Microbial Genetics Study Notes
Microbial Genetics Study Notes CONTENTS 1
2
3
4
5
Bacterial conjugation ..................................................................................................................................... 4 1.1
Lederberg and Tatum experiment ........................................................................................................ 4
1.2
Conjugation and the plasmid ................................................................................................................ 4
1.3
Mechanism of conjugation (tra genes function)................................................................................... 5
1.4
Fertility inhibition.................................................................................................................................. 5
1.5
Formation of HFR: Integration of the F plasmid .....................................Error! Bookmark not defined.
1.6
Formation of F’: Removal from Hfr .........................................................Error! Bookmark not defined.
1.7
Transmission and recombination of Hfr plasmid ....................................Error! Bookmark not defined.
1.8
Constructing F’ plasmid gene library.......................................................Error! Bookmark not defined.
Bacteriophage genetics - λ phage....................................................................Error! Bookmark not defined. 2.1
General information................................................................................Error! Bookmark not defined.
2.2
Comparison of lytic and lysogenic cycles ................................................Error! Bookmark not defined.
2.3
Lytic life cycle ..........................................................................................Error! Bookmark not defined.
2.4
Lysogenic life cycle..................................................................................Error! Bookmark not defined.
2.5
Overview of λ gene expression in lytic cycle...........................................Error! Bookmark not defined.
2.6
Gene expression in the λ lytic cycle ........................................................Error! Bookmark not defined.
2.7
Control of lytic and lysogenic cycles in the λ phage................................Error! Bookmark not defined.
2.8
The ‘decision’ between lytic and lysogenic.............................................Error! Bookmark not defined.
2.9
Mutants...................................................................................................Error! Bookmark not defined.
2.10
Replication mechanisms .........................................................................Error! Bookmark not defined.
2.11
Phage induction ......................................................................................Error! Bookmark not defined.
Archaeal genetics.............................................................................................Error! Bookmark not defined. 3.1
The archaea domain ...............................................................................Error! Bookmark not defined.
3.2
Genetic systems ......................................................................................Error! Bookmark not defined.
3.3
Antarctic haloarchaea genetics...............................................................Error! Bookmark not defined.
3.4
The pJWID1 plasmid:...............................................................................Error! Bookmark not defined.
3.5
Pop-in, pop-out gene knockout system in auxotrophic strains ..............Error! Bookmark not defined.
Lateral gene transfer and evolution ................................................................Error! Bookmark not defined. 4.1
Modes of sharing of genetic material between cells ..............................Error! Bookmark not defined.
4.2
Mobile genetic elements involved in lateral gene transfer ....................Error! Bookmark not defined.
4.3
Recombination of MGEs .........................................................................Error! Bookmark not defined.
4.4
MGEs, LGT and evolution........................................................................Error! Bookmark not defined.
4.5
Horizontal gene transfer .........................................................................Error! Bookmark not defined.
Genomics and DNA sequencing.......................................................................Error! Bookmark not defined.
6
7
8
9
5.1
Current DNA sequencing technologies ...................................................Error! Bookmark not defined.
5.2
Application of sequencing technologies .................................................Error! Bookmark not defined.
Cyanobacterial genetics...................................................................................Error! Bookmark not defined. 6.1
Importance of cyanobacteria..................................................................Error! Bookmark not defined.
6.2
Secondary metabolites: toxins vs. antibiotics.........................................Error! Bookmark not defined.
6.3
Non-ribosomal peptide synthesis ...........................................................Error! Bookmark not defined.
6.4
Gene regulation and the role of secondary metabolites ........................Error! Bookmark not defined.
6.5
Applications to water quality ..................................................................Error! Bookmark not defined.
6.6
Gene transfer ..........................................................................................Error! Bookmark not defined.
6.7
Combinatorial biosynthesis.....................................................................Error! Bookmark not defined.
Archaea in the lab – Methanococcoides burtonii............................................Error! Bookmark not defined. 7.1
Methanococcoides burtonii ....................................................................Error! Bookmark not defined.
