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Deinococcus radiodurans: The
Toughest Bacterium on Earth
Klebsiella pneumoniae
Overview
Klebsiella pneumoniae is a short, gram-negative (-) rod-shaped bacterium that can be found in single cells, pairs, or clusters. It belongs to the Enterobacteriaceae family, which includes E. coli. Klebsiella pneumoniae is a normal part of the human microflora, residing in the nasopharynx and gastrointestinal tract. It is also ubiquitous in the environment, found in water, sewage, soil, plants, and animals.
Pathogenicity
Klebsiella pneumoniae is an opportunistic pathogen that can cause hospital-acquired and community-acquired infections. It is an important nosocomial (hospital-acquired) pathogen, causing urinary tract infections, intra-abdominal infections, pneumonia, septicemia, and soft tissue infections. Klebsiella pneumoniae is often treated with the antibiotic ciprofloxacin, but antibiotic resistance is increasing, particularly to penicillin and ampicillin (CRE superbug).
Virulence Factors
Klebsiella pneumoniae produces a thick capsule (K antigen) that protects it from phagocytosis. It also produces siderophores, which are chelators that help it acquire iron from the host cell. Klebsiella pneumoniae is capable of fixing atmospheric nitrogen into ammonia and amino acids.
Other Characteristics
Klebsiella pneumoniae is a facultative anaerobe, meaning it can grow with or without oxygen. It is non-spore forming and non-motile. Klebsiella pneumoniae ferments lactose and grows optimally at 37°C.
Vibrio cholerae
Overview
Vibrio cholerae is a comma-shaped, gram-negative (-) bacterium that is highly motile with a single polar flagellum. It is the causative agent of the disease cholera, although not all strains are pathogenic. Vibrio cholerae is found in aquatic environments, including fish and shellfish.
Pathogenicity
Vibrio cholerae is endemic in areas with poor sanitation, particularly in Asia and Africa. The pandemic strain, V. cholerae O1 El Tor N196961, is a major cause of cholera in third-world countries. Infection occurs through the ingestion of fecally contaminated food or water, where the bacteria infect the intestinal villi, releasing a toxin that causes severe diarrhea, vomiting, and dehydration. Untreated, cholera can be fatal in 50-70% of cases. The cholera toxin gene is believed to have been acquired by Vibrio cholerae through viral transfer. Vibrio cholerae is the second leading cause of death in children under 5 years old.
Other Characteristics
Vibrio cholerae is a facultative anaerobe and does not form spores or a capsule. It requires 2-3% NaCl for growth and has an optimal growth temperature of 37°C. Vibrio cholerae is used as a model organism to study quorum sensing, which enables cells to communicate with each other.
Neisseria gonorrhoeae
Overview
Neisseria gonorrhoeae is a coffee bean-shaped diplococcus (pairs with flattened sides). It is the causative agent of the sexually transmitted disease gonorrhea, also known as "The Clap".
Pathogenicity
Neisseria gonorrhoeae attaches to and infects mucosal epithelial cells through a process called parasite-directed endocytosis. Humans are the only natural host for Neisseria gonorrhoeae.
Deinococcus radiodurans has complex growth requirements and is found in various environments, including ground meat, feces, air, and fresh water. It is a mesophile, growing optimally at 25-35°C, and is not a pathogen.
Borrelia burgdorferi
Overview
Borrelia burgdorferi is a spirochete, a single-celled bacterium with a spiral shape, that requires a darkfield microscope to be observed due to its small diameter. It is the causative agent of Lyme disease.
Pathogenicity
Borrelia burgdorferi is found in the intestinal tract of animals and the oral cavity of humans. The natural reservoir for Borrelia burgdorferi is the white-footed mouse. Lyme disease is a tick-borne illness, spread by the bite of the deer tick (Ixodes). Borrelia burgdorferi invades the blood and tissues of mammals and birds, including humans, white-tailed deer, and dogs. Lyme disease is the most prevalent tick-borne illness in the U.S., with over 20,000 new cases reported annually.
