Horizontal Gene Transfer

Horizontal gene transfer is unique to prokaryotes and describes gene transfer between two mature cells which is different than vertical gene transfer (mother to daughter). The three major mechanisms of gene transfer are transformation, transduction, and conjugation (please see Ch 10). Transformation is gene variation by the direct uptake and insertion of a foreign genetic material outside the cell. Transduction is the process in which foreign DNA is inserted into a cell by a viral vector. Conjugation is the transfer of genetic material via a pilus by direct attachment of the donor cell to the recipient cell. The genetic material is then incorporated into the recipient genome. Such gene transfer processes can be an opportunity for genetic diversity for usual asexual prokaryotes.

1) Madigan, M. T., & Brock, T. D. (2012). Brock biology of microorganisms. Boston: Benjamin Cummings.

Molecular Microbiology – Figure 1.

http://www.phschool.com/science/biology_place/biocoach/transcription/images/proovrvw.gif

Translation

Translation is the process in which an mRNA template is used to synthesize polypeptides. In translation, the ribosome complex is constructed (small 30S subunit + large 50S subunit, for prokaryote) on the mRNA in conjunction with initiator tRNA. The P ribosome binding site is matched with the start codon (AUG), and the methylated initiating tRNA associates with the region. As elongation begins, elongation factors use GTP to help establish the new tRNA into the A site. Peptide bond formation is assisted by the 23S rRNA as the polypeptide chain grows. As the ribosome moves along the mRNA, translocation is powered from one codon to the next by GTP hydrolysis. The leaving tRNA is released from the E site. Translation continues as new tRNA enters the A site. Protein synthesis ends when a stop codon is reached which is recognized by release factors.

In bacteria, 16S rRNA participates in initiation by base pairing with the ribosome binding site. The 23S rRNA participates in translocation besides just structural support.

In regards to defective mRNA in bacteria, bacteria are known to contain tmRNA that frees stalled ribosomes. The tmRNA collides with the stalled ribosome and binds with the faulty mRNA. tmRNA contains a stop codon and stops translation by recognition by release factors.

Fig 1. Central Dogma of prokaryotic cells

Transcription

Transcription is the process in all living organisms that synthesizes RNA from a DNA template. This RNA will later on be used to synthesize proteins through translation. Analogous to DNA polymerase in replication, RNA polymerase creates phosphodiester bonds between ribonucleotides. During elongation, of the RNA chain, ribonucleoside triphosphate is added to the 3’ hydroxyl. This implies that the growth from the 5’ to 3’ end is antiparallel to the DNA template. Initially, the promoter region of DNA is recognized by the sigma factor which signals RNA polymerase to the initiation site. The promotor region found in the much studied E. coli is noted to be TATAAT in the -10 region known as the Prinboiw box. In addition, the TTGACA promoter region is known to be common in the -35 region. The sigma factor is released and transcription continues to grow the RNA chain. Once the termination site (marked by transcription terminators) is reached, transcription ends when RNA polymerase and the newly synthesized RNA is released.

In prokaryotes, polycistronic mRNA is found to encode of several clustered, group of genes in a single mRNA. As a result, several polypeptides are created by a single ribosome. In addition, operons are a group of genes that give rise to these polycistronic mRNA during transcription.

Fig 1. Principle mechanisms of innate and adaptive immune responses

Innate Immune System

After a pathogen has infiltrated barriers of entry such as the skin, the host innate immune system attempts to prevent infection from the foreign invaders. One of the primary innate defenses consists of complement activation. Complement is a group of soluble serum proteins mainly produced by the liver. These complement components become activated during an infection through the classical, lectin, or alternative pathway. In all three of these pathways, proteins (C1, lectin, C3) bind either antibodies or pathogen antigens to induce the complement cascade. This cascade and its terminal complement proteins can untie to form membrane attack complexes, opsonize pathogens to facilitate phagocytosis, and promote an inflammatory response which includes vasodilation and increasing vascular permeability.

The innate immune system also relies on the association of complements with leukocytes which are various white blood cells. The innate leukocytes include: neutrophils, eosinophils, basophils, mast cells, monocytes/macrophages, and dendritic cells.

1) Neutrophils are produced in large quantities in the early stages and migrate quickly to the infection site to phagocytize, and kill pathogens with enzymes and toxic granules.

2) Eosinophils are present in relatively low quantities in blood and have surface receptors for antibodies and C3b that have already binded pathogens. The eosinophils recognize these markers and release cationic proteins to create pores in the pathogen that allow granules, enzymes, lipase, peroxidase, etc. to enter the cell and destroy it.

3) Basophils are also present in relatively low concentration in the blood. They bind antibodies, degranulates, and release toxics. They also release histamine when basophils bind active C3a and C5a fragments.

