Notes for BIO104 Cellular and Microbial Biology Lecture 8 on The Microbial Cell

BIO104 Cellular and Microbial Biology Lecture 8: The Microbial Cell

Presentation Details

Instructor: Dr. Eva Sonnenschein
Contact: e.c.sonnenschein@swansea.ac.uk
University: Swansea University
Date: Lecture 8

Overview of Lecture

This lecture focuses on the study of prokaryotic cells, particularly Escherichia coli. Key topics covered include:

The interior structure of prokaryotic cells
DNA organization in prokaryotes
DNA replication processes in prokaryotes
Transcription and regulatory mechanisms in prokaryotes
Antibiotics that target specific molecular processes
Cytoplasmic structures found in prokaryotes

Prokaryotic Intracellular Structure

Prokaryotic cells, including Bacteria and Archaea, feature two main intracellular structures:

Nucleoid
Cytoplasm

Distinctions of Prokaryotic Cells

Prokaryotes are characterized by the absence of a true nucleus, meaning that prokaryotic DNA is not enclosed within a membrane-bound structure.
The DNA in prokaryotes is compacted within the cytoplasm and is organized into a structure called the nucleoid, which allows for the coupling of transcription and translation processes.

Visualization of Nucleoid

Under electron microscopy, the nucleoid appears as a lighter, irregular blob of DNA amidst the ribosomes within the cytoplasm.

DNA Organization in Prokaryotes

Chromosomal Structure

Prokaryotes organize their DNA into chromosomes similar to eukaryotes.
The chromosomes in prokaryotes are typically circular, with most bacteria having a single chromosome.
To fit into the cell, DNA must be compacted approximately 1000-fold.
DNA in circular chromosomes is folded by DNA-binding proteins into supercoil loops, which protrude from a denser core structure, resembling a bottle brush.

Types of DNA Supercoiling

Supercoils can take on either:**
- Negatively supercoiled: The DNA is twisted in the opposite direction of the double helix.
- Positively supercoiled: The DNA is twisted in the same direction as the double helix.
Most bacterial genomes are negatively supercoiled during standard growth conditions.

DNA Replication in Prokaryotes

Initiation of Replication

DNA replication begins at a specific site called the origin of replication (oriC).
The initiator protein DnaA binds to this origin, separating the two DNA strands, allowing replication forks to form in both directions from oriC towards the terminus region (terC) where replication concludes.

Challenges of DNA Replication

DNA replication encounters three major challenges:

Unwinding the DNA helix.
Replicating each DNA strand simultaneously.
Separating the two resultant daughter chromosomes.

Role of DnaB Helicase

Following DnaA's action, DnaB helicase separates the parental DNA strands, which are then kept apart by single-stranded DNA-binding proteins (SSB).
This unwinding may induce positive supercoiling in unopen regions of the DNA.

Addressing Supercoiling

DNA Gyrase (topoisomerase II) alleviates positive supercoiling by:
- Cutting the double helix at the supercoil crossover, allowing tunneling of the unbroken strand through, and resealing the cut.
- Decatenating sister chromosomes by cutting one of the chromosomes and allowing the other to pass through before resealing.
These processes require ATP and help prevent the blockage of DNA replication due to supercoils.

RNA Primer Synthesis

RNA primase Synthesizes RNA primers for replication.
The leading strand undergoes continuous synthesis, whereas the lagging strand is made in segments known as Okazaki fragments. After synthesis, these fragments are ligated, and the primers are removed.

Resulting DNA Molecules

Each new DNA molecule produced has one template strand from the original and one newly synthesized strand.

Interlocked Chromosomes Post Replication

Immediately after DNA replication, the two daughter chromosomes are interlocked, like two links in a chain, requiring resolution.

Transcription in Prokaryotes

Transcription Process Overview

Prokaryotes contain a single RNA polymerase (RNAP) that operates at the nucleoid-cytoplasm interface, enabling rapid access of ribosomes to emergent transcripts, facilitating coupled transcription and translation.

Coupled Transcription and Translation

As soon as a gene in prokaryotes begins to be transcribed into mRNA, ribosomes attach to the mRNA and translate the information into protein simultaneously.
Multiple ribosomes can target the same mRNA transcript, speeding up protein production compared to eukaryotes.

Steps of Transcription

Initiation: The RNAP associates with a sigma (σ) factor that aids in recognizing and binding to a specific promoter sequence, leading to the formation of a transcription bubble.
Elongation: Upon clearing the promoter, the σ factor is released. The NusA protein assists RNAP with the formation of hairpins in the growing RNA strand, which can impede transcription.
Termination:
- Rho-independent (intrinsic) termination occurs when the RNAP reaches a termination signal, forming a GC-rich region followed by a stretch of A residues, which creates a hairpin structure and stalls the RNAP.
- Rho-dependent termination involves the Rho factor binding to specific mRNA sequences, moving towards the 3’ end, catching up to the RNAP to facilitate the release of mRNA from the DNA template and the RNAP.