7.2
Lipids .......................................................................................................Error! Bookmark not defined.
7.3
tRNA ........................................................................................................Error! Bookmark not defined.
7.4
Genome - composition............................................................................Error! Bookmark not defined.
7.5
Genome - function ..................................................................................Error! Bookmark not defined.
7.6
Proteomics - transcription ......................................................................Error! Bookmark not defined.
7.7
Proteomics - multiplex ............................................................................Error! Bookmark not defined.
7.8
Transcriptomics.......................................................................................Error! Bookmark not defined.
Bacteria and archaea diversity.........................................................................Error! Bookmark not defined. 8.1
History of microbial diversity ..................................................................Error! Bookmark not defined.
8.2
Levels of diversity....................................................................................Error! Bookmark not defined.
8.3
Diversity changing over time and space .................................................Error! Bookmark not defined.
8.4
Environmental metagenomics, metatranscriptomics and single cell genomes.Error! Bookmark not defined.
8.5
Community structures ............................................................................Error! Bookmark not defined.
Quorum sensing regulation .............................................................................Error! Bookmark not defined. 9.1
What is quorum sensing..........................................................................Error! Bookmark not defined.
9.2
Vibrio fischeri: .........................................................................................Error! Bookmark not defined.
9.3
Moral compass in microbiology ..............................................................Error! Bookmark not defined.
9.4
Beyond V. fischeri....................................................................................Error! Bookmark not defined.
10
Antarctic environment ................................................................................Error! Bookmark not defined. 10.1
Environmental microorganisms ..............................................................Error! Bookmark not defined.
10.2
Antarctica................................................................................................Error! Bookmark not defined.
10.3
Studies.....................................................................................................Error! Bookmark not defined.
10.4
Vestfold Hills: ..........................................................................................Error! Bookmark not defined.
11
Archaeal pathogens.....................................................................................Error! Bookmark not defined. 11.1
Abundance of archaea ............................................................................Error! Bookmark not defined.
11.2
Archaea-eukarya interactions .................................................................Error! Bookmark not defined.
11.3
Animal hosts and archaea.......................................................................Error! Bookmark not defined.
11.4
The immune system and archaea ...........................................................Error! Bookmark not defined.
11.5
Pathogenic molecular signatures and archaea .......................................Error! Bookmark not defined.
11.6
Disease and archaea ...............................................................................Error! Bookmark not defined.
12
Metaproteomics..........................................................................................Error! Bookmark not defined. 12.1
Metaproteome vs. metagenome ............................................................Error! Bookmark not defined.
12.2
Proteins ...................................................................................................Error! Bookmark not defined.
12.3
Advantages of metaproteomics:.............................................................Error! Bookmark not defined.
12.4
Metaproteomic methods........................................................................Error! Bookmark not defined.
12.5
Metagenomic methods...........................................................................Error! Bookmark not defined.
12.6
East Antarctica investigation (summer) ..................................................Error! Bookmark not defined.
12.7
West Antarctica investigation (summer vs. winter)................................Error! Bookmark not defined.
13
Diversity, function and microbial processes................................................Error! Bookmark not defined. 13.1
Heard Island ............................................................................................Error! Bookmark not defined.
13.2
Heard’s importance: ...............................................................................Error! Bookmark not defined.
13.3
Investigation of Heard Island ..................................................................Error! Bookmark not defined.
14
Biofilms........................................................................................................Error! Bookmark not defined. 14.1
Community based gene regulation.........................................................Error! Bookmark not defined.
14.2
Biofilms ...................................................................................................Error! Bookmark not defined.
14.3
Quorum sensing signals are diverse........................................................Error! Bookmark not defined.
14.4
Importance of gene regulation and control............................................Error! Bookmark not defined.
14.5
Microbial control strategies using signalling systems.............................Error! Bookmark not defined.
15
Bacteriophage .............................................................................................Error! Bookmark not defined. 15.1
Bacteriophage background .....................................................................Error! Bookmark not defined.