Characteristics
Borrelia burgdorferi can survive without iron, using manganese instead. It is a fastidious, microaerophilic bacterium with a slow growth rate of 12-24 hours per generation. Borrelia burgdorferi responds to penicillin and is weakly gram- negative, not staining well. It does not form spores or a capsule, but is motile due to its axial filaments that allow it to move in a corkscrew fashion.
Chapter 13: Metabolism
Anaerobic Respiration
Anaerobic respiration uses electron carriers other than oxygen, yielding less energy than aerobic respiration due to the less positive redox potential of the alternative electron acceptors.
Carbon Utilization in Microorganisms
Fermentation
Fermentation allows organisms to recycle their finite supply of NAD+ electron carriers by using organic molecules as the terminal electron acceptor, rather than oxygen. This allows organisms to adjust to changes in their environment and continue glycolytic pathways. Different types of fermentation include lactic acid fermentation, alcohol fermentation, and mixed acid fermentation.
Chemolithotrophy
Chemolithotrophs obtain energy by transferring electrons from inorganic molecules to a terminal electron acceptor, usually oxygen, through an electron transport chain. This process generates a proton motive force that is used to synthesize ATP through oxidative phosphorylation. Chemolithotrophy produces much less energy than aerobic respiration, as the reduction potentials of inorganic molecules are less positive than that of oxygen.
Phototrophy and Photosynthesis
Photophosphorylation
Photosynthetic organisms use photosynthetic pigments to capture light energy and convert it into chemical energy in the form of ATP through the process of photophosphorylation. In eukaryotes, the light reactions occur in specialized thylakoid structures, while in prokaryotes they occur in the cell membrane. Photosynthesis combines phototrophy (light capture) and carbon fixation to produce organic carbon compounds.
Oxygenic vs. Anoxygenic Photosynthesis
Oxygenic photosynthesis, carried out by photosynthetic eukaryotes and cyanobacteria, uses water as the electron source and releases oxygen as a byproduct. Anoxygenic photosynthesis, carried out by phototrophic green bacteria, purple bacteria, and heliobacteria, does not use water as the electron source and does not release oxygen.
Bacteriorhodopsin-Based Phototrophy
Some archaea, such as Halobacterium, use a type of phototrophy that involves the membrane protein bacteriorhodopsin, which functions as a light-driven proton pump to generate a proton motive force without an electron transport chain.
Relationship between Catabolism and Anabolism
Anabolism depends on catabolism to provide the small molecules and ATP needed to build up larger molecules. Catabolism breaks down larger molecules to generate the building blocks and energy required for anabolic processes.
DNA Replication and Gene Expression
Major Macromolecules in Cells
Deoxyribonucleic Acid (DNA) is the storage molecule for genetic instructions that guide metabolism and reproduction. DNA must be replicated to make new cells (DNA Replication) and transcribed into RNA to provide instructions for protein synthesis (Transcription and Translation). Cells have mechanisms to deal with damage to the DNA (DNA Repair).
Experiments Demonstrating DNA as the Hereditary
Molecule
Griffith's Experiment: Demonstrated that a "transforming principle" could convert a non-virulent strain of Streptococcus pneumoniae into a virulent strain, suggesting DNA as the hereditary material. Avery, MacLeod, and McCarty Experiment: Showed that the transforming principle was DNA, not protein or RNA. Hershey-Chase Experiment: Confirmed that DNA, not protein, was the genetic material in bacteriophages.
Genome and Genotype/Phenotype
Genome refers to the complete set of DNA present in a cell or virus. Bacteria and Archaea generally have a single, circular chromosome (haploid), while Eukaryotes have multiple, linear chromosomes (diploid). Genotype is the specific set of genes an organism possesses, while Phenotype is the collection of observable characteristics.
