). The genome was published by Oh et al. (2010). methods (2003). An entire Croceibacter genome was also sequenced during the Pseudo-nitzschia sequencing project. Genome characteristics for a diatom-dependent lifestyle ● TonB-dependent Sus system for uptake of complex carbohydrates P. multiseries ● No mechanism for nitrate assimilation, but does have ammonia, amino acid, and peptide transporters culture (HV2) ● Missing genes for Ile, Leu, Val, Arg biosynthesis but does have branched-chain and other amino acid transporters ● Gliding motility genes P. multiseries culture (SA60) ● High density of peptidase-coding genes ● High density of genes coding for carbohydrate activated enzymes (CAZymes)

250-m Sargasso seawater (HTCC2559) The details of diatom-bacteria interactions impact 3000000 ocean biogeochemistry ● The phycosphere is an active site for cycling of fresh organic matter 2500000 ● Epiphytic bacteria modulate how diatoms respond to the environment 2000000 References Amin, S. A., Parker, M. S., & Armbrust, E. V. (2012). Interactions between All Croceibacter isolates have a detrimental effect on Psemu1.scaffold1 SA60 1500000 Pseudo-nitzschia multiseries in co-culture experiments Diatoms and Bacteria. Microbiology and Molecular Biology Reviews, 76(3), 667–684.

Marine broth (negative control) 1000000 Cho, J., & Giovannoni, S. J. (2003). Croceibacter atlanticus gen. nov., sp. 4 Marinobacter sp. SA14 (commensal control) Croceibacter atlanticus HTCC2559 nov., a novel marine bacterium in the family Flavobacteriaceae. Systematic Croceibacter atlanticus SA60 Croceibacter atlanticus HV2 500000 and Applied Microbiology, 26(1), 76–83. Figure 2 Logarithmic growth curves for P. Oh, H.-M., Kang, I., Ferriera, S., Giovannoni, S. J., & Cho, J.-C. (2010). 0 ln(RFU) 0 multiseries in co-culture with different Complete genome sequence of Croceibacter atlanticus HTCC2559T. Journal of 0 500000 1000000 1500000 2000000 2500000 3000000 CroceibacterNegative control isolates versus a commen- Bacteriology, 192(18), 4796–7. Marinobacter sp. SA14 HTCC2559 HTCC2559 I sal SA60γ-proteobacterium. Bacterial cultures HV2 −3 were grown up in marine broth before Figure 5 Nucleotide dot plot showing similarity be- Acknowledgements being transfered into axenic P. multiseries tween the two newly sequenced genomes (y-axis) The Armbrust lab 0 50 100 150 200 250 300 cultures during the exponential growth and the type strain (x-axis). Matching sequence seg- Hours Everyone at the UW Center for Environmental Genomics phase. ments are shown as diagonal lines. Committee members Bob Morris, Jody Deming, and Anitra Ingalls