4) Mast cells differentiate in tissue and are involved in the protection of internal surfaces and also provide histamine for inflammation.

5) Monocytes are phagocytes in the blood that migrate to tissues to differentiate into macrophages. Macrophages phagocytize invading cells and also promote inflammation. Macrophages can also activate adaptive lymphocytes and cleans up after adaptive responses.

6) Dendritic cells are antigen presenting cells that phagocytize pathogens and displays specific antigens on its surface. They are involved in activating T lymphocytes and provide the transition from innate to adaptive response.

Replication

In replication, the DNA double helix is replicated into two copies. Replication is semiconservative and results in two double helixes consisting of a new stand and a parental strand. DNA replication begins with the replication fork. In the initial step of replication, DNA helicase, an enzyme, exposes a segment of the DNA strand by using ATP to unwind the DNA helix. This point is known as the origin of replication. To prevent reforming of the helix, the exposed strand is stabilized by single strand binding proteins. In addition, to prevent positive supercoils caused by unwinding DNA, DNA gyrase counteracts this effect by inserting negative supercoils. Of note, in bacteria, the origin of replication is known as the oriC and is a specific DNA sequence of about 250 bases recognized by DnaA. Two helicases are loaded in opposite directions and pairs of primase and DNA polymerase are loaded onto the DNA behind the helicases.

In DNA replication, replication always proceeds from the 5’ end to the 3’ end. This is due to the need of a hydroxyl group end only found on the 3’ end to continue building the nucleotide chain. Due to this fact, replication consists of a leading strand and a lagging strand. The leading strand continues as expected with no interruptions from the 5’ to 3’ direction. However, the lagging strand creates Ozaki fragments in a discontinuous process. These fragments are later joined together by DNA ligase by excising the RNA primer that was used as a starting guide for the Ozaki fragments.

In prokaryotes, chromosomes are found to be circular in nature and allows for bidirectional replication. This allows the organism to quickly replicate genetic material and overcome the potential limiting factor for growth.

Adaptive Immune System

When the innate immune response is not sufficient in taking care of the invading pathogens, dendritic cells and macrophages initiate the adaptive immune response. Antigen presenting cells phagocytize pathogens and presents antigens through the exogenous pathway. These now activated antigen presenting cells migrate to secondary lymphatic tissues including lymph nodes and spleen where appropriate adaptive cells are activated. Adaptive immune cells include T lymphocytes and B lymphocytes of various types.

T cells are made in the thymus, activated by antigen presenting cells in the lymph node, and are differentiated into various effector T cells including cytotoxic T cells (CD8+), helper T cells (CD4+), and regulator T cells. Cytotoxic T cells function to recognize and kill specific infected cells by releasing cytotoxic compounds from its granules. These compounds induce apoptosis in infected cells. Helper T cells are mainly divided into Th-1, and Th-2 cells. Th-1 cells release cytokines and activates cytotoxic T cells and induces antibody production by plasma cells. Th-2 cells stimulate plasma cells and produce antibodies. In addition, memory T cells are crucial for long-term host defense against repeat invaders.

B cells called plasma cells are made in the bone marrow and activated in the lymph nodes or spleen. They secrete antibodies to the blood and lymphatic tissues which is part of humoral adaptive immunity.

1684- Leuwenhoek’s microscope finds first microorganism

1843- Rumen ciliate protozoa is first reported

1860- Pasteur denies natural occurrence of microorganisms

1875- Ferdinand J. Cohn publishes a journal in which he first classifies bacteria as Bacillus

1876- Robert Koch publishes a paper about a bacterium being the cause of anthrax

1878- Joseph Lister publishes his results of his study on lactic fermentation of milk. His research used the first method developed to isolate pure culture of bacterium

1880- Louis Pasteur attenuates the pathogen that causes chicken cholera

1881- Robert Koch creates a solid culture medium for bacteria

1884- Robert Koch wins a noble prize for The Etiology of Tuberculosis

1885- Louis Pasteur injects a child with rabies virus.

1889- Martinus Beijerinck obtains a pure culture of Rhizohium

1892- Dmitri Iosifovich Ivanovski discovers viruses

1899- Martinus Beijerinck recognizes viral dependence on cells for reproduction

1900- Walter Reed proves mosquitoes carried the yellow fever agent

1910- Paul Ehrich discovers cure for syphilis

1928- Alexander Fleming discovers Penicillin

1977- Gilbert and Sanger develop a method to sequence DNA

1983- Kary Mulls invents Polymerase Chain Reaction

1995- First microbial genomic sequence published

Compound microscopes are light illuminated. The images are 2D. It is the most common microscope. They cost between 150-10,000 dollars. The source of radiation for the image formation is visible light. The medium is air. The specimen are mounted on glass slides. The lenses are glass. The focusing are mechanical. The magnification is adjusted by changing objectives. The specimen contrast is provided by light absorption.

