RCSI: Overview of the Prokaryotic Cell 2023 PDF

Leading the world to better health  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn Session ID:...

Leading the world to better health  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn Session ID: FFP1ML2 Overview of the Prokaryotic Cell Class Year 1 Course Undergraduate Medicine Lecturer Dr Muaaz Ather Dr Safiya Khalaf Date 9th October 2023  LEARNING OUTCOMES 1. Differentiate between Prokaryotes and Eukaryotes 2. Identify clinically important bacteria based on microscopic appearance 3. Define the role of certain cellular structures in the pathogenesis of infection 4. Describe the processes involved in bacterial growth 5. Describe the structure of bacterial DNA and the process of DNA replication 6. Explain bacterial gene expression (transcription and translation) 7. Describe the clinical significance of plasmids and DNA mutation  LEARNING OUTCOME 1. Differentiate between Prokaryotes and Eukaryotes  Bacterial Cell Eukaryotic Cell Prokaryotes Eukaryotes No nucleus Nucleus Cell wall No cell wall No cell organelles Cell organelles e.g.Mitochondria, chloroplasts, endoplasmic reticulum  LEARNING OUTCOME 2. Identify clinically important bacteria based on microscopic appearance  MICROSCOPY Used to examine bacterial cell shape Colour (stain) Shape Size  THE GRAM STAIN Most important differential staining method in Microbiology Gram-positive Gram-negative (staphylococci) (Escherichia coli) 1. Crystal Violet 2. Iodine 3. Alcohol 4. Neutral Red  THE GRAM STAIN Gram-positive Gram-negative (staphylococci) (Escherichia coli) Differential lipid content of G+ve and G-ve cell envelopes Crystal violet- Iodine complex forms within the cells (Blue colour Alcohol treatment G-ve cell envelope has high lipid content which G+ve cell envelope is extracted by alcohol to has low lipid permeabilise the membrane content and is dehydrated by alcohol - making it impermeable Crystal violet-iodine complex diffuses out and neutral red is taken up SOME BACTERIA DON’T STAIN USING THE GRAM METHOD Mycobacteria have a high wax content in their cell envelope and suspected mycobacteria are stained using the Ziehl-Neelsen stain Mycoplasmas, the smallest known bacteria, have no cell wall to stain MORPHOLOGICAL SHAPES OF DIFFERENT BACTERIA Cocci (spherical) Bacilli (rod shaped) Curved or spiral shaped  BACTERIAL CELL STRUCTURE Genome The bacterial genome or chromosome contains the bacterial genetic information. Plasmids may also be present Cytoplasmic Membrane The cytoplasmic membrane surrounds the cytoplasm Cell Wall Rigid layer surrounding the cytoplasmic membrane Outer Membrane of Gram-negative bacteria Covers the cell wall and acts as a molecular sieve TYPICAL GRAM-NEGATIVE AND GRAM- POSITIVE CELL ENVELOPES  CYTOPLASMIC MEMBRANE Composed primarily of lipids and phospholipids 1. Osmotic barrier - Only molecules smaller than glycerol diffuse into the cytoplasm 2. Site of energy production (oxidative phosphorylation) 3. Transport of important molecules via Permeases - Facilitated diffusion (passive) and Active transport 4. Synthesis of new cell wall 5. Anchor the chromosome  BACTERIAL CELL WALL Pepidoglycan: the principal component of the cell wall, is a unique polysaccharide which gives the cell its characteristic shape and prevents osmotic lysis Gram-positive Gram-negative Many peptidoglycan layers One peptidoglycan layer (90% of cell envelope material) (2-20% of cell envelope material) Penicillin disrupts peptidoglycan synthesis Many antigens are presented on cell wall surface Gram-positive cell envelope Gram-negative cell envelope (Walker) Gram-positive cell envelope Multiple layers of peptidoglycan Techoic acids and Lipotechoic acids Extend into the environment around the cell Adherence Antigenic determinants Gram-negative cell envelope Outer Membrane Phospholipid- Lipopolysaccharide (LPS) Bilayer (extra lipid layer =mechanism of the Gram stain) Bacterial cell adhesion Resistance to phagocytosis Molecular sieve - access of some molecules to cell wall and cytoplasmic membrane  LEARNING OUTCOME 3. Define the role of certain cellular structures in the pathogenesis of infection CELL APPENDAGES AND OTHER CELL STRUCTURES Flagella and Pili extend from the cell surface Flagellae rotate and are required for motility (chemotaxis) Pili - adherence Capsules (tightly associated) and Slime (loosely associated) are polysaccharide or protein layers surrounding many bacterial cells Provide protection from phagocytosis and antibiotics Play a role in bacterial