15.2
Marine viruses.........................................................................................Error! Bookmark not defined.
15.3
Phage functions.......................................................................................Error! Bookmark not defined.
15.4
Viruses and microbial diversity ...............................................................Error! Bookmark not defined.
15.5
Phage associated genes ..........................................................................Error! Bookmark not defined.
15.6
Phage influence on bacterial function and evolution .............................Error! Bookmark not defined.
15.7
CRISPRs ...................................................................................................Error! Bookmark not defined.
1 BACTERIAL CONJUGATION 1.1 LEDERBERG AND TATUM EXPERIMENT • • • •
Strain A: met bio thr+ leu+ thi+ (i.e. met and bio auxotroph). Strain B: met+ bio+ thr leu thi (i.e. thr, leu and thi auxotroph). Strains A and B were plated separately on minimal medium: no colonies (auxotrophic cells). A and B were then conjugated and plated: produced prototrophic colonies (met+ bio+ thr+ leu+ thi+).
1.2 CONJUGATION AND THE PLASMID PLASMID •
• •
•
Conjugation is not restricted to Escherichia coli: o Pseudomonas o Streptomyces o Agrobacterium o Sulfolobus o Haloarchaea We will still focus on E. coli conjugation. Plasmid types: o F (fertility factor): self-transmissible plasmid that transfers in all-ornothing manner. o Hfr: plasmid combines with donor chromosome and is only partially transferred with part of the donor chromosome. o F’: plasmid combines with donor chromosome, but ‘pops’ back out to form a plasmid incorporating part of the chromosome that may have broken away. This plasmid is then transferred similarly to the F plasmid. F-plasmid genes/sites and their functions: o ccdAB: inhibits host cell division. o incBCE: incompatibility. o oriT: site of initiation of conjugal DNA transfer. o oriV: origin of bidirectional replication. o parABCL: partitioning. o traABCEFGHKLQUVW: pilus biosynthesis/assembly. o traGN: mating-pair stabilisation. o traI: relaxase. o traY: accessory for nickase. o traJ, finOP: regulation of transfer (fertility inhibition). o traST: entry exclusion. o IS: insertion sequences (same class as transposase).
1.3 MECHANISM OF CONJUGATION CONJUGATION (TRA GENES FUNCTION) • •
Dtr: DNA transfer and replication. Mpf: mating-pair formation.
•
Relaxase covalently binds to Tyr-OH group in the oriT region of the plasmid, and proceeds to transfer the plasmid from the donor to the recipient.
1.4 FERTILITY INHIBITION •
Most self transmissable plasmids (e.g. RP4) transfer at low frequency. o TraJ: activator of tra gene expression. o finP: antisense mRNA to traJ (i.e. mRNA which is transcribed from the same region as traJ, but in the opposite direction). o FinO: protein that stabilises finP. o Immediately after cell entry, traJ is transcribed which activates tra genes (pili and transfer functions).
o The finO gene is directly after the tra genes, so the FinO protein is transcribed and binds to the finP mRNA. o This complex then inhibits traJ transcription.
•
•
•
Frequency of F transfer is high (F+ into F- strain). o finO is interrupted by IS3. o Therefore in the presence of IS3, traJ and hence the tra operon is always expressed. o This results in a higher frequency of transfer. When RP4 does transfer, if there is a high cell density, RP4 can continue to transfer at high frequency. o When RP4 enters a new cell, the concentration of FinO/FinP will initially be zero, but will increase with time. o Therefore, traJ will initially be expressed, but over time will be inhibited by the increasing concentration of FinO/FinP complex. o Since the cell density is high, there is a greater chance of the continuance of conjugation. If RP4 transfers to F+, then F will transfer at the same low frequency as RP4. o It is therefore clear that the RP4 plasmid has an effect on the F plasmid. This is fertility inhibition. o Compatibilty: F: IncE, IncB and Inc C plasmid. RP4: IncP plasmid. o Therefore, these plasmids have different incompatibility groups, which means they are compatible. o Since RP4 encodes the FinO/FinP complex, this complex can then have an effect on the F plasmid.
