What Have We Learned From Unicellular Genomes?

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What Have We Learned From Unicellular Genomes?

• Propionibacterium acnes
• Bacteroides thetaiotaomicron
• Mycoplasma genitalium
• Mimivirus
• Cyanobacteria
• Plasmodium
• Yeast

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Why do I get so many pimples?

• The genome of Propionibacterium acnes was
  sequenced in July of 2004.
• P. acnes lives in sebaceous cysts and sometimes
  stimulates and immune response.
• A group in Paris, along with two groups in
  Germany sequenced P. acnes.
• They found 2,333 genes in its 2.6 Mb genome.
• 68 of these had orthologs in other species.
• 20 had none, and 12 encoded only RNA.

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Anatomy of a pimple
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Genome-wide evaluations

• A first step following bacterial genome
  sequencing is finding the ori and terminus for
  replication.
• GC skewing (non-uniform distribution of Gs Cs
• Oris tend to have the lowest skew, while termini
  have the highest.
• Genes that have originated by horizontal transfer
  are identified using a sliding window to find
  segments with abnormal GC content.
• Codon bias is also used to detect HT. Immunogenic
  and metabolic genes were detected.

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Transcriptional Phase Variation

• During finishing, it was found that P. acnes had
  a variable of Gs associated with some genes.
• It is hypothesized that the initiation of
  transcription depends on the of consecutive
  Gs.
• As rows of Gs are replicated, the will change.
• This leads to a mixed population of bacteria with
  varying degrees of protein production.
• This diverse population is optimized to respond
  differentially to various skin treatments.

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Digesting Our Cells For Food

• P. acnes was found to be able to grow
  anaerobically as well as aerobically.
• Cells produce many enzymes that are able to
  degrade lipids, ester, and amino acids.
• Some of these degradation products increase
  adhesion to our cells.
• Many of the digestive enzymes contain a motif
  (LPXTG) that targets them to the cell wall.
• Hyaluronate lyase is also found on the surface of
  the bacteria, this destroys the extracellular
  matrix that binds our cells together.

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Stimulating the Immune Response

• P. acnes produces 5 CAMP factors (secreted
  proteins that bind antibodies) that can form
  pores in the cell membrane.
• A dipeptide motif (PT) is present in certain
  proteins, this motif is also found in M.
  tuberculosis.
• The bacteria also has at least 7 heat shock
  protein genes.
• Porphyrin is also secreted, which produces toxic
  forms of oxygen, further stimulating the immune
  response.

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Withstanding the Environment

• P. acnes can signal nearby cells that something
  has changed in the environment.
• Sensors called two-component systems (1 to sense
  1 to signal) exist in some bacteria, P. acnes
  has 10 pairs.
• Quorum sensing is the ability to detect
  conditions of overcrowding. The LuxS gene is
  expressed in these instances, which produces a
  universal signal for interspecies communication
  among bacteria.
• Biofilms of meshed-together cells protect
  themselves.

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Are all bacteria living in us bad for us?

• An average human body is composed of about 1013
  cells.
• Our intestines have about 1010 microbes/ml and
  contain at least 1,000 ml.
• A majority of the cells in our bodies may be
  bacteria! (500 - 1,000 different species)
• This accounts for 2-4 million non-human genes
• Bacteroides thetaiotaomicron constitutes a
  substantial portion of our intestinal flora.
• A group from Wash. U. in St. Louis sequenced its
  genome.

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• Overview of the Genome
• B. thetaiotaomicrons genome contains 6.3 Mb, as
  well as 4,779 genes (and a 33 kb plasmid).
• 58 of ORFs have known function, 18 have
  orthologs of no known function, and 24 have no
  homology with known proteins.
• COGs (functional categories of genes) are
  determined following sequencing to create an
  overview of a given genome.
• Many of the genes specialize in sugar uptake,
  cell wall synthesis, environmental sensing and
  signaling, as well as transposition.

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Major COGs

• Sugar metabolism- 170 genes fit into this
  category, most bacteria have a set of 23.
• 61 of these appear to be secreted, this not only
  benefits other bacteria but us as well.
• 163 paralogs of 2 genes (SusC SusD) import
  sugars into the cytoplasm of the microbe.
• Many two-component genes are present for
  signaling, some of these interact with s factors.
• 63 tranposons are present, which may help spread
  antibiotic resistance.

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Does Size Matter?

• The coding capacity for this genome is very high
  (89 coding DNA) but it has a lower ratio of gene
  to genome size than expected.
• This was a paradox until it was determined that
  the ORFs of this microbe are unusually large. It
  is unclear why this is the case.

Summary

• Gut symbionts provide us with predigested sugars,
  stimulated blood vessel formation, crowd out
  pathogens, sequester limited resources, and
  stimulate our mucosal layer.

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Can Microbial Genomes Become Dependent Upon Us?

• In the microbial world, if you dont use it- you
  lose it.
• Mycoplasma genitalium has one of the most reduced
  microbial genomes and the 2nd smallest bacterial
  genome with 580 kb (the smallest is N. equitans
  with 490 kb).
• TIGR sequenced its genome in 1995.
• 470 ORFs were found, 96 of which have no known
  orthologs.
• M. genitalium has an 88 coding capacity.

