Microbial Genomics Learning Materials

Introduction Growth is an essential feature of living organisms. It results in an increase in cell number and cell mass. There are different methods of growth in microorganisms: binary fission, budding, fragmentation, spore formation, etc. Nature of Growth of Microbial Population The nature of growth is studied in a batch culture system, where nutrients are supplied only once at the beginning and there is no replenishment. Batch Culture The growth of bacteria in a batch culture shows four stages: lag phase, log phase, stationary phase, and decline phase. Lag Phase This is a preparatory period for the bacteria to synthesize cellular constituents for reproduction. There is no increase in cell numbers during this phase. The duration of the lag phase varies depending on environmental conditions and the physiological state of the bacteria. Log Phase Once the lag phase is over, cells start dividing. Cellular metabolic activity increases. Cell numbers increase exponentially after each generation time. The growth curve between time and log of the number of cells becomes a straight line. Mathematically, the growth can be represented as: N(t) = N0 * 2^(n*t) N(t) = number of microbes at time t N0 = initial number of microbes n = growth rate Stationary Phase The exponential growth phase cannot continue indefinitely due to several factors: Essential nutrients in the medium get exhausted. Toxic metabolites in the medium get accumulated, which inhibit growth. A critical level of population may be achieved. These factors lead to a decrease in population growth. The growth curve becomes horizontal, indicating that the number of cells formed is balanced by the number of cells dying. There is no change in the total cell number, resulting in no population growth. Spore Formation Spore-forming bacteria produce endospores during this phase. Pathogenic bacteria generate substances that help them survive harsh conditions. Death Phase As nutrients become less available and waste products increase, the number of dying cells continues to rise. The number of living cells decreases exponentially in the death phase.   As dying cells lyse and break open, they spill their contents into the environment, making them available to other bacteria. This helps spore-producing bacteria survive the harsh conditions of the death phase. Spores are able to survive the harsh conditions of the death phase and become growing bacteria when placed in an environment that supports life Discovery Louis Pasteur coined the term "viruses." He demonstrated that viral diseases like rabies exist. Adolf Mayer discovered the mosaic disease of tobacco. D.J. Iwanowski discovered that TMV, causing mosaic disease, is filterable. M. Beijerinck established that viruses like TMV can replicate only in growing plant cells. W.M. Stanley isolated and crystallized TMV, proving the non-living nature of viruses. He is known as the "father of virology." Bowden Spizizen first established that viruses are nucleoprotein particles. General Characteristics of Virus Viruses are acellular and can easily be crystallized. They are ultramicroscopic and filterable through bacterial filters. Viruses are obligate intracellular parasites and infect all the major biological groups: animals, plants, and bacteria. Each virus species is specific and infects a specific host. They are nucleoprotein in nature. Viruses contain either DNA or RNA but not both, as in any cell. They use the enzymes and other machinery of the host cell for replication. They are resistant to chemicals, alcohols, and environmental changes. They are easily transmitted from infected hosts to healthy ones through vectors like insects, nematodes, etc. Viruses that kill their host are called virulent, and those that do not always kill their host are called temperate or moderate viruses. Classification of Viruses (Baltimore Classification) David Baltimore put forth a system of classification of viruses known as Baltimore classification in 1971. Baltimore classification is a classification system that places viruses into one of seven groups depending on a combination of their nucleic acid (DNA or RNA), strandedness (single or double stranded), sense, and method of replication.   Viruses can be placed into one of the following seven groups: Group I: Double-stranded DNA viruses Examples: Adenoviruses, Herpesvirus, Poxvirus Group II: Single-stranded DNA viruses (the Parvovirus) Example: Parvoviruses Group III: Double-stranded RNA viruses Example: Reoviruses Group IV: (+) Sense RNA viruses Examples: Picornaviruses, Togaviruses Group V: (-) Sense RNA viruses Examples: Orthomyxoviruses, Rhabdoviruses Group VI: RNA-RT viruses (+ Sense RNA with DNA intermediate in the life cycle) Example: Retroviruses Group VII: ds DNA-RT viruses (with RNA intermediate) Example: Hepadnaviruses Components of Virus Structure There are three major components of a virus: (a) capsid, (b) nucleic acid, and (c) envelope (in some viruses). When the capsid, nucleic acid, and envelope are found in the virus, it is called a virion. (a) Capsid The protein coat or shell which encloses the nucleic acid. The capsid contains many similar protein units known as capsomeres. Each capsomere is an association of a few protein molecules. All the capsomeres are not only identical in structure but also contain similar sequences of amino acids, forming short chains of polypeptides. The capsid of a virus has two important functions: protection and attachment. It protects the nucleic acid from the adverse effects of the environment and also helps the virus in recognizing its host and attachment to it. (b) Nucleic Acid Viruses contain only one type of nucleic acid: either DNA or RNA but never both, unlike other organisms. Nucleic acids have their normal composition, containing a pentose sugar, nitrogenous bases, and phosphate. The nitrogenous bases adenine, guanine, cytosine, thymine, and uracil are found in RNA and DNA. The arrangement of the nucleotides in single-stranded viruses is different from double-stranded viruses. Some viruses contain single-stranded DNA and double-stranded DNA. The nucleic acid occupies a definite place in the protein coat and is partially attached to it through salt linkages, hydrogen