1. Main points from last lecture 1.Differences between prokaryotes (Bacteria and Archaea) and eukaryotes 2.Differences among Bacteria, Archaea, and Eucarya: organelles, cell walls (peptidoglycan in bacteria), lipids (ester vs ether linkage) 3.Use of small subunit ribosomal RNA as phylogenetic marker (16S rRNA for prokaryotes) 1

2. PropertiesProcaryotesEucaryotes GroupsEubacteria, archaebacteria*Algae, fungi, protozoa, plants, animals SizeGenerally small, usually <2 um in diameterUsually larger, 2 to >100 um in diameter Nuclear structure and function: Nuclear membrane Nucleolus DNA Division Sexual reproduction Introns in genes Absent Single molecule, not complexed with histones (other DNA in plasmids) No mitosis No meiosis Rare Present Present in several chromosomes, usually complexed with histones Mitosis; mitotic apparatus with microtubular spindle Meiosis Common Cytoplasmic structure and organization: Plasma membrane Internal membranes Ribosomes Respiratory system Photosynthetic pigments Cell walls Endospores Gas vesicles Usually lacks sterols Relatively simple; limited to specific groups 70S in size Part of plasma membrane or internal membranes; mitochondria absent In organized internal membranes or chlorosomes; chloroplasts absent Present (in most) composed of peptidoglycan and other components Present (in some) Sterols usually present Complex; endoplasmic reticulum Golgi apparatus 80S, except for ribosomes of mitochondria and chloroplasts, which are 70S In mitochondria In chloroplasts Present in plants, algae, fungi; absent in animals, most protozoa; usually polysaccharide Absent Forms of motility: Flagellar movement Flagella; each flagellum composed of one fiber; flagella rotate Flagella or cilia; composed of microtubular elements arranged in a characteristic pattern of nine outer doublets and two central singlets; do not rotate MicrotubulesAbsentWidespread: present in flagella, cilia, basal bodies mitotic spindle apparatus, centrioles 2

3. Main points, conti. 4. Dividing up microbes into functional groups source of carbon: autotroph vs. heterotroph source of energy: phototroph vs. chemotroph Chemoorganotroph= heterotroph 5. Eukaryotic microbes in various functional groups: primary producers, grazers, and mixotrophy. 6. Connection between phylogeny (“community structure”) and function (metabolism—what microbes do) is a big question in microbial ecology today. 3

4. Terms you need to learn (if you don’t know already) 1.DNA, protein 2.RNA: mRNA, tRNA, and rRNA 3.Ribosomes (proteins + rRNA) 4.Lipids (ester vs. ether) 5.Organelles: Nucleus, chloroplast, mitochondria 4

5. Big pools & fluxes High biomass Large organic carbon pool ca. 50% of primary production 20

6. What primary producers are in the oceans? What are the main types of phytoplankton? Early emphasis on “net phytoplankton” Big enough to catch with large nets Easily visible and distinguishable by light microscopy (electron microscopy needed for species level identification) 21

7. Identifying features of algae Shape and Size Pigments: many more than in land plants –All have chlorophyll a (chl a)  used to estimate phytoplankton biomass –Anoxygenic photosynthesizing bacteria have bacteriochlorophyll a –Many (all?) have “accessory pigments”, which really are main light harvesting pigments –These pigments can be used to quantitatively estimate abundance of specific algal groups 22

8. Why so many different type of pigments 22A

9. Note “attenuation” (shading) at both ends of the spectrum 22B

10. Very simple guide to photosynthesis Light Accessory Pigments  Chlorophyll a ATP and NADH H2OH2O O2O2 CO 2 CH 2 O Light Reactions Dark Reactions 23

11. Characteristic Pigments % Marine* Division Common name Comments Chlorophytagreen algaeChl b13Predecessor to chloroplast Phaeophytabrown algaechl c and fucoxanthin 99Includes macroalgae (e.g. kelp) Rhodophytared algaephycobilins98produce agar; few microbial representatives Chrysophyta (Bacillario- phyceae)** diatomschl c and fucoxanthin 50Diatoms have Si in cell walls and often dominate spring blooms Chrysophyta (Coccolitho- phoridales) coccolitho- phorids chl c and fucoxanthin 90Outer covering made of CaCO3 Cryptophytachl c; xanthophylls; phycoblins 60Motility driven by flagella Pyrrhophytadinoflagellateschl c and peridinin 93some heterotrophic; red tide organisms *% marine refers to their abundance in the oceans vs. freshwaters **There are other members of this division besides diatoms and coccolithophorids. Some important eukaryotic algal groups: large or net phytoplankton 24