2
3
4
5
Bacterial conjugation ..................................................................................................................................... 4 1.1
Lederberg and Tatum experiment ........................................................................................................ 4
1.2
Conjugation and the plasmid ................................................................................................................ 4
1.3
Mechanism of conjugation (tra genes function)................................................................................... 5
1.4
Fertility inhibition.................................................................................................................................. 5
1.5
Formation of HFR: Integration of the F plasmid .....................................Error! Bookmark not defined.
1.6
Formation of F’: Removal from Hfr .........................................................Error! Bookmark not defined.
1.7
Transmission and recombination of Hfr plasmid ....................................Error! Bookmark not defined.
1.8
Constructing F’ plasmid gene library.......................................................Error! Bookmark not defined.
Bacteriophage genetics - λ phage....................................................................Error! Bookmark not defined. 2.1
General information................................................................................Error! Bookmark not defined.
2.2
Comparison of lytic and lysogenic cycles ................................................Error! Bookmark not defined.
2.3
Lytic life cycle ..........................................................................................Error! Bookmark not defined.
2.4
Lysogenic life cycle..................................................................................Error! Bookmark not defined.
2.5
Overview of λ gene expression in lytic cycle...........................................Error! Bookmark not defined.
2.6
Gene expression in the λ lytic cycle ........................................................Error! Bookmark not defined.
2.7
Control of lytic and lysogenic cycles in the λ phage................................Error! Bookmark not defined.
2.8
The ‘decision’ between lytic and lysogenic.............................................Error! Bookmark not defined.
2.9
Mutants...................................................................................................Error! Bookmark not defined.
2.10
Replication mechanisms .........................................................................Error! Bookmark not defined.
2.11
Phage induction ......................................................................................Error! Bookmark not defined.
Archaeal genetics.............................................................................................Error! Bookmark not defined. 3.1
The archaea domain ...............................................................................Error! Bookmark not defined.
3.2
Genetic systems ......................................................................................Error! Bookmark not defined.
3.3
Antarctic haloarchaea genetics...............................................................Error! Bookmark not defined.
3.4
The pJWID1 plasmid:...............................................................................Error! Bookmark not defined.
3.5
Pop-in, pop-out gene knockout system in auxotrophic strains ..............Error! Bookmark not defined.
Lateral gene transfer and evolution ................................................................Error! Bookmark not defined. 4.1
Modes of sharing of genetic material between cells ..............................Error! Bookmark not defined.
4.2
Mobile genetic elements involved in lateral gene transfer ....................Error! Bookmark not defined.
4.3
Recombination of MGEs .........................................................................Error! Bookmark not defined.
4.4
MGEs, LGT and evolution........................................................................Error! Bookmark not defined.
4.5
Horizontal gene transfer .........................................................................Error! Bookmark not defined.
Genomics and DNA sequencing.......................................................................Error! Bookmark not defined.
6
7
8
9
5.1
Current DNA sequencing technologies ...................................................Error! Bookmark not defined.
5.2
Application of sequencing technologies .................................................Error! Bookmark not defined.
Cyanobacterial genetics...................................................................................Error! Bookmark not defined. 6.1
Importance of cyanobacteria..................................................................Error! Bookmark not defined.
6.2
Secondary metabolites: toxins vs. antibiotics.........................................Error! Bookmark not defined.
6.3
Non-ribosomal peptide synthesis ...........................................................Error! Bookmark not defined.
6.4
Gene regulation and the role of secondary metabolites ........................Error! Bookmark not defined.
6.5
Applications to water quality ..................................................................Error! Bookmark not defined.
6.6
Gene transfer ..........................................................................................Error! Bookmark not defined.
6.7
Combinatorial biosynthesis.....................................................................Error! Bookmark not defined.
Archaea in the lab – Methanococcoides burtonii............................................Error! Bookmark not defined. 7.1
Methanococcoides burtonii ....................................................................Error! Bookmark not defined.
7.2
Lipids .......................................................................................................Error! Bookmark not defined.