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Genes that have been lost

• M. genitalium has presumably lost many genes
  involved in the synthesis of amino acids,
  cofactors, cell envelope, and regulatory factors.
  It has only 1 s factor.
• The microbe has retained genes for energy
  metabolism, fatty acid and phospholipid
  metabolism, nucleotide production, replication,
  transcription, and protein transport.
• The only category overrepresented is translation,
  namely rRNA and tRNA genes.

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What is the Minimum of Genes?

• Craig Venter, along with Hamilton O. Smith, is
  trying to construct an organism with the fewest
  possible genes.
• A new field called synthetic biology seeks to
  synthesize a functioning genome de novo.
• A better understanding of evolutionary principles
  and genome circuitry is sought.
• Japanese European scientists have tried to
  identify the essential genes of B. subtilis.
• They have found that only 192 genes are
  indispensable to life.

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Do all Viruses have Small Genomes?

• Most viral genomes are much smaller than
  bacterial ones
• HIV- 9,200 nt
• WNV- 10,962 nt
• SARs- 29,727 nt
• T7- 39,900 nt
• l- 48,502 nt
• In 2003, a new virus that infects amoeba was
  isolated that has 1.2 Mb! A group in Marseille,
  France sequenced Mimivirus, as it is called.

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Mimivirus Genome

• 1,262 ORFs were identified, the coding capacity
  is 90.5.
• Like most viruses, the genome is linear, but it
  has inverted repeats at both ends by which it may
  circularize, perhaps during replication.
• Isoleucine is used twice as often as usual, and
  there is a strong codon bias for codons lacking G
  or C. The genome is 28 GC.
• Mimivirus is overrepresented in genes for
  translation, posttranslational modification, and
  amino acid transport and metabolism.

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Is Mimivirus Alive?

• The genome of Mimivirus resembles bacterial,
  Mimivirus even stains Gram , is it a virus?
• In 1957, the definition of a virus was proposed
• 1) smaller than .2 microns
• 2) possesses DNA or RNA, not both
• 3) not able to synthesize its own proteins
• 4) cannot generate energy from substrates
• 5) cannot grow by binary fission
• Mimivirus only satisfies the 4th category, we are
  not sure about the 5th.

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What is it then?

• Mimivirus has blurred the distinction between
  prokaryotes and viruses.
• It is hypothesized that, like M. genitalium,
  Mimivirus has lost genes over time.
• We will learn of more obligate intracellular
  parasites later in class.
• Mimivirus may resemble some of the earliest forms
  of life that was able to replicate independently
  until it became a parasite.

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Genomes Reflect an Organisms Ecological Niche

• Cyanobacteria are the most productive
  phytoplankton in the world.
• The two most abundant genera of cyano-bacteria
  are Prochlorococcus and Synecho-coccus. 3
  genomes in the former group and 1 in the latter
  were sequenced in 2003.
• Individual cells from both genera are referred to
  using a numbering system to indicate different
  ecotypes. Species designations are difficult to
  assign still, Prochlorococcus was discovered in
  the 1990s.

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Prochlorococcus
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Dot Plot Align-ment
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Prochlorococcus MED4 vs. MIT9313

• These ecotypes share 1,352 orthologs.
• Short diagonal segments indicate synteny.
• A negative slope indicates that the segment was
  inverted in one type relative to the other.
• Segments with positive slope but located off the
  diagonal indicate chromosome recombinations.
• Genes along the axis means they are missing from
  the other ecotype, MED4 has 364 genes not found
  in MIT9313, which has 923 genes not found in the
  other.

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pcb gene family

• A major difference between the ecotypes is in the
  pcb gene family, which encode chlorophyll-binding,
  light-harvesting antenna complex proteins that
  help capture a wider spectrum of light.
• MED4 (high light) has only 1 pcb gene
• MIT9313 (medium light) has 2 (A B)
• SS120 (low light) has 8 (A-H)
• MED4s gene does not respond to changes in Fe3
  but MIT9313s is induced 7-fold and SS120s is
  induced 23-fold.

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MED4s Small Genome

• MED4s genome is the smallest known for a
  photoautotroph and may represent the minimum for
  a photosynthetic organism.
• MED4 appears to have lost genes over time.
• A more stream-lined genome means a narrower
  ecological range that an organism is adapted for.
  Synechococcus has the largest genome of this
  group and the largest ecological range as well.
• People have proposed seeding the ocean with Fe3
  to help stimulate CO2 consumption.

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Gene deletions in Cyanobacteria
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Malaria

• Malaria, although it rarely makes news headlines,
  is a daily threat to the 3 billion people who
  live in tropical climates.
• In 2002, about 500 million people were infected.
  About 2.7 million people die each year (about 90
  of these are lt 5 years old).
• The cause of malaria has been known for 100 years
  but we still cant stop its spread.
• The most lethal form of malaria is caused by
  Plasmodium falciparum.

