Microbial Systematics Flashcards
What is the understanding for the origin of eukaryotes (evolution)?
endosymbiosis hypothesis
What is the endosymbiosis hypothesis?
the hypothesis that supports that the origin of mitochondria and chloroplasts were from a symbiotic association (engulfing) of prokaryotes with another cell
Who first formulated the endosymbiont hypothesis?
Lynn Margulis (previously, Lynn Sagan), an English scientist
Which scientist famously disagreed with Lynn Margulis’s hypothesis of endosymbiotic origin of eukaryotes? what did he do?
Carl Woese
he developed the 16S rRNA method of sequencing bacterial genomes/phylogenetically categorizing prokaryotes
How did Woese, inadvertently, contribute to Margulis’s endosymbiont hypothesis?
he disagreed, but the 16S rRNA method ended up being the best evidence to support Margulis’s hypothesis
What are the 2 hypotheses of the endosymbiont origin of eukaryotes?
eukaryotic cells originated containing a nucleus and endosymbiosis to attain mitochondria and chloroplasts occurred later
eukaryotic cells came from an intracellular association of an O2-consuming bacterium into an Archaeal host to produce the mitochondria
On a basic phylogenetic tree, how are bacteria, eukarya, and Archaea related?
from some unknown ancestor, bacteria and archaea split off
from the archaea branch, eukaryotes split off
= eukarya are more closely related to archaea than bacteria
What do both hypotheses of endosymbiosis suggest about the eukaryotic cell?
it’s chimeric (origins from both Archaea and Bacteria)
What supports the suggestion that eukaryotic cells are chimeric?
eukaryotes have lipids and metabolisms close to bacteria
eukaryotes have transcription and translational machinery close to Archaea
What did Woese contribute to phylogenetic studies in 1970s?
the use of 16S rRNA to
establish that prokaryotes are actually bacteria and archaea (not monophyletic)
create a universal framework for studying bacterial phylogeny
Why did Woese choose the 16S ribosomal subunit of RNA?
it was practical and plays a key role in bridging the functions of both subunits
it’s in the small subunit with 20s which binds mRNA
Describe the ribosomal RNA subunits
large subunit: 23s and 5s
small subunit: 16s + proteins
What makes small subunits of rRNA ideal for studying phylogeny?
they are in all domains of life
in all domains, they have the same function
they evolve slowly (are conserved)
they have enough nucleotides to study and determine reasonable significant differences
What is the small rRNA subunit in prokaryotes? eukaryotes?
pro: 16S rRNA
euk: 18S rRNA
When looking at the nucleotide sequence of a small rRNA subunit, what do the V regions mean? what are the white regions?
V = variable, indicate different prokaryotes
white regions are the conserved, essential regions that make prokaryotes prokaryotes
What is phylogeny?
the estimated evolution of organisms predicted from nucleotide sequencing
What is the universal phylogenetic tree based on?
SSU rRNA genes
Approx how many phyla are in bacteria according to Bergey’s manual?
24
What is LUCA?
the origin of life, the unknown ancestor of bacteria, archaea and eukarya
Pre-Darwinian evolution
How many phyla of Archaea in Bergey’s?
2
Crenarchaeota
Euryarchaeota
How many phyla of Eukaraya in Bergey’s?
How many phyla of Bacteria are known? (not just in the bergey’s)
there’s 80 known
Are most of the bacteria phyla cultured or uncultured?
most are uncultured and just defined by environmental sequences
What does it mean for bacteria to be phenotypically diverse?
physiology and phylogeny not necessarily linked
How many known phyla of Archaea? Not just in Bergey’s. What are they?
7
Crenarchaeota
Euryarchaeota
Nanoarchaeota
Korarchaeota
Thaumarchaeota
and 2 that are not culturable species
Where did eukaryotic organelles originate?
bacteria
mitochondria from proteobacteria
chloroplasts from cyanobacteria
What eukaryotic organelles came from proteobacteria?
mitochondria
What eukaryotic organelles came from cyanobacteria?
chloroplasts
What characterizes the 3 domains of life (Bacteria, Archaea, Eukarya)?
phenotypic properties
What is comparative rRNA?
molecular sequences by:
amplifying genes that encode SSU rRNA
sequencing amplified gene
analyzing sequence by comparing and referencing other sequences
What can be used to amplify SSU rRNA genes?
