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1. How to get changes in the genome? Mutation (Endogenous) Horizonal gene Transfer (Exogenous) -Transformation (Getting it from the environment) Griffith's Experiment, Occur Naturally in Environment. -Tranduction (Getting it from the virus) -Conjugation (Gettig it straight from another cell, through cell-to-cell contact) How mutations occur in genomes. Changes in the DNA sequence, if not repaired, can be passed to the RNA and, from there, to proteins. Some changes may be innocuous and have no effect on protein function; others lead to phenotypic variation. Because bacteria and archaea usually reproduce quickly and to high numbers when conditions are favorable, they can accumulate changes in their DNA sequence and rapidly pass them to progeny cells. Any change in the DNA sequence inherited by the progeny is called a mutation. Small Mutation are 1bp to 10 bp Large mutations are anthign thing larger then 10 bp
Auxotroph: - Requires an exogenous building block or growth factor Condition lethal: - Unable to grow in a particular envirnonement, can grow in some and may not in others. To recognize mutations:- Enrichment method: - Kill/Stop growth of the cells without mutation. Replica Plating :- Kill/Stop the growth of the cells with mutation. Mutation can be Exogenous (UV light, mutagens) or Endogeonous (DNA Replication, Reactive byproducts, Transposons)
2. What is the difference between DnaC, DnaG, and Gyrase? DnaC:- DnaC with the help of DnaA helps lod the DnaB (helicase) onto teh single strand of DNA DnaG (RNA Primase):- Synthesizes short RNA primers on Dna strand one in Leading and multiples in Lagging strand to provide a starting point for DNA Polymeraze III. DNA polymerase III cannot start synthesis on its own and needs these RNA primers to add the nucelotides. Gyrase:- Type of topoisomerase. Introduces negative supercoils ahead of the replication fork, alleviating the tension generated as helicase unwinds the DNA. This makes it
Type VI sectretaion system is not sec dependent (use ATP and PMF) Transports both DNA and proteins from bacteria to other cells (can also inject into host cells).
- Evolved form bactreiophage injection mechanism.
- (can target Prok + Euk) 5. Post-translational Modifications (PTMs) Post-translational modifications (PTMs) are chemical changes made to a polypeptide chain after translation, transforming it into a functional protein. Peptide chain is not necessarily functional yet, Covalent Modifications: Changes to the protein molecule itself Cleaving amino acids: This includes the removal of the N-terminal formylmethionine (fMet) or other N-terminal signal peptides. ○ N-terminal modifications: Often involve removal of signal peptides Addition of other molecules: ○ Assembly reactions: Permanent chemical additions/changes such as glycosylation (adding sugars), phosphorylation (adding phosphate groups), acetylation (adding acetyl groups), and more. ○ Modulation reactions: These are reversible changes, to regulate the protein’s activity.
Protein Folding
Importance of Folding : ○ Protein folding is crucial because a protein's function is directly tied to its 3D structure. Even small misfolding can lead to loss of function or diseases. Chaperones : ○ Not all proteins can fold properly on their own, so chaperones (specialized proteins) help other proteins fold correctly. They assist in preventing misfolding and aggregation. Types of Chaperones :
○ Trigger factor : A chaperone that binds to nascent (newly synthesized) peptides to prevent premature folding. ○ DnaK : Helps refold misfolded proteins by fixing hydrophobic regions that incorrectly interact with each other. ○ GroEL/GroES (USE ATP) : A Large folding machine that protects peptides from protease while they are properly folded.
**6. Stress response
- Lac-Operon** A model for regulating gene expression, The lac operon is a system that allows bacteria to efficiently metabolize lactose only when it is present in the environment, while saving energy when other carbon sources, like glucose, are available. Prokaryotes have Gyrase, eukaryotes DO NOT have gyrase
The stringent response helps bacteria survive unfavorable conditions by slowing down processes like ribosome activity and DNA replication, and shifting the bacterial cell's energy toward stress survival mechanisms.
