1. Lecture 21: Bacterial diversity and Microbial Ecology Dr Mike Dyall-Smith Haloarchaea Research Lab., Lab 3.07 mlds@unimelb.edu.au Ref: Ref: Prescott, Harley & Klein, 6th ed., parts of chapters 21-24 (refer to these notes). Also p590-1 (lichens)

2. Main Topics That, along with the Archaea, the Bacteria are very widespread in nature That the Domain Bacteria contains many, different groups, with considerable metabolic diversity How microbial diversity is studied

3. Microbial habitats Archaea and Bacteria are found wherever there is: Water Energy source C, N, P, S, etc. Within physicochemical limits ( ℃, pH, salt,...)

4. Ecological characteristics of bacteria live almost anywhere there is liquid water occur in large numbers Most bacterial cells are relatively small Species diversity is very large (and growing) Most of the ~35 phyla are poorly understood Can be studied to some extent without cultivation

5. Ecological characteristics of bacteria Most of the 10 30 bacterial cells are relatively small * exceptions soil bacteria0.3 × 0.5 µm marine bacteria0.3 × 1 µm Escherichia coli1 × 3 µm Epulopiscium*50 × 600 µm Thiomargarita*750 µm

6. Microbial habitats Within physicochemical limits: TEMP: –10 to 113 ℃ (20 - 40  ℃  ) pH: 0 to 11 (3 - 5 units for any one species) [NaCl]: 0 to 6 M (~saturation) depending on species Others: Oxygen (toxicity), pressure, radiation

7. Phylogeny of Bacteria (using 16S rRNA) At least 35 phyla Groupings and relationships are very informative: e.g. Gram positives cluster together in Firmicutes Cyanobacteria: photosynthetic Spirochaetes: helical cells; motility by axial filaments placement of a new isolate into a phylogenetic grouping can be highly predictive

8. Phylogeny of Bacteria (using 16S rRNA) Most of the studied bacteria belong to just 4 phyla - - Proteobacteria - Bacteroidetes - Firmicutes - Actinobacteria Some phyla have no cultured representatives (white wedges) These are detected by 16S rRNA sequences directly from natural samples, but cannot be grown in pure culture in the laboratory

9. Cultivation dependent - ideal, but has problems! Cultivation independent: Sequence information - eg. 16S rRNA sequences, genome sequences rRNA targeted probes, eg. FISH (Fluorescent In Situ Hybridization) Allows a visual inspection of phylogenetic groups of cells in a natural sample How to study microbial diversity and ecology

10. Cultivation dependent Pure cultures are the basis of the traditional way of studying bacteria Usually only 1% of cells in a natural sample will form colonies on plates Different bacteria have different abilities to be cultured; from easy to difficult Known examples that cannot be cultured

11. Bacteria: examples that have not yet been cultured Mycobacterium leprae (leprosy) Treponema pallidum (syphilis) Epulopiscium fishelsoni All members of the TM7 phylum (a major lineage of Bacteria)

12. Mycobacterium leprae Treponema pallidum Epulopiscium

13. Cultivation independent: Sequence data 16S rRNA sequences, specific genes, mRNAs, whole genome sequences, metagenomes Discovered many new groups of Bacteria - but physiologies yet unknown Can use sequence information to directly visualise specific bacteria in situ (in their natural state) Fluorescent In Situ Hybridization (FISH)...

14. Cultivation independent: Sequence data 16S rRNA sequences, specific genes, mRNAs, whole genome sequences, metagenomes HOW ? Take sample, extract DNA (or RNA) a) PCR amplify 16S rRNA genes clone individual genes, sequence b) Sequence DNA directly (metagenomics) - usually difficult to reconstruct individual microbial genomes as too many species present

15. Permeabilize cells so that the DNA probe can enter Allow it to find its matching sequence on rRNA - short DNA sequence - complementary to rRNA - specific sequence (eg. to genus) - fluorescent tag attached rRNA FISH - Fluorescent In Situ Hybridisation