PCR
How has PCR amplification been useful for molecular sequencing?
PCR amplification helps show features of microbial community structure and interactions
What are the steps for using 16s RNA to culture bacteria?
- isolate DNA
- use PCR to amplify 16s RNA
- run amplified gene on agarose gel to check nucleotide length (size)
- Sequence
- align sequences to generate a phylogenetic tree by comparing sequences with other species
What is different about culturing bacteria from sequencing ex. for Crenarchaeota?
there is no cloning step in culturing because it’s a pure culture
Why is 16s rRNA useful in culturing and making a phylogenetic tree?
16s rRNA has a highly conserved region = bacteria
and hypervariable regions = species
How are the hypervariable regions of 16s rRNA used to distinguish species of bacteria?
can look at either the similarity or the distance (Difference) coefficient
aligning the sequences with other species to count the # of different nucleotides divided by the total number of nucleotides
what is the first step of sequence analysis?
aligning the cultured sequence with sequences from homologous genes from other strains or species
why is it important to align sequences?
lining up the homologous genes show the accurate differences
ex. unaligned had 11 differences vs. aligned had 3
What is a phylogenetic tree?
an illustration of the relationships between sequences
What does the branch length of a phylogenetic tree represrent?a
the number of changes that have occurred on that branch
What is an unrooted tree?
a phylogenetic tree that has no unknown outgroup (ex. the universal tree)
What is a rooted tree?
a phylogenetic tree that has an outgroup
How does evolutionary analysis make phylogenetic trees?
cladistics (character-state) methods
What are cladistic methods?
methods of creating a phylogenetic tree based on character-state methods
phylogenetic relationships are determined by changes in individual nucleotide positions = characters are used to define monophyletic groups
what are the 2 common cladistic methods?
algorithms
optimality criteria
Describe how algorithms are used as a cladistic method?
algorithms are a programmed series of steps that produces a matrix to display difference coefficients
lowest distance coefficients make a clade
What are 2 types of algorithms used for cladistic methods?
unweighted pair group and arithmetic mean
neighbour joining methods
What is optimality criteria? What 3 things does it assume/is it based on?
a common cladistic method of developing a phylogenetic tree that picks the best possible tree
based on parsimony, maximum likelihood, and Bayesian analysis
What are the 3 steps in making a phylogenetic tree based on cladistics?
- align sequences
- calculate distance coefficients and make a matrix
- add nodes to join lineages with lowest distance coefficients
What is the largest contributor to genetic diversity?
gene mutation
what are mutations?
changes to the nucleotide sequence of a genome
How do mutations occur?
caused by errors in replication
UV radiation
How can gene mutations lead to diversity?
adaptive mutations improve fitness and increase survival and chances of passing on the gene
What are other types of genetic changes that don’t involve mutations?
gene duplication
horizontal gene transfer (ex. plasmid or transformation)
gene loss (streamlining genome to reduce redundancy)
What are the 3 steps of the evolutionary process?
recombination
selection
genetic drift
what is recombination?
the physical exchange of DNA between chromosomes
what is selection?
how evolution occurs
based on the fitness of a gene type
generally increases chance of survival and is heritable
what is genetic drift?
an event that causes random gene frequencies to change over time
What is transformation?
when bacteria uptake DNA from their environment
What is conjugation?
bacteria to bacteria transfer of genetic material
usually through the plasmid
most common
What is transduction?
when a virus (bacteriophage) infects a bacteria and the virus incorporates part of the host’s DNA
What traits would natural selection be favouring for in bacteria?
ones that improve growth
or optimal activity (ex. increasing enzyme catalytic speed)
T or F: genetic drift is a random event that effects gene frequencies randomly
true
Why is defining microorganism species difficult?
species can be comprised of a range of individuals with different traits
this can conflict with the Biological Species Concept
Why does the speciation of microorganisms conflict with the Biological Species Concept?
because the BSC requires sexual reproduction as a means for speciation in eukaryotes, but bacteria do not sexually reproduce
bacteria are haploid
also because some species have individuals with a wide variety of different traits
What can be used to define speciation for microorganisms?
molecular clocks (chronometers)
What are molecular clocks (chronometers)?
genes and proteins that are measures of evolutionary change
What is a major assumption of the molecular clock method to determine speciation for microorganisms?
that nucleotide changes (mutations) are:
- occurring at a constant rate
- neutral
- random
How can 16s rRNA be used to determine species?
if 97% or more of the nucleotide sequence is similar, they are likely the same species
if more than 3% are different, they are likely different species
How long is the nucleotide sequence of 16s rRNA ?
