Dr S.

Brown
 Molecular Phylogeny
 The evolutionary history of a group of organisms is
called its phylogeny

 Phylogeny is inferred indirectly from nucleotide

sequence data

 Premise is that bacteria ( and all organisms) are

related by descent and that the sequence of a
bacterium’s genome is a record of the
bacterium’s ancestry
 Molecular Phylogeny
 Evolution is a process of inherited nucleotide
sequence change

 Analysing DNA sequence differences among

bacteria allows the reconstruction of their
phylogenetic history
 Genes used in Phylogenetic Analysis

 16S rRNA

 18S rRNA (eukaryotes)

 These are called small subunit ribosomal RNA

(SSU rRNA)
 Genes used in Phylogentic Analysis
SSU rRNA genes are used extensively for sequenced
based evolutionary analysis because they are:
 Universally distributed

 Functionally constant

 Sufficiently conserved

 Adequate length (provides good view of evolution)

 Genes used in Phylogentic Analysis

 Carl Woese used SSU rRNA to establish the three

domains of life

 Received the 2003 Craford Prize

 Ribosomal Database Project II contains over

440,000 SSU rRNA sequences
 Genes used in Phylogenetic Analysis

 23S large –subunit rRNA (LSU rRNA) is

phylogenetically highly informative

 Protein synthesis elongation factor Tu (Ef-Tu)

 Heat shock protein Hsp60

 tRNA synthetases
 Genes used in Phylogentic Analysis

 The amount of variation present in SSU rRNA gene

sequence is not enough to provide good
descrimination between bacterial species
 Genes used in Phylogenetic Analysis
 The use of single gene to study bacterial evolution
has drawbacks as that given gene may not be
present in all microorganism eg recA

 RecA is only present in bacteria

 Can remedy by using multiple genes

 Evolutionary Analysis
( Analytical methods)
 1. Obtaining DNA sequences

 2. Sequence alignment

 3. Phylogenetic Trees

 4.Tree reconstruction
 Universal phylogenetic tree determined from comparative
rRNA gene sequence

Analysis of over 30 genes from 190 species of Bacteria, Archaea and Eukarya
Dr S. Brown
MICR2211
 Learning Outcome
 12.8 The Species Concept in Microbiology

 12.9 Taxonomic Methods in Systematics

 12.10 Classification and Nomenclature

 Reference: Brock Biology of Microorganism 14th ed
 Pg 393-399
The Species Concept in Microbiology

 Biological species concept not meaningful

 This is because prokaryotes are haploid and

do not undergo sexual reproduction
 The Species Concept in Microbiology

 Phylogenetic species concept is an alternative

 Prokaryotic species is a group of strains

that, based on DNA sequences of multiple
genes,

 cluster closely with others phylogenetically

and are distinct from other groups of strains
 The Species Concept in Microbiology

 Current definition of prokaryotic species

 Collection of strains sharing a high degree of

similarity in several independent traits

 Most important traits include 70% or greater

DNA–DNA hybridization

 and 97% or greater 16S rRNA gene sequence

identity
The Species Concept in Microbiology

 16S rRNA gene sequences are useful in

taxonomy; serve as "gold standard" for the
identification and description of new
species

 Proposed that a bacterium should be

considered a new species if its 16S rRNA
gene sequence differs by more than 3%
from any named strain, and a new genus if
it differs by more than 5%
 Microbial Systematics
 Systematics is the study of the diversity of
organisms and their relationships

 It links phylogeny with taxonomy

 Organisms are characterized, named, and

placed into groups according to their natural
relationships
 Microbial Systematics
 Traditionally taxonomy was based on
phenotypic characteristics

 Now incorporating DNA sequences

 Polyphasic approach :
 genotypic
 Phenotypic
 phylogenetic
 Microbial Systematics
 Phenotypic: morphological, metabolic,
physiological, and chemical characteristics of
cell