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Lifecycle of Plasmodium
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RBC Infection

• The most vulnerable time for Plasmodium is during
  the RBC infection stage.
• The parasite must force its way into a RBC
  without rupturing any plasma membranes.
• Three structures are important during infection
• 1) extracellular coating to make cells sticky
• 2) apical end of cell must be oriented downward
• 3) apicoplast is an internalized algal symbiont

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Plasmodium Genomes

• Plasmodium actually has three genomes nuclear,
  mitochondrial, and apicoplastic.
• Pulse-field gel electrophoresis to separate
  chromosomes, followed by shotgun genome
  sequencing was used on Plasmodium.
• This proved to be the most AT-rich genome
  sequenced so far (19.4 GC).
• The 22.9 Mb genome has 52.6 coding capacity and
  5,268 ORFs (60 of which have no known function,
  the largest of any genome).

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Tricking the Immune System

• The genes of Plasmodium that are responsible for
  binding to RBCs and for avoiding the immune
  system are located near the telomeres of this
  eukaryote.
• Genes located near Plasmodium telomeres are
  replicated many times, all three gene families in
  these categories (var, rif, stevor) are
  polymorphic.
• There are 59 var paralogs, 149 rif, and 28
  stevor. This may account for our immune systems
  lack of ability to deal with this parasite

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The Plasmodium Proteome

• 1 of proteins are used for host cell invasion
• 4 help evade the immune response
• 31 are integral to the membrane
• 14 are enzymes (about 4x lt most proteomes)
• 10 are transported to the apicoplast
• 60 have unknown function
• The Krebs cycle is present, but the organism
• grows anaerobically and only uses this cycle for
• heme biosynthesis (which it could get from us)

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Apicoplast Proteome

• Similar to a chloroplast in origin but used for a
  different purpose now.
• Only two photosynthetic orthologs remain.
• This organelle synthesizes fatty acids,
  isoprenoids, and heme groups.
• Nuclear proteins sent here assist in DNA
  replication repair, transcription, translation,
  posttranslational glycosylation, protein import,
  and protein degradation.

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Comparing Plasmodia

• The Plasmodium sequencing project took 45 people
  6 years to complete.
• At the same time, other groups were working on P.
  yoelii, which infects rats and is used as a model
  organism for malaria research.
• Unfortunately, this latter genome was never
  finished, making comparisons difficult.
• P. yoelii has 600 additional ORFs, and the two
  have 3,310 genes in common (56).
• Is this similar enough to make a good model
  organism?

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Malaria Treatment Options?

• Recently, a German American team used reverse
  genetics (starting with a gene sequence and
  deducing its function) to target a gene in the
  production of a knock-out strain. This strain is
  expected to be less pathogenic than wild type.
  Mice injected with this strain were protected for
  30 days.
• Even if a better drug were produced, funding and
  health care infrastructure are lacking in many
  problem areas. Very little is spent on malaria
  research.

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Yeast
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Yeast Genome

• The S. cerevisiae genome was sequenced in 1996.
• It took over 600 scientists in Europe, North
  America, and Japan working together to seqeunce
  the 12 Mb genome.
• Yeast has a 70.3 coding capacity, higher than
  Plasmodium but lower than all bacteria.
• There is a gene every 2 kb in yeast, one every 6
  kb in C. elegans, and one every 30 kb in humans.
  Eukaryotes have more junk DNA than prokaryotes
  and enhancers, promoters, and introns add
  substantially to the size of eukaryotic genes.

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Chromosome Structure in Yeast

• The 4 smallest chromosomes in yeast have a
  unique structure. It was known from using YACs
  that chromosomes smaller that 150 kb were not
  stable in yeast. These chromosomes are
  relatively gene-poor and undergo recombination at
  high frequencies, perhaps to protect the larger
  ones from the same fate.
• Transcriptionally silent genes are found in the
  sub-telomeric regions of many chromosomes, this
  may help identify the right and left sides of a
  chromosome.

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Yeast Chromosomes
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Evolutionary History of Yeast

• There were a substantial number of genes found in
  duplicate copies in yeast.
• It was proposed that yeast had undergone
  duplication events at some point in time.
• Many regions of chromosomes are syntenic with
  regions on other chromosomes. Such paralogs are
  seen as evolutionary experiments where one gene
  can drift to provide new specialized functions.
• Some genes were initially thought to be extra
  copies but experiments proved their difference

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Predictions for the Future

• The authors of the landmark 1996 yeast sequencing
  publication made the following predictions
• 1) they described plans to produce a collection
  of single, double, and even triple KO mutations
• 2) they addressed the value of making all genome
  sequences publicly available.
• 3) They felt WGS sequencing of large genomes was
  not feasible.
• 4) They looked forward to comparing yeast with
  the S. pombe as well as the human genome.

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Better Annotation

• A number of yeast genomes have been sequenced
  since 1996. With these, the need to annotate
  genes based on GO, Gene Ontology, became clear.
• Improvements in computers, search algorithms, and
  the increased volume of genes in the databases
  lead to better annotation.
• The original 5,885 ORFs annotated has been
  increased to 6,672, many below the original
  cutoff of 100 codons