Study Aid for Exam 2

General Properties of Viruses
Virus Characteristics
A virion is the extracellular form of a virus and contains either an RNA or a DNA genome.
Only nucleic acid and protein are present, with the nucleic acid on the inside.

The virus genome is introduced into a new host cell by infection.

The virus redirects the host metabolism to support virus replication.

The whole unit, genome and capsid, is called the nucleocapsid.

Classification of Viruses
By Genome: Either RNA or DNA
By Number of strands: Either single strand or double strand for both DNA and RNA
Retroviruses contain single stranded RNA which is changed to double stranded DNA by the enzyme reverse transcriptase

Capsid and Membrane
Capsids consist of capsomeres
Some Viruses have membranes composed of lipid bilayer from host and protein from virus
Membrane makes initial contact with host cell receptor.

Plaques are clear zones that develop on lawns of host cells. Each plaque results from infection by a single virus particle.

Viral Replication
The virus life cycle can be divided into five stages:
attachment (adsorption)
penetration (injection)
protein and nucleic acid synthesis
assembly and packaging
virion release.
Attachment

Bacteriophage
Virus that attacks bacterial cell
Structure
Head
Collar
Tail
Tail pins and fibers
Replication
For example, a virus that has a single-stranded RNA genome with the same orientation as its mRNA is said to be a positive-strand RNA virus.

A virus whose single-stranded RNA genome is complementary to its mRNA is said to be a negative-strand RNA virus.

Viral Genome Replication
Double stranded DNA: transcription of - strand mRNA to + to DNA

Positive single stranded mRNA: transcription of + mRNA

Minus single stranded mRNA: transcription of - stranded mRNA to +

Retrovirus: single stranded + mRNA that is transcribed to DNA by reverse transcriptase

Virulent Bacteriophages
A virion of attaches to a host cell and the DNA penetrates the cytoplasm.

The viral genes redirect the host machinery to the reproduce viral nucleic acid and protein. Virus then assembles and releases new virions by lysing the cell

Temperate Bacteriophage
Host cells can harbor viral genomes without harm if the expression of the viral genes can be controlled. This is the situation found in lysogens and the virus is called a provirus or prophage.

If control is lost, the virus enters the lytic pathway, produces new virions, and lyses the host cell.

Retroviruses
Retroviruses are RNA viruses that replicate through a DNA intermediate. The retrovirus HIV causes AIDS.

The retrovirus contains an enzyme, reverse transcriptase, that copies the information from its RNA genome into DNA, a process called reverse transcription
Retrovirus The DNA becomes integrated into the host chromosome in the same way as it does in a temperate virus.

The retrovirus DNA can be transcribed to yield mRNA (and new genomic RNA), or it may remain in a latent state.

Viroids and Prions
Viroids are small, circular, single-stranded RNA molecules that do not encode proteins and are completely dependent on host-encoded enzymes.

Prions consist of protein but have no nucleic acid.

Prions and viroids are the smallest known pathogens.

Influenza
The flu genomes is highly variable, especially with respect to the envelope components, hemagglutinin (HA) and neuraminadase (NA)
The changing antigens guarantee that there will be susceptible people-Antigenic shift and drift
Antigenic Shift: When two genetically different strains infect the same cell and the RNA in reassorted.
Antigenic Drift: alteration of hemaglutinin and neuraminidase

Chapter 10
Mutation
Mutation is a heritable change in DNA sequence that can lead to a change in phenotype (outer characteristics)

By definition, a mutant differs from its parental strain in genotype, the nucleotide sequence of the genome.

Types of Mutation
A point mutation, occur due to base-pair substitutions leading to a phenotypic change
Types of mutations:
Missense: Changes in amino acid sequence causing inactive polypeptide
Nonsense: Formation of stop codon
Silent: Polypeptide not affected
Wild Type: Non-mutated genome
Insertion/Deletion: Shifts order of codons

Mutagens
Radiation
Ionizing radiation – breaks in chromosomes
Nonionizing radiation – induces thymine dimers

Chemical Mutagens

SOS Repair
A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage

Initiates a number of DNA repair processes, both error-prone and high-fidelity
Genetic Recombination
Homologous recombination: when closely related DNA sequences from two distinct genetic elements are combined in a single element.

Recombination that occur in prokaryotes involve DNA transfer during the processes of transformation, transduction, and conjugation

Transformation: Process by which free DNA is incorporated into a recipient cell and brings about genetic change.

Competence: the ability to receive DNA and become transformed. A state in which cells are able to take up free DNA released by other bacteria

Transfection: when bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium.


Transduction
Transduction involves the transfer of host genes from one bacterium to another by bacterial viruses.

