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Marrying the Classic and Contemporary in Microbial Genetics
Joel GriffittsBrigham Young University
Brigham Young UniversityUndergraduate Enrollment: 29,000Life Science: 2,600Health and Human Performance: 2,500Nursing: 900
NH4+
N N
Sinorhizobium meliloti(wt) on alfalfa
Mutant R3H4on alfalfa
(Fix+) (Fix-)
The classical approach to studying the genetics of symbiosis
A new kind of mutant
Naturally incompatible strain inoculated on M. praecox
Gain-of-compatibility mutant
DivyeshChoudhri
Daniel Lindsay
Ryan VanYperen
LejdjaBeshku
Joanna Ryther
Matt Crook
Rebecca Elliott
Elise Scoggin
Clarice Harrison
Why are we here?...
Discover (v)To obtain knowledge or awareness of something not known before; to make visible; bring to view
1. Appreciation for seminal discoveries of the past
Attributes of a successful discoverer:
2. Proficiency with the tools of discovery3. Familiarity with the current frontiers of discovery4. Passionate, observant, contemplative, imaginative
1. Appreciation for seminal discoveries of the past2. Proficiency with the tools of discovery3. Familiarity with the current frontiers of discovery4. Passionate, observant, contemplative, imaginative
Attributes of a successful discoverer:
Avery, Oswald (1877-1955)Benzer, Seymour (1921-2007)Brenner, Sidney (b. 1927)Chargaff, Erwin (1905-2002)Chase, Martha (1927-2003)Crick, Francis (1916-2004)Delbruck, Max (1906-1981)Franklin, Rosalind (1920-1958)Hershey, Alfred (1908-1997)Jacob, Francios (b. 1920)Kornberg, Arthur (1918-2007)Lederberg, Joshua (1925-2008)Luria, Salvador (1912-1991)Meselson, Matthew (b. 1930)Monod, Jacques (1910-1976)Nirenberg, Marshall (b. 1927)Pauling, Linus (1901-1994)Perutz, Max (1914-2002)Sanger, Frederick (b. 1918)Stahl, Franklin (b. 1929)Tatum, Edward (1909-1975)Watson, James (b. 1928)Yanofsky, Charles (b. 1925)
Leaders in the Revolution in Molecular Biology (1943-1963)
rIIA
rIIB
1. Appreciation for seminal discoveries of the past2. Proficiency with the tools of discovery3. Familiarity with the current frontiers of discovery4. Passionate, observant, contemplative, imaginative
Attributes of a successful discoverer:
Our HeritageThe Genetic Code
DNA replicationMutation
RecombinationTransposition
TransformationConjugation
TransductionGene regulation
Plasmids
Our Tool KitChemical mutagensPCR cloningPCR mutagenesisMobilizable plasmidsSuicide/Ts plasmids
Internal fragment disruptionMarker exchange by +/- selection
Lambda-Red recombinationMini-transposonsTransductional mappingInducible expression plasmidslacZ/Fluorescent protein reportersMicroarraysMassively parallel sequencing
1. Appreciation for seminal discoveries of the past2. Proficiency with the tools of discovery3. Familiarity with the current frontiers of discovery4. Passionate, observant, contemplative, imaginative
Attributes of a successful discoverer:
Bacterial Genetics at the Cutting Edge• Structure/Function of the Cell
• Shape• Envelope maintenance
• RNA-mediated Regulation• Riboswitches• Small RNAs
• Defense Against Insults• CRISPR• Persisters• Programmed cell death/TA systems
• Intercellular communication• Cooperation• Territoriality• w/ host cells
• Horizontal Gene Transfer• Visualization• Regulation
• Functional Genomics• Single-gene knock-out collections• Genetic interaction arrays
• Synthetic Genomics• In vitro genome synthesis• Genome transplantation
• Metabolic Engineering
• More esoteric, but no less interesting:• Circadian (clock) biology• Appendage-less swimming• Magnet biosynthesis• Bdellovibrio predation• etc.
Bacterial Genetics at the Cutting Edge• Structure/Function of the Cell
• Shape• Envelope maintenance
• RNA-mediated Regulation• Riboswitches• Small RNAs
• Defense Against Insults• CRISPR• Persisters• Programmed cell death/TA systems
• Intercellular communication• Cooperation• Territoriality•w/ host cells
• Horizontal Gene Transfer• Visualization• Regulation
• Functional Genomics• Single-gene knock-out collections• Genetic interaction arrays
• Synthetic Genomics• In vitro genome synthesis• Genome transplantation
• Metabolic Engineering
• More esoteric, but no less interesting:• Circadian (clock) biology• Appendage-less swimming• Magnet biosynthesis•Bdellovibrio predation• etc.
Structure/Function of the Cell:Life without walls
Pxyl-murE+ xylose
no xylose
Some wall-less progeny (L-forms)able to survive and proliferate
Making a wall-less strain:
Wild typeWall-less
Defense Against Parasites: CRISPR
(Streptococcus thermophilus)
CRISPR=Clustered Regularly Interspersed Palindromic Repeat
(CRISPR in Strep thermophilus)
Select for resistance to phage Φ858 or Φ2972
CRISPR cassettes can block all sorts of parasitic DNAs
Streptococcus thermophilus
Escherichia coli
Staphylococcus epidermidis
Intercellular Communication:The Extracellular Death Factor
ToxinAntitoxin
Plasmid loss Toxin
unstablestable
Toxin Antitoxin
Toxins/Antitoxins in Plasmid Addiction
mazEF
The Riddle of Chromosomal Toxin-antitoxin Systems
Low dose of rif.Dense culture 90% deadI
mazEF Low dose of rif.Dilute culture 0% deadII
ΔmazEF Low dose of rif.Dense culture 10% deadIII
mazEF Low dose of rif.Dilute culture 95% deadDense culture+supernatant Contains Death Factor
IV
N N W N N
The Extracellular Death Factor (EDF) ModulatesProgrammed Cell Death Via the mazEF TA System
Horizontal Gene Transfer: Conjugation Made Explicit
(1946)
dam-
seqA-yfp
dam+
tetR/Ptet-RFP
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..Hfr
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=RFP (leaky) =SeqA-YFP
Visualizing Gene Transfer: The Strategy
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Synthetic Genomics
Phase 2: Re-booting a cell
Phase 1: Synthesizing a whole chromosome
Phase 1: Synthesizing a whole chromosome
Phase 2: Re-booting a cell
Mycoplasma mycoides Mycoplasma capricolum
Mycoplasma capricolum/mycoides
Mycoplasma mycoides
1. Appreciation for seminal discoveries of the past2. Proficiency with the tools of discovery3. Familiarity with the current frontiers of discovery4. Passionate, observant, contemplative, imaginative