adherence Spores Some Gram-positive bacteria can form Spores which provide protection from adverse conditions Gram-negative bacteria cannot form spores  BACTERIAL BIOFILMS Many bacterial infections treated by clinicians involve biofilms Pseudomonas aeruginosa infections in cystic fibrosis patients Staphylococcus epidermidis intravascular catheter related infection Biofilms form when bacteria adhere to surfaces and excrete slimy glue-like substances which anchor the cells  Why are biofilm infections difficult to treat? Antibiotic Doses 1 1000 Planktonic Biofilm cells cells  LEARNING OUTCOME 4. Describe the processes involved in bacterial growth GROWTH AND METABOLISM In the laboratory Liquid broths and Nutrient Agar plates  BACTERIA DIVIDE BY BINARY FISSION Binary Fission Chromosome divides to produce two identical copies 1 2 4 8 16 32 64 128 256 512 Contributes to the remarkable adaptability of bacteria Growth in a hostile environment can create a selective pressure for mutant cells which can persist. One mutant cell which can survive will rapidly grow and take over.  GROWTH REQUIREMENTS For Bacterial Cells to Grow they require: 1. Energy 2. The building blocks required for the construction of cellular machinery 3. Appropriate environmental conditions ENERGY AND GROWTH REQUIREMENTS Energy Derived from the enzymatic breakdown of organic substrates (carbohydrates, lipids, proteins) in a process called catabolism Growth requirements (vary depending on organism) Nutrients (water, carbon, nitrogen, inorganic salts, iron) Temperature pH 02 (Anaerobic vs Anaerobic) WHY IS BACTERIAL GENETICS RELEVANT TO CLINICAL MICROBIOLOGY Emergence of antibiotic resistant pathogens and pathogens with enhanced virulence are driven by genetic variation processes Some antibiotics target genetic processes Genetic methods have been developed that facilitate early detection of pathogens allowing more timely treatment of patients with infection  LEARNING OUTCOME 5. Describe the structure of bacterial DNA and the process of DNA replication  BACTERIAL GENOME Bacterial genome is the total collection of genes carried by a bacterium both on its chromosome and on plasmids. Genome contains genetic information required for all cellular processes Circular molecule of double stranded DNA (helix) Contains approx. 4000 genes; 5 million DNA base pairs  DNA – DEOXYRIBONUCLEIC ACID Genes are composed of DNA Basic building blocks of DNA are nucleotides. – They consist of Base: Guanine (G), Adenine (A), Cytosine (C), or Thymine (T) Sugar: deoxyribose One or more phosphate groups Bases extend from the sugar phosphate backbone to form a helix or ‘ladder’ structure. The double stranded DNA is wound around its axis to form the double helix.  SUPERCOILING DNA gyrase is a topisomerase enzyme (Type II Topoisomerase) Catalyses the negative supercoiling of DNA which releases tension in the structure This activity accommodates replication and transcription  DNA REPLICATION The process of generating an identical set of genes during cell division DNA replication is a semi-conservative process 4 steps in the process: – Initiation, Elongation, Proofreading, Termination One strand serves as template for the second strand  DNA REPLICATION: INITIATION Replication begins at a region on the chromosome called the ‘origin of replication’ (oriC) An enzyme Helicase unwinds the dsDNA at the origin to expose ssDNA which can be replicated. Replication occurs in both directions from what are termed growing forks.  DNA REPLICATION: ELONGATION The enzyme DNA polymerase attaches to DNA and catalyses the formation of the new strand. It adds bases that are complimentary to the bases in the parent strand However, this enzyme works in one direction (5’ to 3’) and remember the two DNA strands are oriented in opposite directions The leading strand The lagging strand Okazaki fragment DNA polymerase RNA primase lays down an RNA primer when the single strand DNA becomes available. copies the DNA and the DNA polymerase copies the DNA and the RNA primer (in the 5’ RNA primer to 3’ direction), these are called Okazaki fragments DNA ligase links the Okazaki fragments. (in the 5’ to 3’ direction) Replication is discontinuous on this strand, the lagging strand DNA REPLICATION: PROOF-READING AND TERMINATION Proof-reading Errors can occur during DNA replication. – These ooccasional inaccuracies give rise to a slightly altered nucleotide sequence - a mutation DNA polymerase has proofreading activity. – It can remove bases inserted incorrectly and replace them with the