Generalized transduction: defective virus particles incorporate fragments of the cell's chromosomal DNA randomly. The efficiency is low.

Specialized Transduction: when DNA becomes integrated into the host DNA at a specific site.

Conjugation
Involves cell to cell contact. A donor and a recipient cell.

Rolling cell replication

F plasmid: unintegrated fertility plasmid

Hfr plasmid: A chromosome-integrated F plasmid.

Transposition
Segments of DNA that move from one location to another in the same or different molecule

Microbial Growth Control
Sterilization
Sterilization: killing of all organisms, including viruses. Heat is the most widely used method of sterilization.

The temperature must eliminate the most heat-resistant organisms, usually bacterial endospores

Pasteurization: does not sterilize but reduces microbial load, inhibiting the growth of spoilage microorganisms.

Autoclave
An autoclave permits application of steam heat under pressure at temperatures above the boiling point of water, killing endospores
Temperature is 121oC using steam under pressure

Antimicrobials
These agents are termed bacteriocidal, fungicidal, and viricidal agents, killing bacteria, fungi, and viruses, respectively.

Cidal: kills organism but cell lysis does not occur
Static: Injures organism. Frequent inhibitors of protein synthesis.
Lytic: induces killing by cell lysis
Testing Methods

A broth dilution assay: a standard concentration of the organism tested against different concentrations of the antimicrobial in broth. Antimicrobial activity is measured by determining the smallest amount of agent needed to inhibit the growth of the test organism

Disk-Diffusion assay: a standardized concentration of the organism is spread over Mueller-Hinton medium; paper disks containing concentrations of various antimicrobials are added to the surface of the medium. After 24 hr of incubation, the zone of inhibition around each disk is measured and represents whether the organism is susceptible or resistant.

Chemical Agents for External Use
Sterilants, sterilizers or sporicides: kill all forms of microbes. Compounds used to decontaminate nonliving material.

Cold sterilization: process that uses chemicals for sterilization

Disinfection: the elimination of organisms from inanimate objects or surfaces. May not kill endospores
External Agents
Sanitizers: reduce but may not eliminate, microbial numbers.

Antiseptics and germicides: chemical agents that kill or inhibit growth or organisms. Non-toxic to living tissues.

Agent Classification
Classification:
Structure,
mechanism of action
Spectrum of antimicrobial activity.

Two Categories
Synthetic agents and antibiotics

Antimicrobial Action
Selective toxicity: Method of action of antimicrobial agent

Inhibition of Cell Wall Synthesis
Inhibition of Protein Synthesis
Disruption of Cytoplasmic Membranes
Inhibition of Metabolic Pathways
Inhibition of Nucleic Acid Synthesis
Naturally Occurring Antimicrobials
Produced by wide range of fungi and bacteria
Inhibits or kills other microorganisms

Semisynthetic: structurally modified by pharmaceutical company

Broad Spectrum: Effective against both Gram-positive and Gram-negative organisms

Narrow Spectrum: Effective against only certain groups of organisms.
Inhibition of Protein Synthesis
Prokaryotic ribosomes are 70S (30S+50S)

Eukaryotic ribosomes are 80S (40S+60S)

Effect organism at level of translation
Initiation
Elongation

Inhibition of Cell Wall Synthesis
Beta-lactam agents: all contain beta lactam ring structure. Penicillins and Cephalosporins

Interferes with the peptides that link carbohydrates of peptidoglycan

Cell bursts from osmotic pressure

Microbial Resistance
Preexisting Resistance - resistant before exposure to antibiotics
Intrinsic resistance - born bad
Genetic mutation of chromosome
Transfer of genetic material: transformation, transduction, conjugation
Resistance Mechanisms
Reduced cell permeability
Efflux of agent from organism
Inactivation of agent
Alteration of target
Development of resistant biochemical pathway

Industrial Microbiology
Industrial Microbiology
Industrial microbiology uses microorganisms, typically grown on a large scale, to produce commercial products.

Biocatalysis: The actual reactions carried out by microorganisms in industrial microbiology

Microbial Biotechnology: Methods for gene manipulation used to yield new microbial products

Properties of Organism
Capable of large scale inexpensive liquid culture
Should produce spores or other reproductive cell for inoculation into large fermentors.
Must grow rapidly
Produce the desired product in short time period
Should not be pathogenic
Must be amenable to genetic manipulation

Metabolites
Primary metabolites are produced during active cell growth (exponential phase)
Secondary metabolites are produced near the onset of stationary phase

Secondary Metabolites
Growth and reproduction not essential
Dependent on growth conditions
Produced as a group of closely related compounds
Can get overproduction of secondary metabolites

Characteristics of Large-Scale Fermentations
Fermentation: any large-scale microbial process whether or not it is biochemically a fermentation.

