Microbial Phylogeny

Dr Jai Tree
School of Biotechnology and Biomolecular Sciences
Learning objectives

q Briefly describe taxonomy, phylogeny and phylogenetics, and understand

the how each is different.

q Explain why the small subunit of ribosomal RNA is used to calculate the
evolutionary distance between microorganisms.

q Briefly describe the characteristics of notable bacterial phyla including the

Firmicutes, Proteobacteria, Tenericutes, Planctomyces, Thermotogae, and
Aquificae.

q Briefly describe how DNA sequencing and mass spectrometry can be used
to identify microbes.
 Microbes in time

First microorganisms?

Formation of
Cyanobacterial microfossils O2 algae,
(oxygenic photosynthesis) atmosphere marine invertebrates
Earth formed insects
mammals

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

No free O2
Billion Years Ago
Phylogeny of Life

~8.7 million species (8.7x106)

~1 trillion species of Bacteria and Archaea (1012) - includes many microbes
(fungi, yeasts, algae,
protozoa)

Locey & Lennon PNAS 2016 Mora et al PLoS Biology 2011

 Taxonomy
The science of defining groups of biological
organisms on the basis of shared characteristics
and giving names to those groups
(ie. binning)
 Taxonomy
The science of defining groups of biological
organisms on the basis of shared characteristics
and giving names to those groups
(ie. binning)

Intrinsic part of the Aristotle Andrea Cesalpino Carl Linnaeus

human condition First to classify all The first taxonomist Systema Naturae
(pre-history) living things (1519-1603) Binomial naming system
(384–322 BC) (384–322 BC)
 Hierarchical classification scheme

Domain Kingdom Phylum Class Order Family Genus Species

Binomial naming system

Escherichia coli and thereafter E. coli

 Phylogeny
The study of evolutionary relationships among
groups of organisms
(ie. family trees)

Phylogenetics is phylogeny based on analysis of sequences of DNA

What features would be required to compare all life?

www.zeetings.com/jtree
Some cellular structures are conserved in all life
The ribosomal small subunit (SSU)

Carl Woese (1928-2012)

Why base phylogenetics on the SSU rRNA gene?
Why base phylogentics on the SSU rRNA gene?

1. SSU rRNA provides sufficient sequence information (variable and invariant

regions) to permit statistically significant comparisons between homologues.
SSU rRNA Databases 2. ALL
SSUliving
rRNAorganisms
is also highly abundant in the cell
2. contain ribosomal RNAand the
EZ Taxon quantity reflects the capacity of an organism to make protein
sequences
RDP and therefore do work.
Greengenes
SILVA 3. SSU rRNA is also the subject of large curated databases.
 A species definition for bacteria

Members of the same bacterial species

a)  Share phenotypic characteristics
b)  Have >70% total genome similarity
c)  Have >97% sequence identity in the 16S rRNA gene

But

Just because two cells belong to the same species

doesn’t mean they are identical
Domain • Kingdom • Phylum • Class • Order • Family • Genus • Species • Strain
For example Escherichia coli K12 is harmless. Escherichia coli O157:H7
01H4:H4 can kill you
Bacteria are evolving fast
 Phylogenetic trees represent evolutionary distance

Commonly ribosomal SSU sequences

More detail on constructing phylogenetic trees in Lecture 4 with Prof Mark Tanaka
 Predictions suggest that up to 1 trillion (1012) microorganisms exist

Locey & Lennon PNAS 2016

Viruses are missing (Lectures 19, 21, and 22) … WHY?