Dissection microscopes are light illuminated. The images are 3D. It is used to look at large specimen. They cost between 100-1500 dollars. The source of radiation for the image formation is visible light. The medium is air. The specimen are not mounted. The lenses are glass. The focusing are mechanical. There is only one objective. The specimen contrast is provided by light scattering or light reflection.

Confocal microscopes use laser light. They cost between 20,000-100,000 dollars. The source of radiation for the image formation is laser light. The medium is air. The specimen are mounted on glass slides with dyed samples. The lenses are glass with dichromatic mirrors. The focusing is a digital computer focusing mechanism. The magnification is adjusted digitally enhancing. The specimen contrast is provided by laser light with dichromatic mirror concentrated at pinhole.

Scanning Electron Microscope use electron illumination. The image is 3D. They cost more than 50,000 dollars. The source of radiation for the image formation is electrons. The medium is vacuum. The specimen are mounted on aluminum stubs and coated with gold. There is one electrostatic lens with a few electromagnetic lenses. The focusing are electrical. The magnification is adjusted electrically. The specimen contrast is provided by electron scattering.

Transmission Electron Microscope are electron illuminated. The image is 2D. They cost more than 50,000 dollars. The source of radiation for the image formation is electrons. The medium is vacuum. The specimen are mounted on films of collodion. There is one electrostatic lens with a few electromagnetic lenses. The focusing are electrical. The magnification is adjusted electrically. The specimen contrast is provided by electron scattering.

Gram negative bacteria cells, unlike gram positive cells, cell walls consist mostly of outer membrane, not peptidoglycan, which is responsible for the strength of the cell. It’s like a second lipid bilayer but composed of lipid and polysaccharide, instead of protein and phospholipid. It’ made of one layer of lipopolysaccharide layer or LPS and the other is peptidoglycan. The polysaccharide in the lipopolysaccharide is made from O- specific polysaccharide and core polysaccharide. A unique characteristic of the lipopolysaccharide is its toxicity to animals. The lipid portion of the LPS layer is made of Lipid A, which contains an endotoxin that is responsible for its toxicity to animals. Another unique characteristic of the LPS layer in gram negative bacteria would be that the LPS acts like an anchor and types peptidoglycan to itself, creating a bilayer distinct from the cytoplasmic membrane. Between the cytoplasmic membrane and the outer membrane, is the periplasm. This space is filled with fluid that has a gel- like consistency due to all the proteins located there. Its purpose it to make sure that proteins that have activities outside the membrane do not diffuse away. The outer membrane is semipermeable to small molecules due to the presence of porins. It will not let in large molecules and proteins. Microbiologists are able to differentiate between gram positive cells and gram negative cells due to gram staining. Due to the structure of gram negative bacteria cell walls, it does not retain the purple color of the crystal violet iodine complex that forms in the cell, due to it being able to be extracted by alcohol. Thus it has to be counterstained with safranin, and appears pink. The thin layer of peptidoglycan is responsible for this occurrence.

Precursor Metabolites (Intermediates)-

• Glycolysis
  • Glucose 6-Phosphate
    • Results from glucose being phosphorylated by ATP
  • Fructose 6-phophate
    • Results from Glucose 6- P being isomerized
  • Fructose 1,6- Phosphate
    • Results from Fructose 6- P being phosphorylated
  • Glyceraldehyde 3-phophate and dihydroxyacetone phosphate
    • Results from the enzyme aldose splitting fructose 1,6- bisphosphoglyceric acid
  • 1,3- Biphosphoglycerate
    • Results from the oxidation of glyceraldehyde 3- phosphate
  • 3-P-Glycerate
    • ATP synthesized when 1,3- Bisphosphoglyceric acid is converted to 3-phosphoglyceric acid
    • Glycine and Cysteine amino acids
  • Citric acid cycle intermediates
    • α- ketoglutarate and Oxaloacetate are precursors of amino acids
      • Proline, Glutamine, and Arginine
    • Oxaloacetate
      • also a precursor to phosphoenolpyruvate, precursor of glucose
      • Asparagine, Lysine, Methionine, Threonine, Isoleucine
    • Succinyl-CoA
      • Precursor of cytochromes and chlorophyll
    • Acetyl-CoA
      • Precursor of fatty acid biosynthesis
    • Glyoxylate cycle
      • Glyoxylate intermediate
    • Adenosine diphosphoglucose
      • Precursor for glucose biosynthesis
    • Uridine diphosphoglucose
      • Precursor of some glucose derivatives needed for biosynthesis of polysaccharides