 Genotypic: comparative aspects of cells at the

level of genome

 Group organisms based on similarities

 Complemented by phylogenetic analysis

 Organisms habitat and ecology is also
important
Microbial Systematics
Phenotypic Analysis
 Phenotypic Analysis
FAME (Fatty Acid Methyl Ester) Analysis
 Identifies the types and proportions of fatty
acids present in the cytoplasmic membrane
and outer membrane
 Major phenotypic trait

 Used in clinical, public health, food and water

inspection labs
 Widely used in characterizing new species
FAME (Fatty Acid Methyl Ester) Analysis
FAME (Fatty Acid Methyl Ester) Analysis
FAME (Fatty Acid Methyl Ester) Analysis

Drawbacks
 Rigid standardization required because fatty
acid profiles vary as a function of temperature,
growth phase, growth medium

 FAME analysis limited to those organisms that

can be grown under the same conditions as
those in the database
 Microbial Systematics
Genotypic Analysis

 Provides insight at the DNA level

 Several methods of genotypic analysis exist

 Genotypic Analysis
DNA-DNA hybridization
 If two organisms share many similar
(identical ) genes, their DNA should be
expected to hybridize to one another

 This should be in approximate proportion to

the similarities in the gene sequence

 Provides a rough index of similarity to each

other

 Complements 16S rRNA sequencing

Genomic hybridization as a taxonomic tool
 DNA profiling methods
Repetitive extragenic palindromic PCR (rep-
PCR)

 Based on the presence of highly conserved

repetitive DNA elements interspersed
randomly around the bacterial genome

 Number and positions of these elements vary

between strains of a species that have diverged
in genomic sequence

 Amplification of these sequences results in a

profile (banding pattern) that is strain -
specific
Repetitive extragenic palindromic PCR (rep-PCR)
 DNA profiling methods
Multilocus sequence typing (MLST)

 Involves sequencing several different

housekeeping genes from an organism

 And comparing their sequences with sequences of

the same genes from different strains of the same
organism

 Restricted to strain differentiation due to

sensitivity
Multilocus sequence typing (MLST)
 Bacterial Various "house-
chromosome keeping" genes Analyze alleles.
Strains
1–5
New isolate or
clinical sample New strain
Strain 6
Isolate DNA. Amplify 6–7 Sequence. Compare with
target genes. other strains
and generate Strain 7
tree.

Figure 12.27
 DNA profiling methods
Phylogenetic analysis

 Analysis of individual gene

 Mulitigene sequence analysis

 Whole-genome sequence based analysis

 Definition of prokaryotic species
 A prokaryotic species is defined operationally
as:

 A collection of strains sharing a high degree of

similarity in several independent traits
 DNA-DNA hybridization of 70% and greater

 16S rRNA gene sequence of 97% or greater

 Microbial systematics
 Nomenclature is the application of formal
rules for naming organisms

 Following the binomial system of

nomenclature, prokaryotes are given genus
names and species epithets:
 Eg Bacillus subtilis (slender)
 Bacillus cereus (waxen)
 Bacillus megaterium (big beast)
 Microbial systematics

 The assignment of names for species and

higher groups of Bacteria and Archaea is
regulated by the Bacteriological Code-

 The International Code Of Nomenclature of

Bacteria
 Microbial systematics

 Because taxonomy is largely a matter of

scientific judgement, there is no ‘official’
classification of Bacteria and Archaea.

 Presently the classification system most widely

accepted is the ‘Taxonomic Outline of the
Prokaryotes’ in the Bergey’s Manual of
systematic Bacteriology
Microbial systematics
Culture Collections
Classification and Nomenclature

 Formal recognition of a new prokaryotic

species requires:
 Deposition of a sample of the organism in
two culture collections

 Official publication of the new species

name and description in the International
Journal of Systematic and Evolutionary
Microbiology (IJSEM)
Classification and Nomenclature

 The International Committee on Systematics

of Prokaryotes (ICSP)

 is responsible for overseeing nomenclature

and taxonomy of Bacteria and Archaea