Bacterial Phyla

In 1987 Carl Woese listed 11 bacterial phyla…

In 1987 Carl Woese listed 11 bacterial phyla…
… there are now more than 29 cultured phyla
In 1987 Carl Woese listed 11 bacterial phyla…
… there are now more than 29 cultured phyla
and >70 that have never been cultured
 There are currently 100 Bacterial phyla

AC1 Cyanobacteria Kazan-3B-28 OP9 TM6

Acidobacteria Deferribacteres KB1 OP11 TM7
Actinobacteria Thermi KSB3 PAUC34f TPD-58
AD3 Dictyoglomi LCP-89 Planctomycetes Verrucomicrobia
AncK6 Elusimicrobia JL-ETNP-Z39 Poribacteria VHS-B3-43
aquifer1 EM3 JS1 Proteobacteria WCHB1-60
aquifer2 EM19 LD1 RsaHF231 WD272
Aquificae FBP Lentisphaerae S2R-29 WPS-2
Armatimonadetes FCPU426 MAT-CR-M4-B07 SAR406 WS1
Bacteroidetes Fibrobacteres MVP-21 SBR1093 WS2
BD1-5 Firmicutes MVS-104 SBYG-2791 WS3
BHI80-139 Fusobacteria NC10 SC4 WS4
BRC1 GAL08 Nitrospirae SHA-109 WS5
Caldiserica GAL15 NKB19 SM2F11 WS6
Caldithrix Gemmatimonadetes NPL-UPA2 Spirochaetes WWE1
CD12 GN01 OC31 SR1 ZB3
Chlamydiae GN02 OctSpA1-106 Synergistetes
Chlorobi GN04 OD1 TA06
Chloroflexi GOUTA4 OP1 Tenericutes
Chrysiogenetes H-178 OP3 Thermodesulfobacteria
CKC4 Hyd24-12 OP8 Thermotogae

Demonstration purposes only – don’t memorise this list

Characteristics Bacterial phyla

q 3.5 billion years has given rise to incredible diversity of microorganisms

q Some phyla are better characterised than others

q We will highlight:
q Proteobacteria
q Firmicutes
q Tenericutes
q Planctomycetes
q Thermotogae & Aquificae

Interesting because they’re odd

q Huge amount of diversity we won’t discuss

Proteobacteria
q By far the largest phylum of Bacteria
q Contains more than a third of characterised
Bacteria
q Majority of medical, industrial, and
agriculturally important Bacteria

q All gram negative

q Morphologically and metabolically diverse

q Six major sub-groups termed

Ø Alpha
Ø Beta
Ø Delta
Ø Epsilon
Ø Gamma
Ø Zeta

Brock Figure 15.2

Gammaproteobacteria

q Contains nearly half of the species in

Proteobacteria
q Highly diverse metabolism (photo-,
chemoorgano-, chemolithotrophs)

q Enterobacteriales
q Contains many human pathogens
q Facultative aerobes
q Well characterised - easy of ID
q Oxidase negative & catalase positive

q Many can be differentiated on sugar

fermentation
q Mixed-acid fermentation (MR test)
q Butanediol fermentation (VP test)

More on biochemical testing in Week 3 tutorial

Alphaproteobacteria

q Second largest Class of Proteobacteria

q Mostly obligate and facultative aerobes

q Many oligotrophic – low nutrient environments

q Includes Rhizobales
q named after polyphyletic rhizobia
q Fix N2 gas in root nodules

q Includes Rickettsiales
q Intracellular parasites
q Rickettsia spp. cause human disease
q Arthropod bites
q Rocky Mountain Spotted fever &
thyphus
 Alphaproteobacteria: Wolbachia

q Rickettsiales also contains Wolbachia

q Found in ~70% insects

q Intracellular parasite

q Wolbachia spp. cause distinct reproductive

defects
q Convert male à female
q Cytoplasmic incompatibility

q Used to control Dengue

q Prevents Dengue replication
q Spreads through wild mosquito popn
q Cytoplasmic incompatibility

q worldmosquitoprogram.org
q Dengue, Chikungunya, Zika, Yellow
Fever
Herron et al Nature Review Micro 2008
Firmicutes
q Gram positive Bacteria

q With Actinobacteria and Tenericutes ~ half of all

characterised Bacteria

q Firmicutes have low G+C genomes

q Some are able to form endospores

q Bacillus
q Clostridium

q Many medically and industrial important species

q Lactobacillus – used in dairy fermentation
q Some cause disease
q Staphylococcus
q Listeria
q Streptococcus
q Clostridium