Industrial fermentors can be divided into two major classes:
anaerobic processes
aerobic processes (majority of processes)

Industrial Microbiology
Grow organisms
Make products from gene-directed metabolites.

Antimicrobials
Must be produced in large scale industrial fermentors.
Must be purified
Need high yielding strains
Involves mutagenesis of the initial culture
Plating of mutant types and testing of mutants for antibiotic production

If penicillin fermentation is carried out without addition of side-chains, natural penicillins are produced.

A side chain can be added to the broth toproduce desired penicillin: biosynthetic penicillin.

All of these antibiotics are typical secondary metabolites.

Microbial biotransformation employs microorganisms to biocatalyze a specific step or steps in a strictly chemical synthesis.


Brewing and Distilling
Alcoholic beverages are produced by yeast and sugar

Vinegar Production
The active ingredient in vinegar is acetic acid, which is produced by acetic acid bacteria oxidizing an alcohol-containing fruit juice.

Adequate aeration is the most important consideration in vinegar process.

Genetic Engineering and Biotechnology

Biotechnology is the use of living organisms to carry out chemical processes for commercial use.

Genetic engineering is based on molecular cloning, in which a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host.

Cloning vectors include plasmids and bacteriophages.


Definitions
Cloning Vector - Element into which genes can be recombined and replicated

Genetic Map -The arrangement of genes on a chromosome

Genotype -The precise genetic makeup of an organism; both chromosome and plasmids

Molecular Cloning -Isolation and incorporation of a fragment of DNA into a vector where it can be replicated

Shuttle Vector -A cloning vector that can stably replicate in two different organisms.

Expression Vector - a cloning vector that contains the necessary regulatory sequences to allow transcription and translation of cloned genes to express the protein coded by the gene.

Integrating Vector -a cloning vector that becomes integrated into a host chromosome

Nucleic Acid Probe -a strand of nucleic acid that can be labeled and used to hybridize to a complementary DNA molecule

Reporter Gene -A gene incorporated into a vector because the product it encodes is easy to detect

Transfection –The introduction of DNA into mammalian cells.

Reverse Translation -using a codon table and amino acid sequence of a protein to obtain a possible sequence of the mRNA or gene that encoded the protein.


Reverse Transcriptase - enzyme which copies DNA from an RNA template

Repressor Protein -A regulatory protein that binds to specific sites on DNA and blocks transcription

Recombination -The process by which parts or all of the DNA from two separate sources are exchanged or brought together into a single DNA molecule
Wild Type -The bacterial strain isolated from nature

Molecular Cloning
Cloning: purpose is to isolate multiple copies of specific genes in pure form.

Steps in Cloning:
Isolation and fragmentation of the source DNA
Joining the DNA fragments to a cloning vector with DNA ligase
Introduction and maintenance of the clone DNA in a host organism using a vector like a plasmid

Site directed mutagenesis: insertion of synthetic DNA at precisely determined sites in genes in vitro.

Uses of cloned genes
To produce a protein product, like human growth hormone.

To prepare many copies of the gene itself

To determine the gene's nucleotide sequence

An ideal host:
Grows rapidly in an inexpensive culture medium.
Host=nonpathogenic
Capable of taking up DNA
Genetically stable in culture
Has appropriate enzymes to allow replication of the vector.

Prokaryotic Vectors
Plasmid
Bacteriophage
Can be placed in host by transformation
transduction or conjugation
The cloned gene can be harvested or the protein product of the gene can be made and collected.

Transfection of Eukaryotic Cells
Phagocytosis –precipitating DNA for take-up by cell
Electroporation –exposing host cells to pulsed electrical fields in the presence of cloned DNA. The electris treatment opens small pores in host membrane.
Particle Gun –Fires nucleic acid-coated particles into target cells.
Microinjection-Injection of DNA into host using micropipets.
Finding the Right Gene Clone
Special procedures are needed to detect
the foreign gene in the cloning host.

If the gene is expressed, the presence of the gene is detected using hybridization (reaction with specific radiolabeled nucleic acid strand complementary to cloned gene)


Hybridization=nucleic acid probe.

The protein produced by cloned gene can be identified using a tagged antibody


Grow cloned cells in culture. To find colonies/cells with the cloned DNA or protein, use nucleic acid hybridization (DNA) or antibody assay (protein)

Hybridization depends on base-pairing between the gene and a complementary sequence.

The probe or antibody will bond specifically to complementary gene or protein.

Specialized Vectors
Shuttle vector:
Allows cloned DNA to be moved between unrelated organisms (eukaryotic and prokaryotic)
Is a cloning vector that can stably replicate in two different organisms.