Central Dogma of Molecular Biology
The Central Dogma describes the flow of genetic information from DNA to RNA to Protein, which is conserved in all cellular life forms. Transcription is the process of making an RNA copy of a specific gene. Translation is the process of using the information in mRNA to synthesize a polypeptide, involving tRNA and rRNA.
Structure of DNA
DNA is composed of nucleotides, each containing a 5-carbon sugar (deoxyribose), a phosphate group, and a nitrogenous base (Adenine, Guanine, Cytosine, or Thymine). The sugar-phosphate backbone is formed by phosphodiester bonds, and the two strands are complementary and antiparallel. Bacteria have a single, circular chromosome, while Archaea and Eukaryotes have their DNA packaged around histone proteins.
DNA Replication
DNA replication is a semi-conservative process, where each new daughter cell inherits one original and one newly synthesized strand. Replication occurs at the replication fork, where the two strands separate, and DNA polymerase synthesizes the complementary strands. Replication is highly accurate, with only about 1 mistake per 10^9 base pairs, and it is also very fast, at 750-1000 base pairs per second. The leading strand is synthesized continuously, while the lagging strand is synthesized discontinuously in short Okazaki fragments.
Okazaki Fragments and DNA Replication
Helicase and DNA Replication
Helicase is not a cutting enzyme; it simply unwinds and separates the DNA strands. During DNA replication, once the replication fork forms, DNA polymerase III adds nucleotides to the initial RNA primers. This process results in the formation of a continuous leading strand and a discontinuous lagging strand (forming Okazaki fragments). The process of DNA replication is virtually identical in both bacteria and eukaryotes. DNA polymerase I removes the RNA primers and fills in the gaps with new nucleotides. DNA ligase seals the sugar-phosphate backbone between the Okazaki fragments on the lagging strand.
Telomerase and Proofreading
Telomerase extends the telomeres in eukaryotes by adding bases at the end, providing a template for DNA polymerase to bind to. Proofreading is carried out by DNA polymerase III, which removes mismatched bases from the 3' end of the growing strand through its exonuclease activity. This proofreading activity is not 100% efficient, but the same molecule that is adding the bases can look back to ensure the right base has been added, preventing many mutations.
Promoter Region and RNA Polymerase Functions
The promoter region is where RNA polymerase will attach to begin transcription. The promoter region contains the Pribnow box, which is the recognition/binding site for RNA polymerase. The promoter region is not transcribed, and it has a high AT content to facilitate the unwinding of the DNA when RNA polymerase binds. RNA polymerase functions include unwinding the DNA, moving along the template, and synthesizing mRNA.
Leader Sequence and Terminator
The leader sequence is transcribed into mRNA but is not translated into amino acids. The Shine-Dalgarno sequence is part of the leader sequence and is important for the initiation of translation. The terminator is where transcription ends.
Transcription in Bacteria
Sigma Factors and RNA Polymerase
In bacteria, sigma factors bound to the RNA polymerase core enzyme direct the combined holoenzyme to a promoter. Once the RNA polymerase is situated, the sigma factor dissociates. Different sigma factors can direct the core RNA polymerase enzyme to different genes as needed. The core RNA polymerase enzyme is composed of 5 chains and catalyzes RNA synthesis, while the sigma factor has no catalytic activity but helps the core enzyme recognize the start of genes.
Transcription Elongation and Termination
During transcription elongation, the RNA polymerase unwinds the DNA, and a transcription bubble is produced, which moves with the polymerase as it transcribes mRNA from the template strand. Transcription termination can be Rho-dependent, where the Rho protein follows the RNA polymerase and pops it off the DNA when it reaches a termination sequence, or Rho-independent, where the DNA sequence transcribed forms an RNA hairpin loop structure that causes the RNA polymerase to dissociate from the DNA.