1500 nucleotides
What are core genes?
genes that exist within all members of a species
What are pan genes?
the core genes + the genes that are different from other members of the same species
What type of mutations play a key role in microbial genome dynamics? why?
deletions
because bacteria are streamlining their DNA
As more genomes are analyzed, how does the number of genes in the core genome v the number of genes in the pan genome change?
the core genome stablizes and plateaus as less genes are added to the core genome from looking at new genomes
the pan genome continues to increase because new different genes are being added to the analysis with each new genomes
What is an alternative to the biological species concept for microbiology?
phylogenetic species concept
What is the phylogenetic species concept?
a prokaryotic species is a group of strains cluster closely with others phylogenetically based on their DNA sequences of multiple genes and cluster distinctly from other groups of strains
T or F: there is a universally accepted concept of species for prokaryotes
false there is not
What is the current definition of a prokaryotic species?
a prokaryotic species is a group of strains that have a high degree of similarity for multiple different traits
key traits include > 70% DNA-DNA hybridization
and
> 97% 16s rRNA nucleotide sequence
What is the ‘golden standard’ for identification and description of new prokaryotic species?
16s rRNA sequence analysis and phylogenetic analysis
When is a new species defined?
if more than 3% of its 16s rRNA gene sequence differs from other named strains
When is a new genus defined?
if more than 5% of its 16s rRNA gene sequence differs from other named strains
What is DNA-DNA hybridization?
when 2 genomes are hybridized to look at the similarities in gene sequences
Describe the method of DNA-DNA hybridization - what do the results mean?
PREP:
extract genomic DNA from 2 different organisms
organism 1 will be the probe DNA - cleave and label with P
organism 2 will be the target DNA - cleave DNA
denature DNA with heat
EXPERIMENT:
mix DNA on nylon membrane, adding excess probe DNA
a) control: probe + probe = 100% hybridization
b) treatment: probe + target = 25% hybridization
the results for the control means that > 70% = same species
the results for the treatment: 25% hybridization < 70% = different species and genus
What is the purpose of DNA-DNA hybridization?
to provide a rough estimate for the similarity between 2 organisms
When does hybridization suggest the strains are of the same species? genus?
> 70% hybridization (similarity) = same species
> 25% hybridization = same genus
When is hybridization useful?
in combination with 16s rRNA sequencing and if trying to distinguish 2 very similar organisms
How many species of bacteria and archaea are currently known?
~7000, but not all are culturable
How many phyla of bacteria have been accepted into the Bergey’s manual?
24
How many phyla of bacteria are there if not considering the Bergey’s manual guidelines (required to be culturable)?
> 80 phyla
How many phyla of Archaea accepted by Bergey’s?
2
How many phyla of Archaea are known (not necessarily in the Bergey’s)?
7
What are the 2 phyla of Archaea accepted by Bergey’s?
Crenarchaeota
Eukaryoata
What is taxonomy?
the science of identification, classification and nomenclature of organisms
What is systematics?
the study of the diversity of organisms and their relationships = connects phylogeny with taxonomy
the framework for evolution
When was the phylogeny of bacteria established? by who?
in the 1970s by Woese
T or F; there’s only one way to identify and describe new species of bacteria
false there’s multiple
What approach is most often used to ID and describe new species of bacteria?
polyphasic approach to taxonomy
What are the 3 methods of the polyphasic approach to taxonomy?
phylogenetic analysis
genotypic analysis
phenotypic analysis
What is phylogenetic analysis in taxonomy?
analyzing the 16s rRNA gene sequence
Why is phylogenetic analysis limiting for identifying different species?
phylogenetic analysis focuses on the 16s rRNA sequence which helps identify that the genome is bacterial, but does not help distinguish between species of bacteria because of the high degree of similarity
What can be used to support phylogenetic analysis in defining species?