7.3
tRNA ........................................................................................................Error! Bookmark not defined.
7.4
Genome - composition............................................................................Error! Bookmark not defined.
7.5
Genome - function ..................................................................................Error! Bookmark not defined.
7.6
Proteomics - transcription ......................................................................Error! Bookmark not defined.
7.7
Proteomics - multiplex ............................................................................Error! Bookmark not defined.
7.8
Transcriptomics.......................................................................................Error! Bookmark not defined.
Bacteria and archaea diversity.........................................................................Error! Bookmark not defined. 8.1
History of microbial diversity ..................................................................Error! Bookmark not defined.
8.2
Levels of diversity....................................................................................Error! Bookmark not defined.
8.3
Diversity changing over time and space .................................................Error! Bookmark not defined.
8.4
Environmental metagenomics, metatranscriptomics and single cell genomes.Error! Bookmark not defined.
8.5
Community structures ............................................................................Error! Bookmark not defined.
Quorum sensing regulation .............................................................................Error! Bookmark not defined. 9.1
What is quorum sensing..........................................................................Error! Bookmark not defined.
9.2
Vibrio fischeri: .........................................................................................Error! Bookmark not defined.
9.3
Moral compass in microbiology ..............................................................Error! Bookmark not defined.
9.4
Beyond V. fischeri....................................................................................Error! Bookmark not defined.
10
Antarctic environment ................................................................................Error! Bookmark not defined. 10.1
Environmental microorganisms ..............................................................Error! Bookmark not defined.
10.2
Antarctica................................................................................................Error! Bookmark not defined.
10.3
Studies.....................................................................................................Error! Bookmark not defined.
10.4
Vestfold Hills: ..........................................................................................Error! Bookmark not defined.
11
Archaeal pathogens.....................................................................................Error! Bookmark not defined. 11.1
Abundance of archaea ............................................................................Error! Bookmark not defined.
11.2
Archaea-eukarya interactions .................................................................Error! Bookmark not defined.
11.3
Animal hosts and archaea.......................................................................Error! Bookmark not defined.
11.4
The immune system and archaea ...........................................................Error! Bookmark not defined.
11.5
Pathogenic molecular signatures and archaea .......................................Error! Bookmark not defined.
11.6
Disease and archaea ...............................................................................Error! Bookmark not defined.
12
Metaproteomics..........................................................................................Error! Bookmark not defined. 12.1
Metaproteome vs. metagenome ............................................................Error! Bookmark not defined.
12.2
Proteins ...................................................................................................Error! Bookmark not defined.
12.3
Advantages of metaproteomics:.............................................................Error! Bookmark not defined.
12.4
Metaproteomic methods........................................................................Error! Bookmark not defined.
12.5
Metagenomic methods...........................................................................Error! Bookmark not defined.
12.6
East Antarctica investigation (summer) ..................................................Error! Bookmark not defined.
12.7
West Antarctica investigation (summer vs. winter)................................Error! Bookmark not defined.
13
Diversity, function and microbial processes................................................Error! Bookmark not defined. 13.1
Heard Island ............................................................................................Error! Bookmark not defined.
13.2
Heard’s importance: ...............................................................................Error! Bookmark not defined.
13.3
Investigation of Heard Island ..................................................................Error! Bookmark not defined.
14
Biofilms........................................................................................................Error! Bookmark not defined. 14.1
Community based gene regulation.........................................................Error! Bookmark not defined.
14.2
Biofilms ...................................................................................................Error! Bookmark not defined.
14.3
Quorum sensing signals are diverse........................................................Error! Bookmark not defined.
14.4
Importance of gene regulation and control............................................Error! Bookmark not defined.
14.5
Microbial control strategies using signalling systems.............................Error! Bookmark not defined.
15
Bacteriophage .............................................................................................Error! Bookmark not defined. 15.1
Bacteriophage background .....................................................................Error! Bookmark not defined.
15.2
Marine viruses.........................................................................................Error! Bookmark not defined.