16. Fluorescent DNA probe will bind to rRNA in the cells only if it exactly matches complementary sequence of rRNA target region Many different coloured fluors, so can do simultaneous probes for different genera, families.... View cells (in situ) under fluorescent microscope, and see what cells fluoresce, showing they have bound the probe FISH - Fluorescent In Situ Hybridisation

18. Growth in Laboratory media versus natural conditions

19. Oligotrophy is the rule in nature Most of the biosphere has low available nutrients (or at least one limiting nutrient) oligotrophy (‘small feeding’) is growth at low nutrient concentrations Dissolved organic C NATUREcoastal waters0.1 - 1 mg/l soil5 - 20 mg/l LAB.M9 minimal800 mg/l

20. Surface area to Volume ratio Small cell size is a way to cope with low substrate availability. It increases the surface area to volume ratio. Substrate uptake is via cell membrane proteins. Increasing SA/Vol improves the ability to supply nutrients to the cytoplasmic volume

21. natural microbial populations Large numbers of small cells Nutrient levels usually very low Population size controlled and limited by nutrient availability Low growth rate (as nutrients removed rapidly), and just matches the death rate Energy mainly used for cell maintenance

22. Microcystis bloom in Matilda Bay, Swan-Canning Estuary, Western Australia. an un-natural cyanobacterial bloom due to excessive nutrients (pollution) Photo by Tom Rose (WA Waters and Rivers Commission)

23. Phylum: Cyanobacteria Largest and most diverse group of photosynthetic bacteria (24 genera) carry out oxygenic photosynthesis: similar to eukaryotes. Fix CO 2 Cells contain thylakoid membranes Significant proportion of marine plankton (and marine microbial food web)

24. Phylum: Cyanobacteria Cells contain thylakoid membranes

25. Phylum: Cyanobacteria photolithoautotroph: energy from light, inorganic electron source, carbon from CO 2 many filamentous forms possess heterocysts, where nitrogen fixation occurs Typical gram -ve cell wall structure diverse modes of reproduction some show gliding motility Can form symbiotic relationships with fungi = lichens. heterocyst

26. Lichens: an association between two partners: an ascomycete (fungus) and a cyanobacteria (or alga). Partnership forms when both are nutritionally deprived Cyanobacteria provide organic compounds via photosynthesis, and can fix nitrogen Fungus provides protection, water retention, extracts minerals and nutrients from substrate

27. Summary 16S rRNA gene sequence comparisons allow a phylogenetic framework to be discerned. Useful for taxonomy, ecological and evolutionary research Domain Bacteria has 35 phyla, and are species rich Great metabolic and genetic diversity within phyla Many phyla are poorly studied because no members have yet been cultivated Despite this, useful information can be obtained using cultivation independent methods (e.g. 16S rRNA sequences, genome sequences, FISH) One example given, phylum Cyanobacteria

28. Summary 50 - 90% of all biomass on Earth is microbial bacteria widespread, only limited by: free water, energy source, components of biomass, and where biomolecules can be stable oligotrophy is the most common state in nature cell size is small in order to increase the surface area to volume ratio, hence improving the ability to take up nutrients at low concentration

29. final note: the vast majority of bacteria are not pathogens. They work for us, in the environment

30. PICTURE CREDITS Treponema pallidum: http://www.brooksidepress.org/Products/OperationalMedicine/DATA/operatio nalmed/Manuals/GMOManual/clinical/Dermatology/Treponema%20pallidum5 00.jpg http://www.brooksidepress.org/Products/OperationalMedicine/DATA/operatio nalmed/Manuals/GMOManual/clinical/Dermatology/Treponema%20pallidum5 00.jpg Mycobacterium leprae: www.wissenschaft-online.de Cyanobacterial bloom in Swan river: http://www.ozestuaries.org/indicators/econ_cons_algal_blooms.jsp Photo by Tom Rose (WA Waters and Rivers Commission) http://www.ozestuaries.org/indicators/econ_cons_algal_blooms.jsp Others from Prescott, Harley and Klein.