correct base Termination At the completion of the replication process two identical daughter helices have been made  DNA REPLICATION – SUMMARY Four stages: Enzymes involved 1. Initiation include: 2. Elongation helicase, 3. Proofreading DNA polymerase, 4. Termination ligase, gyrase  LEARNING OUTCOME 6. Explain bacterial gene expression (transcription and translation)  GENE EXPRESSION How do bacteria decode the genetic information contained in DNA to produce the proteins they require? GENE STRUCTURE DNA contains the genetic information (genes) Open-reading Frame (ORF) required for all cellular processes Genes can occur individually or in groups (operons) (rare in eukaryotes)  GENE EXPRESSION Transcription Initiated at the promoter region upstream of the gene RNA polymerase copies the DNA and produces an RNA transcript (messenger RNA/mRNA) Translation mRNA is decoded by ribosomes and transfer RNA (tRNA) molecules to specify the exact sequence of amino acids in a protein  TRANSCRIPTION RNA polymerase binds to promoter regions and unwinds dsDNA ahead of it in short segments RNA polymerase transcribes DNA into RNA  TRANSLATION mRNA template (from transcription) is threaded through the ribosome for decoding of information to the amino acid sequence Amino acyl transfer RNAs (tRNA) transports amino acids to the ribosome to form peptide chains  (Turning Point Session ID: FFP1ML2) WHICH ENZYME ADDS NUCLEOTIDES TO THE GROWING STRAND DURING REPLICATION? A.Helicase B.Ligase C.Polymerase D.Topoisomerase II  LEARNING OUTCOME 7. Describe the clinical significance of plasmids and DNA mutation  PLASMIDS Small circular extrachromosomal DNA molecules Replicate independently, can be copied and transferred between cells Can confer phenotypic advantages to the host cell Plasmids with multiple antibiotic resistance genes predominate within hospital bacteria – this makes the host bacterium resistant to a wide range of antibiotics. Plasmids res id e To a m xi n o n ge lp h n e Su Plasmid genes: Antibiotic resistance genes (often multiple) Virulence genes (e.g. toxins) Metabolic genes n ce s i sta i ll i n re p ic Am  GENE MUTATIONS Most common source of genetic variation Spontaneous or induced (mutagens) Mutation rates of 10-3-10-9 per cell division Three types: – Substitution – Deletion – Insertion  SUBSTITUTION Usually only affect the amino acid encoded by that triplet codon and no changes beyond that point Effects on protein may be significant if in a region important for functionality, but may be a ‘silent’ mutation with no effect on protein function Bases TTG AAA ATT CGA Codons Leu Lys Ile Arg Bases TTC AAA ATT CGA Codons Phe Lys Ile Arg  DELETIONS Deletions can additionally affect amino acid sequence beyond the point of the deletion, as they cause misreading of triplet codons beyond the deletion point This is referred to as a frameshift Bases TTG AAA X CGA ATT GAT CAA Codons Leu Lys Ile Arg Asp Gln Bases TTG AAA ATC GAG ATC AA Codons Leu Lys Ile Glu Ile  INSERTIONS Deletions and insertions (frameshift mutations) often result in premature termination of translation as a STOP codon can be generated by the frameshift. Premature termination results in truncated proteins which may be non-functional. STOP codons: C TAA Ochre TAG Amber TGA Opal Bases TTG AAA GAT AAG ATT --- Codons Leu Lys Asp Lys Ile Bases TTG AAA CGA TAA GAT T-- Codons Phe Lys Arg OCH ANTIBIOTIC OVER USE AND MISUSE LEADS TO ANTIBIOTIC RESISTANCE (AMR) BECAUSE … A. Antibiotics can increase mutation rates B. People develop immunity to antibiotics over time C. Bacteria with AMR genes can survive and predominate  SUMMARY Different genera of bacteria can be differentiated from one another on the basis of their cell shape, how they are arranged when viewed by microscopy and Gram stain result Gram-positive and Gram-negative bacteria differ from another in the structure of their cell envelope (cell envelope = cytoplasmic membrane, cell wall (thicker in Gram-positives) and outer membrane (only in Gram-negatives) Bacteria can possess pilli to aid in attachment, flagellae for movement, some can produce spores for survival and most can produce a biofilm. All important factors in pathogenesis of infection and for treatment DNA structure and replication, important to be able to describe these The processes must be carefully controlled Plasmids contribute to dissemination of antibiotic resistance Importance of mutations and genetic exchange mechanisms. Roles in clinical infection, e.g. evolution of virulence and antibiotic resistance Thank you