Exospores
● Formed by: Some fungi, algae, and actinobacteria. ● Location: Formed outside or at the surface of the cell. ● Purpose: Typically for reproduction rather than extreme survival. ● Structure: Usually thinner and less resistant than endospores, making them more vulnerable to environmental conditions. ● Release: Bud off from the parent cell and can grow into new organisms if they find suitable conditions. Endospore (Develop inside the parent bacterial cell) :- Endospores are a highly resistant, dormant cell type produced by certain bacteria, notably Bacillus and Clostridium species. Endospores are formed in response to adverse environmental conditions, allowing bacteria to survive extreme stresses such as heat, radiation, desiccation, and exposure to chemicals. Entry into sporulation:- when nutrients are limited but still sufficient to allow sporulation. Stage I: - Axial Filament Stage II:- Septum forms Stage III:- When Mother cell engulf Forespore Stage IV:- Cortex formation
- – Mostly murein
- – May have a protein spore coat (exosporium)
- Internal changes:
- Small Acid-Soluble
- Proteins (SASPs) Stage V/VI:- further refinement Stage VII :- Release
- Complete endospore
- Contains no detectable metabolism Transcription initiation:- when Dna binds to sigma factor and is attached to the promoter. The promoter consists of key sequence elements, such as the -10 and -35 consensus sequences. These sequences are recognized by the sigma factor, which helps position RNA polymerase correctly at the transcription start site. Biofilms provide a multifaceted survival advantage for bacteria, offering protection from environmental stresses, promoting nutrient exchange, facilitating genetic exchange, enhancing adhesion, supporting cooperative behaviors, and enabling persistence in chronic infections. These benefits make biofilms a key factor in bacterial pathogenicity and survival in various environments. The different phases of growth? Stationary and exponential phase. Stationary phase is a type of stress response, there are many ways to get to the stationary phase. Any type of stress sRNAS is usually the first one to integrate multiple (stress factors) into a single response (cell does not waste its energy to create multiple responses for each stress). RpoS regulates (make) genes that help bacteria prepare for and survive the stationary phase during stress. Transposons;- can spread antibiotic resistance Major contributors to evolution are the so-called mobile elements (or transposable elements), such as transposons. Transposons are pieces of DNA that move from one location to another, and they are abundant. All organisms—bacteria, archaea, and eukaryotes—carry such “jumping genes.” There are different kinds of transposons that “hop” by different mechanisms (Table 10.5), but in all cases their movement is catalyzed by a transposase encoded in the transposon. All transposons carry terminal inverted repeats that serve as recognition sites for the transposase enzyme. Depending on the type of transposon, the transposase either cuts the element and pastes it in another location (the “cut and paste” mechanism) or makes a copy to be inserted elsewhere (the replicative mechanism; Fig. 10.3). The insertion site is not random: the transposase recognizes and cuts specific sequences, usually direct repeats, in the target DNA Lytic cycle:- Replicates rapidly Lysogenic cycle:- Integrate its own DNA into bacterial Chromosomes and become dormant (prophage) and gets passed to all the descendents, as it replicates and aligns with the bacteria genome. Lysogeny can last indefinitely until something like stress triggers the phage to enter the lytic cycle.