12. Characteristic Pigments DivisionCommon nameComments Chrysophyta (Bacillariophy ceae) diatomschl c and fucoxanthin Diatoms have Si in cell walls and often dominate spring blooms Chrysophyta (Coccolitho- phoridales) Coccolitho- phorids chl c and fucoxanthin Outer covering made of CaCO3 Pyrrhophytadinoflagellateschl c and peridinin some heterotrophic; red tide organisms 25

13. Evidence that the oceans have more than just “net phytoplankton” Lots of chlorophyll and 14CO2 fixation in <1 um size fraction Epifluorescence counts of auto-fluorescencing cells Cells were too small (ca. 1 um) and without internal structures, i.e. they are bacteria. (But there are some small eukaryotic phytoplankton— poorly understood. 26

14. Coccoid cyanobacteria are abundant and important in the oceans! 1. Well known in lakes and reservoirs 2.Importance in oceans discovered in 1980 (Synechococcus) and Prochlorococcus (1986) 3.Another important cyanobacterium: Trichodesmium 27

15. Synechococcus and Prochlorococcus are both cyanobacteria and are distantly related Some separate Prochlorococcus from “cyanobacteria” and equate “cyanobacteria” with Synechococcus, but not true 29

16. Selected papers about marine coccoid cyanobacteria Li, W. K. W. and others 1983. Autotrophic picoplankton in the tropical ocean. Science 219: 292-295. Chisholm, S. W., R. J. Olson, E. R. Zettler, R. Goericke, J. B. Waterbury, and N. A. Welschmeyer. 1988. A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature 334: 340-343. Palenik, B. and others 2003. The genome of a motile marine Synechococcus. Nature 424: 1037-1042. Rocap, G. and others 2003. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424: 1042-1047. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus, p. 71-120. In T. Platt and W. K. W. Li [eds.], Photosynthetic Picoplankton. Department of Fisheries and Oceans. 30

17. Schematic of epifluorescence microscope Stage with sample Excitation light Objective (100X) Dichroic mirror Ocular (10x): emission 31

18. 32

19. Sample is excited by lower wavelength light (say 400 nm) and the emitted light (“emission”) is at a higher wavelength (say 600 nm) Final magnification= 1000X 33

20. Autofluorescencing cells = autotrophs= phototrophs Must have pigment, with few exceptions Usually chlorophyll, but can excite different pigments with different wavelenghts of light Heterotrophic cells (heterotrophic bacteria) Need to add fluorogenic stain (DAPI and acridine orange) to stain DNA or other cellular material 34

21. Red color due to fluorescence from chl a 35

22. 35A

23. PropertySynechococcusProchlorococcus Size1.0 um0.7 um Chlorophyll aYesModified Chlorophyll bNoYes PhycobilinsYesLess, variable Visible in microscope?Yes Difficult HabitatWidespreadOpen oceans 36

24. From Campbell et al. 1994 L&O Biomass in North Pacific Gyre 37

25. HBACT=heterotrophic Bacteria; Pro=Prochlorococcus; Syn=Synechnococcus; PEUK=picoeukaryotes Cells per ml From Landry and Kirchman, DSR 2002 38

26. Numbers worth remembering Viruses: 10 7 ml -1 Heterotrophic Bacteria: 10 6 ml -1 Cyanobacteria: 10 5 ml -1 Protists (grazers): 10 4 ml -1 Large (>3 um) phytoplankton: 10 3 ml -1 39

27. In oligotrophic waters, coccoid cyanobacteria account for >90% of Phytoplankton biomass (chlorophyll a) Primary production 40

28. Global estimates: Roughly 50% of total marine primary production If marine is 50% of total production---> Cyanobacteria account for about 25% of global primary production!! 41

29. From Madigan et al. “Brock Biology of Microorganisms” 42

30. Another main type of cyanobacteria: Trichodesmium (formally known as Oscillatoria) Filaments of several cells, common in Sargasso Sea Can form macroscopic tuffs of cells Do NOT have heterocysts More about Tricho and heterocysts when we talk about N2 fixation. 43

31. Other algae: note the weird and wonderful shapes!

32. Not all algae are “nice”