Chapter 9 Microbial Genetics and Gene Regulation

The central dogma outlines the way in which genetic information is transferred to different levels of the organism (i.e., faithful replication). Yet, it is important to note that variations in the genetic code can still occur:

Central Dogma Flowchart

Figure 9.1: Central Dogma Flowchart

A mutation is a heritable change in an organism’s DNA sequence that is inherited by its offspring. Mutations can give rise to phenotypic variation and in some cases, even maximize the offsprings’ chance for survival.

9.1 Types of Mutations

Mutations can be phenotypic (i.e., they affect the phenotype of the organism).

There are also different types of mutations (know the table):

Types of Mutations

Figure 9.2: Types of Mutations

Furthermore, there are also many sources of mutations. Spontaneous mutations (i.e., mutations that occur due to random error) have a \(\frac{1}{1000000}\) chance of occuring!

Sources of Mutations

Figure 9.3: Sources of Mutations

9.1.1 Penicilin counterselection for selecting mutants

Since penicilin kills growing cells, non-growing histidine auxotrophs will survive (i.e., these cells will be enriched).

Penicilin Selection

Figure 9.4: Penicilin Selection

In this method, one merely identifies the His auxotrophs that they have enriched for!

9.1.2 Mutations by transposition

The general mechanism for transposition is as follows:

Tranposition in Action

Figure 9.5: Tranposition in Action

There are also several transposable elements (know the table) and the following method for determining transposed elements:

Transposible Elements

Figure 9.6: Transposible Elements

9.2 Mechanisms of Gene Exchange in Prokaryotes

There are three different mechanisms of genetic exchange:

Mechanisms of Genetic Exchange

Figure 9.7: Mechanisms of Genetic Exchange

Prokaryotic genetic exchange doesn’t depend on reproduction, involves small amounts of DNA, and haploid organisms can directly express their alleles.

9.2.0.1 Griffith experiment

Observe the famous Griffith experiment:

Griffith Experiment

Figure 9.8: Griffith Experiment

Natural transformation is often induced in dense cultures via quorum sensing (e.g., biofilms).

9.2.1 Genetic exchange via competence pili

Competence Pili

Figure 9.9: Competence Pili

First, the pill bind to eDNA and translocates the DNA across the outer membrane via retraction. Then, the outer membrane’s secretin pore allows for DNA to diffuse across the outer membrane.

9.2.2 Life cycle of a phage

Observe the life cycle of a bacteriophage:

Bacteriophage Life Cycle

Figure 9.10: Bacteriophage Life Cycle

DNA in a bacteriophage can also be packaged in concatameric, rolling circle replications of phage genomes:

Concatameric Storage of DNA

Figure 9.11: Concatameric Storage of DNA

There are cleavage at pac sites by phage enzymes (TerSL), and cleavage at similar pac sites in the host genome can also package non-viral DNA too!

Furthermore, there is also site-specific recombination and crossovers in bacteriophage DNA:

DNA Crossover in Bacteriophages

Figure 9.12: DNA Crossover in Bacteriophages

Gene transfer can also be transferred in one of two ways: specialized transduction or lateral transduction.

Types of Transductions

Figure 9.13: Types of Transductions

9.2.2.1 Lateral Transduction

Lateral Transduction

Figure 9.14: Lateral Transduction

In step 1, there is the replication of the DNA. In step 2, there is the initiation of packaging from the pac site by ter.

All of the above occurs while prophage excision occurs and headfuls of host DNA are packaged!

9.2.2.2 Conjugation

The F Plasmid

Figure 9.15: The F Plasmid

The integration of a F plasmid into a bacterium’s genome results in the creation of Hfr strains (i.e., strains with a high frequency of recombination).

Conjugation of an F plasmid

Figure 9.16: Conjugation of an F plasmid

These Hfr strains, when mobilized, can drag along chromosomal DNA (which would take ~100 minutes to transfer to an entire E. coli genome).

Conjugation in *E. faecalis*

Figure 9.17: Conjugation in E. faecalis

E. faecalis also has a conjugation mechanism (shown above), albeit it relies on pheromones and AS-mediated pumping.

9.2.3 CRISPR

CRISPR Schematic

Figure 9.18: CRISPR Schematic

CRISPR is a family of DNA sequences that are found in the genomes of prokaryotic organisms (e.g., bacteria and archaea) and are derived from the DNA fragments of bacteriophages that have previously infected the prokaryote.