Eukaryotic Transcription
In eukaryotes, the RNA is modified after transcription, including the addition of a 5' cap and a poly(A) tail, as well as the splicing out of introns and joining of exons. The TATA box at the promoter regions helps RNA polymerase know where to begin transcription.
Translation
Initiation, Elongation, and Termination
Translation involves a series of events, including initiation, elongation, and termination, and is the most energy-intensive process, so it is tightly controlled and regulated. The direction of protein synthesis is from the N-terminus to the C- terminus, and it is very fast, with up to 900 amino acids per minute in E. coli.
Ribosomes and rRNA Functions
Ribosomes are the site of translation, and in bacteria and archaea, transcription and translation are coupled due to the lack of a nuclear membrane. Bacterial ribosomes are 70S, composed of 30S and 50S subunits. Polyribosomes are complexes of mRNA with several ribosomes. The rRNA functions in translation include contributing to the structure of the ribosome, providing the ribosomal binding site for mRNA (16S rRNA), and catalyzing peptide bond formation.
Codons, Anticodons, and the Genetic Code
Codons are 3-base sequences in mRNA that specify individual amino acids. The anticodon on tRNA is complementary to the codon and binds to it. The start codon is always AUG, which codes for methionine. Sense codons are the 61 codons that specify amino acids, while stop (nonsense) codons are the 3 codons used as translation termination signals. The genetic code exhibits degeneracy, where up to six different codons can code for a single amino acid, and the last base of the codon (the "wobble" base) is not as critical for specifying the amino acid.
Transfer RNA (tRNA)
tRNA has a cloverleaf secondary structure due to base pairing within the molecule. The anticodon on the tRNA is complementary to the codon and binds to it. The 3' end of the tRNA binds the amino acid.
Amino Acid Activation
Aminoacyl-tRNA synthetases are enzymes that attach the correct amino acid to the corresponding tRNA using a high-energy bond from ATP. If the wrong amino acid were attached, it could lead to the incorporation of the wrong amino acid into the growing polypeptide chain.
Protective Mechanism and Refolding of
Denatured Proteins
Mutations and Genetic Variation
Mutations are not always bad - some occur in non-coding regions and do not affect the final protein. Mutations are important for evolution, as they introduce genetic variation. Most mutations are deleterious and result in non-functional proteins.
Types of Mutations
Point Mutations
Most common type of mutation, involving the alteration of a single nucleotide pair. Can be spontaneous (low rate) or induced (caused by agents that damage DNA).
Larger Mutations
Insertions, deletions, inversions, duplications, and translocations.
Causes of Mutations
Base analogs (e.g. 5-bromouracil) can be mistakenly incorporated during DNA replication. DNA modifying agents can alter bases, causing them to mispair. Intercalating agents can distort the DNA structure, leading to single nucleotide pair insertions and deletions. UV light can produce thymine dimers, which are one of the easiest mutations to repair.
Effects of Mutations
In Protein-Coding Genes
Silent mutations: change the nucleotide sequence but not the encoded amino acid. Missense mutations: change a single amino acid in the encoded protein. Nonsense mutations: convert a sense codon to a stop codon, resulting in a truncated protein. Frameshift mutations: insertions or deletions of one or two base pairs, shifting the reading frame and likely producing a non-functional protein.
Other Types of Mutations
Conditional mutations: expressed only under certain environmental conditions. Auxotrophic mutants: unable to synthesize an essential macromolecule, but can survive if the deficient nutrient is provided. Mutations in regulatory sequences: can affect gene expression without directly altering the encoded protein. Mutations in tRNA and rRNA genes: can disrupt protein synthesis.
Bacterial Genetic Analysis
Advantages of Bacteria for Genetic Research
Single chromosome makes it easy to detect mutations. Auxotrophic mutations (nutritional requirements) can be easily identified.
Mutation Detection and Selection
Observation of Phenotypic Changes
Positive selection process, looking for something that grows.
Replica Plating Technique
Used to detect auxotrophic mutants, a negative selection process looking for something that cannot grow.