15.3
Phage functions.......................................................................................Error! Bookmark not defined.
15.4
Viruses and microbial diversity ...............................................................Error! Bookmark not defined.
15.5
Phage associated genes ..........................................................................Error! Bookmark not defined.
15.6
Phage influence on bacterial function and evolution .............................Error! Bookmark not defined.
15.7
CRISPRs ...................................................................................................Error! Bookmark not defined.
1 BACTERIAL CONJUGATION 1.1 LEDERBERG AND TATUM EXPERIMENT • • • •
Strain A: met bio thr+ leu+ thi+ (i.e. met and bio auxotroph). Strain B: met+ bio+ thr leu thi (i.e. thr, leu and thi auxotroph). Strains A and B were plated separately on minimal medium: no colonies (auxotrophic cells). A and B were then conjugated and plated: produced prototrophic colonies (met+ bio+ thr+ leu+ thi+).
1.2 CONJUGATION AND THE PLASMID PLASMID •
• •
•
Conjugation is not restricted to Escherichia coli: o Pseudomonas o Streptomyces o Agrobacterium o Sulfolobus o Haloarchaea We will still focus on E. coli conjugation. Plasmid types: o F (fertility factor): self-transmissible plasmid that transfers in all-ornothing manner. o Hfr: plasmid combines with donor chromosome and is only partially transferred with part of the donor chromosome. o F’: plasmid combines with donor chromosome, but ‘pops’ back out to form a plasmid incorporating part of the chromosome that may have broken away. This plasmid is then transferred similarly to the F plasmid. F-plasmid genes/sites and their functions: o ccdAB: inhibits host cell division. o incBCE: incompatibility. o oriT: site of initiation of conjugal DNA transfer. o oriV: origin of bidirectional replication. o parABCL: partitioning. o traABCEFGHKLQUVW: pilus biosynthesis/assembly. o traGN: mating-pair stabilisation. o traI: relaxase. o traY: accessory for nickase. o traJ, finOP: regulation of transfer (fertility inhibition). o traST: entry exclusion. o IS: insertion sequences (same class as transposase).
1.3 MECHANISM OF CONJUGATION CONJUGATION (TRA GENES FUNCTION) • •
Dtr: DNA transfer and replication. Mpf: mating-pair formation.
•
Relaxase covalently binds to Tyr-OH group in the oriT region of the plasmid, and proceeds to transfer the plasmid from the donor to the recipient.
1.4 FERTILITY INHIBITION •
Most self transmissable plasmids (e.g. RP4) transfer at low frequency. o TraJ: activator of tra gene expression. o finP: antisense mRNA to traJ (i.e. mRNA which is transcribed from the same region as traJ, but in the opposite direction). o FinO: protein that stabilises finP. o Immediately after cell entry, traJ is transcribed which activates tra genes (pili and transfer functions).
o The finO gene is directly after the tra genes, so the FinO protein is transcribed and binds to the finP mRNA. o This complex then inhibits traJ transcription.
•
•
•
Frequency of F transfer is high (F+ into F- strain). o finO is interrupted by IS3. o Therefore in the presence of IS3, traJ and hence the tra operon is always expressed. o This results in a higher frequency of transfer. When RP4 does transfer, if there is a high cell density, RP4 can continue to transfer at high frequency. o When RP4 enters a new cell, the concentration of FinO/FinP will initially be zero, but will increase with time. o Therefore, traJ will initially be expressed, but over time will be inhibited by the increasing concentration of FinO/FinP complex. o Since the cell density is high, there is a greater chance of the continuance of conjugation. If RP4 transfers to F+, then F will transfer at the same low frequency as RP4. o It is therefore clear that the RP4 plasmid has an effect on the F plasmid. This is fertility inhibition. o Compatibilty: F: IncE, IncB and Inc C plasmid. RP4: IncP plasmid. o Therefore, these plasmids have different incompatibility groups, which means they are compatible. o Since RP4 encodes the FinO/FinP complex, this complex can then have an effect on the F plasmid.