Expression vectors:
Vector that can be used to clone the desired gene but can also contain the necessary regulatory sequences to manipulate the expression of the gene

Translation of Cloned Gene
Expression vectors must ensure mRNA translation
Bacterial ribosome binding site; must be engineered into eukaryotic vector
The correct codons must be present either naturally, or by synthetic DNA or site-directed mutagenesis

Eukaryotic Vectors
Eukaryotic Vectors should have:
Origins of Replication-specific sequence that is recognized by specific initiation proteins, including protein that opens up helix

Selectable Marker-factor that allows identification of mutated cells (i.e. antibiotic resistance)

Multiple cloning sites- a short segment of DNA containing many different restriction enzyme cut sites.

Virus vectors or yeast artificial chromosome

Integrating Vectors-designed to maintain clones in very low but stable copy numbers

Reporter genes
Are incorporated into vectors because they encode proteins that are readily detected. Can be used to signal the presence or absence of a particular genetic element or its location. Can also be fused to other genes or to the promoter of other genes so that expression can be studied.

Reverse Translation
Using the amino acid sequence of a protein to synthesize an oligonucleotide probe that encodes it.
The codons that represent the amino acid of the protein are obtained.
The nucleotides that represent the codons are selected for use in designing the probe.

Protein Folding
Cloned gene made to contain a fusion protein in the vector.
Fusion protein can be engineered to contain bacterial signal peptide that enables transport of the clone across the cytoplasmic membrane
Cloned protein is released from the fusion protein by special enzymes

Microbial Evolution and Synthesis
Exam Questions
Study the Working Glossary on page 300
Early Earth
Early Earth was anoxic and much hotter than the present Earth

UV energy was dominant

The first biochemical compounds were made by abiotic syntheses that set the stage for the origin of life.

The first life forms may have been self-replicating RNAs (RNA life).
Primitive Life: Energy and Carbon Metabolism
Primitive metabolism was anaerobic and likely chemolithotrophic (oxidation of inorganic compounds), exploiting the abundant sources of sulfides present

Carbon metabolism may have included autotrophy. (Use of CO2 for metabolite)

Mitochondria and chloroplasts, the principal energy-producing organelles of eukaryotes, arose from the symbioticassociation of prokaryotes of the domain Bacteria within eukaryotic cells, a processcalled endosymbiosis

Mitochondria arose from Proteobacteria

Phylogeny
The phylogeny of microorganisms is their evolutionary relationships.

Comparisons of sequences of ribosomal RNA and differences in nucleotide or amino acid sequence of similar macromolecules are a function of their evolutionary distance.

Ribosomal Database
SSU (small subunit) RNA: the sequencing of the 16S(prokaryote) or 18S(eukaryote) ribosomal unit.

Phylogenetic trees based on ribosomal RNA have now been prepared for all the major prokaryotic and eukaryotic groups.

A huge database of rRNA sequences exists. The Ribosomal Database Project (RDP) contains a large collection of such sequences, now numbering over 100,000.


RNA Sequencing
Ribosomal RNA sequencing involves the amplification of the gene encoding 16S ribosomal RNA, sequencing it, and analyzing the sequence in reference to other bacterial 16S sequences.

16 S subunit is part of bacterial 30 S and 50 S subunits

The evolutionary distance is calculated as the percentage of nonidentical sequences between the RNAs of any two organisms
Domains
Life on Earth evolved along three major lines, called domains, all derived from a common ancestor.

Each domain contains several phyla. Two of the domains, Bacteria and Archaea, remained prokaryotic

The third, Eukarya, evolved into the modern eukaryotic cell

Characteristics of the Domains of Life
The domains of living organisms were originally defined by ribosomal RNA sequencing, but studies have shown that they differ in many other ways.

In particular, the Bacteria and Archaea differ extensively in cell wall and lipid chemistry and in features of transcription and protein synthesis.

Taxonomy
Bacterial taxonomy places emphasis on analyses of phenotypic properties of the organism.

Taxonomy: The science of classification.
Consists of two major subdisciplines, identification and nomenclature

Phylogeny: evolution of organisms.
Groups of genera (singular: genus) are collected into families, families into orders, orders into classes, classes into phyla (singular: phylum), and so on up to the highest-level taxon, the domain.

Speciation
The model for speciation is based solely on the assumption of vertical (mother to daughter) gene flow.

However, bacterial speciation is also affected to some degree by lateral (horizontal) gene transfer.

Lateral flow is the transfer of genes between species by conjugation, transduction, and transformation

Nomenclature and Bergey's Manual
Following the binomial system of nomenclature, prokaryotes are given genus and species name.