9.3 Genes and Regulation of Cellular Processes

9.3.1 Evidence for coordinated regulation of metabolic reactions

E. coli cultured in minimal medium and 14C-glycerol from 14CO2 (as the carbon source) had 14C present in its macromolecules (including amino acids).

Similarly, E. coli cultured in minimal medium and histidine using the same carbon source had 14C present in all of its macromolecules but histidine!

9.3.2 Metabolic regulatory devices

Some Metabolic Regulatory Devices

Figure 9.19: Some Metabolic Regulatory Devices

Some other microbial themes include:

  1. Substrate limitations
  2. Short life span of mRNAs
  3. Operons and coordinated expressions
  4. Coupled tln and txns

9.3.2.1 Allostery

An allosteric site is a regulatory site that the effector binds to and is separate from the active site of the enzyme.

Note that the allosteric effector does not have to look anything like the substrate (or the product for that matter).

9.3.2.2 sRNA

sRNA Control of Protein Activity

Figure 9.20: sRNA Control of Protein Activity

The 6S RNA of E. coli regulates the transition to the stationary phase (of the cell cycle of the bacterium) by modifying the RNAP-$$70 housekeeping transcription activity (hence preventing the normal expression of genes in the exponential growth phase of the bacterium).

9.3.3 lac operon

lac operon

Figure 9.21: lac operon

There are obviously many regulatory control points in the lac operon.

9.3.3.1 Positive transcriptional control

Positive Transcriptional Control

Figure 9.22: Positive Transcriptional Control

Here, the binding of a regulatory protein at a regulatory region on DNA promotes transcription initiation.

An inactive protein can be activated by an inducer and inactivated by an inhibitor.

9.3.3.2 Trp attenuation

Trp Attenuation

Figure 9.23: Trp Attenuation

The trp operon is a group of genes that encode for biosynthetic enzymes for the amino acid tryptophan; these genes are also found in E. coli.

When tryptophan levels are low, the trp operon is turned on; otherwise, trytophan blocks the expression of the operon.

Such an amino acid synthesis is also regulated by attenutation (based off the coupling of transcription and translation).

9.3.3.3 Antisense sRNAs (in translation)

Antisense sRNAs

Figure 9.24: Antisense sRNAs

Antisense sRNAs have also been shown to exhibit regulatory effects on protein translation.

9.3.4 Two-component regulatory and phosphorelay systems

Many genes can be turned “on” or “off” in response to environmental conditions (e.g., temperature, osmolarity, O2 levels, etc). The regulatory proteins responsible for the latter are part of a two-component signalling system that links external events to the regulation of gene expression.

All three domains of life have these systems - bacteria and archaeal organisms have many two component signal transduction systems. Though, some of these systems regulate protein activity instead of gene transcription.

9.3.4.1 Sensor kinase

Sensor Kinase

Figure 9.25: Sensor Kinase

These pathways span the entire cytoplasmic membrane and have an extracellular receptor for signals. They also play a role in intracellular communication pathways.

9.3.4.2 Response-regulator proteins

Response Regulator Proteins

Figure 9.26: Response Regulator Proteins

These are activated by a sensor kinase and a DNA-binding protein. An activator enhances transcription whereas a repressor inhibits transcription (unless needed).

9.4 Global Regulations

Catabolite repression is the regulation of transcription by both repressors and activators.

Catabolite Repression

Figure 9.27: Catabolite Repression

Diauxic growth has two patterns: a biphasic growth phase and a lag phase.

A biphasic growth pattern involves the preferential use of one carbon source over another when both sources are available in the environment.

As witnessed in the above graphic, lag occurs after a preferred substrate is exhausted - microbe growth then continues (using the second carbon source) after a brief moment of time.

Catabolite Repression Example

Figure 9.28: Catabolite Repression Example

Catabolite repression plays a role in this pattern of growth!

9.4.1 Stringent response

Stringent Response

Figure 9.29: Stringent Response

When cells are starved for amino acids, protein synthesis is unable to proceed (for obvious reasons).

Consequently, tRNA and rRNA production is decreased, and the protein ReIA downregulates the synthesis of (p)ppGpp - an alarmone. Cells also increase the biosynthesis of the amino acids needed.