Staphylococcus
Tenericutes: gram positives without a cell wall

q sole Class is Mollicutes aka Mycoplasmas

q Mycoplasmas do not have a cell wall

q What the? Won’t they burst?

q Stabilise the cytoplasmic membrane with:

q Sterols
q Lipoglycans (kinda like LPS)

q Can’t be killed with cell wall inhibiting antibiotics

q eg: penicillin and cephalosporins
Planctomycetes: bacteria with a nuclear membrane

q Prokaryote a that lacks a membrane enclosed nucleus

and other organelles Bacteria or Archaea

q Gram negative

q Cell walls lack peptidoglycan

q Use an S-layer (see Lecture 8 on Archaea)
q Resistant to penicillins and cephalosporins

q Planctomyces are unique – they contain membrane

bound compartments
q Gemmata have a membrane that encloses the
nucleoid
Thermotogae and Aquificae

q Phyla of hyperthermophilic bacteria – growth at

>80oC
q Found in terrestrial and marine thermal
springs/vents

q Branch deep within the phylogenetic tree – near

the root

q ~20% of Thermotoga genes are closely related

to Archaeal genes
q Acquired by HGT

q Many are H2 chemolithotrophs

q Like many thermophilic Aquificae,
Thermotogae and Archaea
q May suggest H2 was a key electron donor
in primitive organisms
Mitochondria and chloroplasts

q Mitochondria are energy producing organelles of

eukaryotic cells
q Chloroplasts are photosynthetic organelles of plant cells

q Both contain ribosomes with SSUs consisting of 16S rRNA

q Eukaryotes use 18S rRNA SSU ribosomes

q Suggests that mitochondria and choroplasts were

endosymbionts

q SSU rRNA sequences indicate

q Chloroplasts related to Cyanobacteria
q Mitochondria related to Alphaproteobacteria
 Identifying microorganisms
The problem: How to identify bacteria at a genus and species level?

Equus caballus Equus quagga

Morphological differences are often sufficient for macroscopic plants and animals
 Identifying microorganisms
The problem: How to identify bacteria at a genus and species level?

Escherichia coli Salmonella typhimurium

Normal flora Food poisoning

 Identifying microorganisms
Sample with microbes of interest
Week 3 Tutorial + Prac

Culture and isolate:

Nutrient requirements
Phylogenetics (Prac)

Morphological Molecular fingerprinting Genotyping:

characterisation: MALDI-TOF Mass rRNA SSU profiling
Cell shape spectrometry Whole genome sequencing
Gram stain Select loci (eg: MLST)

Biochemical Can identify

characteristics uncharacterised
microorganisms

Identify microbe
Mass spectrometry for identifying microorganisms
q Measures molecular weight of proteins
in culture samples
q Protein profile is molecular signature
for bacterial species
q Abundant proteins 2-20kDa
q Many ribosomal proteins
q MALDI-TOF most commonly used

q Compared to a database of protein profiles

q ~5mins identification time (cf 24-36hours)
q Improved patient treatment in clinical settings

Mass/charge (m/z) peaks

corresponding to bacterial
proteins
What you may have learned

q Taxonomy is the classification of life into categories or “bins”

q Phylogeny is evolutionary relationships between living organisms
q Phylogenetics uses DNA to determine the relationships between living organisms

q Ribosomal RNAs can be used to measure the distance between ALL life

q 3.5 billions years is a long time to diversify – microorganisms are diverse!

q Most Bacteria have never been cultured – but we know they’re there!

q Some characteristics of the common bacterial phyla:

q Proteobacteria
q Firmicutes

q How biochemical characterization, phylogenetics, and mas spectrometry can be used to identify
microorganisms
Further Reading

Chapter 16 Brock Biology of Microorganisms 15th edition