multigene sequence analysis
examines complete sequences and makes comparisons using cladistic methods
provides greater accuracy for separating species
What is multilocus sequence typing (MLST)?
a taxonomic method used in systematics that sequences different ‘housekeeping genes’ from an organism
Why is MLST a helpful taxonomic method?
it is accurate and specific enough to differentiate between closely related strains
What are housekeeping genes?
genes that are consistently expressed in different tissues in an organism
essential genes
conduct cellular maintenance
conserved within a species*
Describe the process of MLST method
isolate DNA from bacterial chromosome
amplify target genes (housekeeping genes)
sequence the amplified genes
analyze alleles and compare with other strains to create a tree
How was MLST used to distinguish Shigella flexneri from E. coli?
housekeeping genes were isolated, amplified, sequenced and analyzed and found that there 7 different housekeeping genes related to DNA replication, metabolism, and signal transduction that differed from E. coli
What is ribotyping?
a taxonomic method in systematics used to identify microbes through analyzing DNA fragments produced using restriction enzyme digestion of genes that encode for 16s rRNA
it focuses on a single gene
What are the pros of ribotyping?
it’s highly specific and very fast sequencing
What are the practical applications of ribotyping?
used for bacterial ID in clinical diagnostics, microbial analyses of food, water and drinks
What is genome fingerprinting?
the generation of DNA fragment patterns for analysis of genotypic similarity between strains
What are 3 types of genome fingerprinting?
- ribotyping
- repetitive extragenic palindromic PCR (rep-PCR)
- amplified fragment length polymorphism (AFLP)
what is the major difference between ribotyping and rep-PCR and AFLP?
ribotyping looks at one gene whereas rep-PCR and AFLP look at many genes located randomly in a genome
What type of taxonomic method for systematics is becoming more common?
whole genome sequencing
What are benefits of whole genome sequencing?
analyses of the entire genome tell us about
- structure (Size, number of chromosomes G:C ratio)
- gene content
- gene order
Would a parasitic or free-living bacteria have a larger genome? why?
larger genome = free-living
a parasitic bacteria would be able to streamline their genome by deleting genes that they don’t need cause they’re receiving inputs from their host
what are pseudogenes?
non-protein coding genes
How does the amount of protein coding genes in M. tuberculosis compare to M. leprae? pseudogenes?
protein-coding: much less in M. leprae
pseudo: much more in M. leprae
both have similar sized genomes
what is phenotypic analysis?
a taxonomic method used in systematics to examine the morphological, physiological and chemical characteristics of a cell
What is fatty acid analysis (FAME)?
FAME = fatty acid methyl ester
examine the type and proportion of fatty acids in a membrane lipid for specific prokaryotic groups
What is challenging about fatty acid analysis?
FAME profiles can vary depending on temperature, growth phase, and growth medium
so strict standardization for assessment is required
What are 5 classes of FAs in bacteria?
saturated
unsaturated
cyclopropane
branched
hydroxy
Describe the process of fatty acid analysis in identifying an organism
FAs extracted from a bacterial culture
FAs are derivatized into methyl esters (derivates of FAs)
gas chromatography is used to look at the FAME profile
the peaks and patterns are compared to described patterns in database
What are API strips?
an example of phenotypic analysis (conventional)
that looks at 20 biochemical characteristics and is tested to be positive or negative
similarities are compared between two organisms
What is BIOLOG?
a phenotypic analysis method with 90+ tests
what is classification?
the organizing of organisms into more and more inclusive groups based on phenotypic or evolutionary similarities/relationships
how are prokaryote species and genera named?
the binomial nomenclature system of biology
What regulates the naming of prokaryotes?
International Code of Nomenclature of Bacteria
What are the 3 major references for bacterial diversity?
Bergey’s Manual of Systematic Bacteriology - phylogenetic classification
The Prokaryotes
NCBI website (GenBank)
what are bacteria phyla in the Bergey’s manual are starred to know?
Chloroflexi
Cyanobacteria
Chlorobi
Proteobacteria
Firmicutes
What does formal recognition of a new prokaryotic species need?
a sample of the organism in 2 culture collections
an official publication of the new species name and description in the “International Journal of Systematic and Evolutionary Microbiology”
What body governs the nomenclature and taxonomy of Bacteria and Archaea?
the International Committee on Systematics of Prokaryotes