Regulation of Gene Expression in Prokaryotes

Prokaryotic genomic DNA comprises structural genes, encoding products serving as structural components or enzymes, and regulatory genes that modulate gene expression. Regulation prevents unnecessary protein production when not required.
Related structural proteins are typically grouped in blocks called operons.

Operon Structure

An operon is defined as a single transcriptional unit containing multiple genes, governed by a shared promoter and terminator. Most prokaryotic mRNA transcripts are polycistronic, encoding several peptides.
The regulatory region of an operon encompasses necessary sequences that influence transcription, including the promoter and nearby regulatory sequences.

Regulatory Genes and Transcription Factors

Regulatory genes encode proteins that control transcription by binding specific DNA sites known as transcription factors.
These factors significantly influence the binding of RNA polymerase to the promoter, facilitating transcription progress of structural genes.

Mechanisms of Gene Expression Regulation

Cis-acting factors affect gene expression in proximity, whereas trans-acting products, like transcription factors, can influence distant genes by acting on cis-regulatory elements.

Regulatory Mechanisms

Negative regulation: The operator serves as the binding site for regulatory proteins. When a repressor binds to this site, RNA polymerase cannot initiate transcription.
Positive regulation often necessitates that an activator transcription factor bind to the promoter to enable RNAP initiation.

Example of Operon Model - Lac Operon in E. coli

In E. coli the genes lacZ, lacY, and lacA are essential for lactose metabolism, regulated by the repressor LacI.

Antibiotics Targeting Molecular Processes

Antibiotics can inhibit cellular processes, focused on:

DNA replication
Transcription
Translation

Antibiotics Targeting DNA Replication

Many broad-spectrum antibiotics target DNA gyrase, blocking replication by:
1. Preventing gyrase-DNA binding.
2. Impairing DNA re-ligation post-gyrase action.
3. Inhibiting ATPase activity crucial for gyrase function.

Antibiotics Targeting Transcription

Rifampin specifically targets prokaryotic transcription mechanisms, as:
1. Prokaryotes have a singular RNAP essential for viability.
2. Prokaryotic RNAP is highly conserved and distinct from eukaryotic RNAP, reducing toxicity risks.

Antibiotics Targeting Translation

Broad-spectrum antibiotics like Tetracycline and Chloramphenicol inhibit ribosome functionality by:
1. Stopping the formation of functional 70S ribosomes.
2. Impairing aminoacyl-tRNA translocation to the A site or the peptide bond formation.
3. Hindering ribosome recycling.

Cytoplasmic Structures in Prokaryotes

I. Plasmids

Plasmids are extrachromosomal, double-stranded DNA molecules found in many prokaryotes that replicate independently and often encode critical genetic information.

II. Inclusion Bodies or Storage Granules

Inclusion bodies store nutrients like polysaccharides, nitrogen, sulfur, and phosphates, especially in environments lacking constant nutrient availability.
They are notably involved in producing poly-β-hydroxybutyrate (PHB) in response to nutrient stress, an example of a carbon polymer useful in biodegradable plastics.

III. Gas Vesicles

Found in aquatic prokaryotes that harness light for photosynthesis or energy, gas vesicles help these microbes maintain buoyancy, ensuring optimal lighting and salinity.
Gas vesicles are tubular structures surrounded by a permeable protein shell, regulating gas content while remaining impermeable to water.

IV. Thylakoids

Membrane-bound compartments in chloroplasts and cyanobacteria, thylakoids form stacks of membranes organized as concentric shells.
They contain pigments and proteins essential for light energy capture, enhancing the surface for photosynthesis in cyanobacteria.

V. Carboxysomes

Found in many autotrophic bacteria, carboxysomes consist of polyhedral protein shells filled with enzymes, primarily RuBisCO, crucial for CO2 fixation in phototrophs and chemolithotrophs.

VI. Magnetosomes

Present in motile aquatic bacteria, magnetosomes are intracellular structures surrounded by membranes containing iron crystals that act as magnets, allowing these bacteria to orient themselves in magnetic fields.

Summary

Upon completion of this lecture, students should be able to:

Describe the interior of prokaryotic cells
Explain the organization and replication of DNA in prokaryotes
Discuss transcription and its regulation in prokaryotes
Identify different types of antibiotics and their molecular targets
Describe various cytoplasmic structures in prokaryotic cells.