Phenotypic Selection
Using growth media that will inhibit microbes lacking the desired genes, such as antibiotic resistance.
Repair of Mutations
Proofreading
DNA polymerase's 3'-5' exonuclease activity corrects errors during replication.
Excision Repair
Nucleotide excision repair and base excision repair remove and replace damaged DNA.
Direct Repair
Photoreactivation and direct repair of alkylated bases.
Transposition: Segments of DNA that Move
About the Genome
Jumping Genes
Transposition is a process where segments of DNA move about the genome and can be integrated into different sites in the chromosome. These DNA segments are called "jumping genes" or transposable elements.
Insertion Sequences
The simplest transposable elements are just insertion sequences, which carry no genes.
Composite Transposons
Transposable elements that contain genes other than those used for transposition are called composite transposons. Composite transposons were discovered in the 1940s by Barbara McClintock.
RecA and Antibiotic Resistance
Transposable elements can use RecA for double-stranded breaks, allowing them to use new genetic information for both strands. They can also carry other genes like antibiotic resistance, which can be transferred quickly between cells.
Replicative Transposons
Replicative transposons have a resolvase gene, which allows them to leave a copy behind and move around, amplifying the transposon quickly.
Insertion Sequence (IS) Elements
Insertion sequence (IS) elements are short sequences of DNA (700-1600 bp) that contain only the gene for the enzyme transposase and have inverted repeats (15-25 bp) at each end. Their only function is to recognize and insert at complementary places in the chromosome. IS elements move by simple (cut and paste) transposition.
Transposons
Transposons have the transposase gene plus other genes, such as antibiotic resistance genes.
They usually consist of two IS elements on each side of a gene with inverted repeats and move by simple (cut and paste) transposition.
Replicative Transposons
Replicative transposons have additional genes, including a transposase and a resolvase gene, and move by making copies of themselves.
Simple Transposition
Simple or "cut and paste" transposition involves transposase-catalyzed excision and cleavage of a new target site, followed by ligation.
Replicative Transposition
Replicative transposition involves two genes coding for enzymes (transposase and resolvase), where the original transposon remains at the parental site, and a copy is inserted in the target DNA.
Effects of Transposable Elements
Transposable elements can insert within a gene to cause a mutation. Some carry stop codons or termination sequences to block transcription or translation. Some may carry promoters and activate genes near the insertion points. Some can turn genes on or off. Some can participate in plasmid fusion, insertion of plasmids into chromosomes, and plasmid evolution.
Role of Transposons in Plasmid Evolution
Plasmids have several different transposon target sites, so transposons frequently move between plasmids. This is a concern with the transfer of antibiotic resistance genes between plasmids and between plasmids and chromosomes. Plasmid genes are also passed during bacterial conjugation, allowing antibiotic-resistant transposons to spread (conjugative transposons). The Tn3 transposon, which carries an R plasmid, can confer resistance to β-lactam antibiotics like penicillin.
Bacterial Plasmids
Bacterial plasmids are small, autonomously replicating DNA molecules that can exist independently or integrate reversibly into the host chromosome as episomes. Conjugative plasmids, such as the F plasmid, can transfer copies of themselves to other bacteria during conjugation.
Lytic vs. Lysogenic Cycle
In the lytic cycle, the virus replicates and destroys the host cell. In the lysogenic cycle, the viral DNA integrates into the host genome, and the organism makes more copies of the phage genome as it divides.
Generalized and Specialized Transduction
Generalized transduction occurs during the lytic cycle of virulent phages, where fragments of host DNA are mistakenly packaged into the phage head. Specialized transduction is carried out by temperate phages that have established lysogeny, where only specific portions of the bacterial genome are transferred when the prophage is incorrectly excised.
Defective Transducing Particles
Defective transducing particles take up some of the host DNA by accident and transfer that genome fragment to a new host, as they lack the ability to make more of themselves.