Conjugation:- Transfer of F plasmids Normal Excision (in the lysogenic cycle): In a normal excision, when the prophage (the phage DNA integrated into the host's genome) is induced to enter the lytic cycle, it excises precisely from the host genome. The excised phage DNA is then used to replicate new phages, and no bacterial genes are included in the phage particles. This process results in the production of only phage DNA being packaged in the phage capsids and no transfer of host DNA occurs. Abnormal Excision (in specialized transduction): In specialized transduction, abnormal excision occurs during the transition from the lysogenic cycle to the lytic cycle. The phage excises from the host chromosome imprecisely, which means that a small portion of the host’s genome, located near the integration site, is excised along with the phage genome. As a result, the phage particles that are produced now carry both phage DNA and host DNA. When these phages infect a new bacterial cell, they can transfer the host genes (which were mistakenly excised) into the recipient bacterium. Antibiotics:- Any agent that slows or stops growth of microorganisms
- Inhibitors of essential processes Bacteriostatic – stops bacteria from dividing Bactericidal – kills bacteria 3 major mechanisms Antibiotic works
- DNA replication (quinolones, etc)
- Protein production (tetracyclines, macrolides, etc)
- Cell wall synthesis (penicillins, etc) Other mechanisms
- RNA transcription (rifamycins)
- Antimetabolites (sulfonamides)
- Depolarize membrane (lipopeptides) Many antibiotics are natural products of secondary metabolism → many produced by Streptomyces spp. (genus of Actinobacteria) → other sources: fungi, insects Alexander Fleming – Doctor in WWI, saw many patients die of sepsis He was Studying Staphylococci, found a plate contaminated with mold, he noticed Bacteria did not grow near the mold, but could grow a certain distance away. That's when he discovered Fungus penicillium notatum, which had natural antibiotic properties Penicillin could kill many gram-positive, Not good at killing Gram-negative bacteria. This antibiotic did not require physical contact → fungus produced a secreted substance “mold juice” AKA penicillin Penicillins and sulfonamides used with great effect in WWII Antibiotics developed as potential drugs rapidly after discovery
B-lactam antibiotics
Bind and inhibit the activity of penicillin binding proteins (transpeptidase)
B-lactam antibiotic resistance:- Decreased influx (porins)
increased efflux (multidrug pumps)
TB
Asymptomatic Reservoirs :
Mobility and Copying :
● These mobile elements can move within the genome or replicate ,
creating multiple copies within the same bacterial cell. This increases
genetic diversity and can lead to new traits (like antibiotic resistance)
spreading throughout the population.
Transposons and Antibiotic Resistance :
● Transposons are specific segments of DNA that can carry antibiotic
resistance genes. They can insert themselves into various parts of the
genome, and in some cases, they can move onto plasmids.
Plasmids as Vectors for Resistance :
● When a plasmid contains a transposon with resistance genes, it can
transfer between different strains of C. difficile or even to other bacterial
species. This movement allows for horizontal gene transfer , spreading
antibiotic resistance genes rapidly across a bacterial population.
Implications :
● Because of these mobile elements, C. difficile can not only acquire
resistance to multiple antibiotics but can also evolve rapidly through
mutations in these elements, making treatment more difficult as resistance
traits spread between strains.
To combat antibiotic resistance:
1. New Antibiotics: Develop drugs with new targets or modify existing ones
for increased effectiveness.
2. Vaccines: Create vaccines to prevent infections and reduce antibiotic
need.
3. Extend Current Drug Use:
○ Limit and control antibiotic use to prevent overuse.
○ Alternate antibiotics or use adjuvants to reduce treatment time.
○ Educate on correct antibiotic practices to avoid misuse.
Bacillus spores are extremely resistant to heat
Heat shock REsponse:- Key points about the Heat Shock Response:
1. Sensor: The sensor for heat shock response in E. coli is mRNA encoding an σ-factor, specifically RpoH (also called σH or σ32). ○ Under normal temperatures, the mRNA has a hairpin structure that sequesters the Shine-Dalgarno (SD) sequence, preventing translation. ○ Heat stress causes the hairpin to melt, exposing the SD sequence, allowing for translation. 2. Role of RpoH (σH): ○ RpoH (σH) is a sigma factor that helps RNA polymerase (RNAP) bind to specific promoters, leading to the expression of heat shock genes. ○ RpoH activates the transcription of more than 250 genes in E. coli, which include genes encoding membrane proteins and chaperones. 3. Chaperones: DnaK chaperon and FtsH protease control this process. The primary function of chaperones in the heat shock response is to assist in the proper folding of proteins that may have become misfolded due to heat stress. Heat causes proteins to unfold, which can lead to aggregation and loss of function. Chaperones help prevent this DnaK is one of the chaperones that is produced or translated during heat stress. Which also helps protein to fold which gets unfold in heat shock. Genomics: You can read The complete DNA sequence of an organism (the genome), including all genes and non-coding regions. Metagenomics:- sample from environment and look at the whole genome. Transcriptomics: - what are all of the RNA in a single organism under certain conditions. Proteomics: - all the proteins in a single organism, under certain conditions.
● Synthesis: DNA polymerase fills in the gap using the complementary strand as a template. ● Ligation: DNA ligase seals the backbone, completing the repair. Double-strand breaks are more complex and require specialized repair pathways. The two major repair mechanisms for DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR)