bonds, and other seen forces. Enveloped Viruses The viral nucleic acid enters the host cell as infective unit but not the capsid in most cases. The viral nucleic acid contains the codes for viral proteins, which are synthesized only after it enters the host cell. Enveloped viruses have a lipoprotein layer with embedded proteins in the phospholipid bilayers. Some spike-like structures composed of glycoproteins are found in the envelope, which help in hemagglutination activities. Since enveloped viruses are flexible, they do not exhibit a fixed shape and size and are called pleomorphic. The envelope sometimes arises from the nuclear or plasma membrane of the host cell. Protein components of the envelope are of viral origin or mixed origin with host proteins. Some enzymes like DNAse, reverse transcriptase in retroviruses, protein kinase in herpesvirus, DNA-dependent RNA polymerase in poxviruses are found in the envelope. They help in viral replication mechanisms. When the envelope is present, the virus is called an enveloped virus, and when it is absent, it is called a naked virus. Naked virus does not mean the lack of a capsid. Morphology of Viruses The arrangement of capsomere units forming the capsid gives the morphology of a virus. It is known that the structure of a virus has one of the following three basic symmetries as proposed by Goodheart: (a) Cubical Symmetry (b) Helical Symmetry (c) Complex Symmetry Cubical Symmetry These are also known as sidedod, quasi-spherical, polyhedral or more specifically, icosahedral. Icosahedral means many-sided. The filaments of these viruses range from 26-30 nm in size. A typical icosahedron has 12 vertices, 20 faces, and 30 edges. Many of the viruses like herpes virus, adenovirus, polyoma virus, polio virus have icosahedral structure. Helical Symmetry These viruses are also known as elongated viruses (10-25 nm). They are more than 1000 nm in length. The capsomeres show a helical-like arrangement like the arrangement of beads on a string. The helical arrangement occurs around an imaginary axis. There are two fundamental types of helical viruses: rigid rod-shaped and flexible rod-shaped. The viruses TMV, TRV, and potato virus X are examples of helical viruses that may be naked or surrounded by an envelope. Complex Symmetry This group of viruses exhibit complex symmetry or a combination of helical and cubical symmetry. They can be divided into two groups: one with the combination of cubical symmetry and helical symmetry, and the other with a mixed symmetry that does not exist in a clear definition. The mixed symmetry of bacteriophage T4 has a head-like structure with the tail containing a hollow tube surrounded by a helical contractile protein sheath. Replication of Viruses Replication of viruses refers to the formation of copies of the viral genome and the formation of new viruses inside the host cell after the infection process. It includes the following steps: Adsorption Penetration & Entry Uncoating Replication of viral genome Assembly of virions Maturation & Release Adsorption or Attachment It is the first step in virus replication where the virus becomes able to attach to the target or correct cell. It is a passive process and does not require energy. Some specific proteins of the viral capsid get recognized on the cellular surface receptors and the virus gets attached or adsorbed to the surface of the cell. These cellular surface receptors include proteins, carbohydrates, glycoproteins, lipids, etc. Other factors that influence the efficiency of virus attachment include density of the receptors present on the cell surface, density of the ligands on the virus surface, concentration, pH of the virus and the host cells. Penetration or Entry into the Host Cell After attachment, the virus must enter the cell, crossing the cell membrane in such a way that the cell is neither killed nor the viral genome is disturbed. The virus entry for enveloped and non-enveloped viruses can occur through several pathways: For enveloped viruses, the lipid bilayer of the envelope merges with the host cell membrane either by ligand-mediated fusion or receptor-mediated endocytosis. In ligand-mediated fusion, the viral ligand interacts with the cellular receptor, leading to a fusion event. The viral envelope fuses with the cell membrane, releasing the nucleocapsid inside the cell. The remaining viral envelope remains as a patch on the cell membrane. In receptor-mediated endocytosis, the attachment of the virus to the plasma membrane of the host cell initiates the formation of an endocytic vesicle. The virus enters the cell within this vesicle, which fuses with a lysosome containing acidic pH. The acidic environment facilitates conformational changes in the viral envelope proteins, leading to the fusion of the viral membrane with the endocytic membrane. The nucleocapsid gets released into the cytoplasm. Uncoating This step refers to the removal or degradation of the capsid. This uncoating may occur during the penetration process or may occur completely after penetration. In some cases, there is no role of the capsid as the capsid proteins play some role in viral transcription and replication. It is found that most of the RNA viruses replicate in the cytoplasm, whereas most of the DNA viruses replicate in the nucleus of eukaryotic cells. Replication of Viral Genome After the viral genome has been uncoated, transcription and translation of the viral genome are initiated. There is a huge difference between DNA and RNA viruses with respect to transcription and replication strategies. In DNA viruses, transcription and translation are not coupled; transcription occurs in the nucleus, and translation occurs in the cytoplasm except for poxviruses.   All RNA virus replication occurs in the cytoplasm of the host cell. Both DNA and RNA viruses may be single or double-stranded. This may play a role in the de novo synthesis of viral proteins and genome. Assembly After the de novo synthesis of viral genome and proteins, which can be post-transcriptionally modified, viral proteins are packed with newly replicated viral genome into new virions. These virions are matured and ready for release from the host cell. This process can also be referred to as maturation.