Microbial GeneticsInheritance and

Variability

Microbial genetics is concerned with the transmission of hereditary characters in microorganisms. Microbial genetics has played a unique role in developing the fields of molecular and cell biology and also has found applications in medicine, agriculture, and the food and pharmaceutical industries.

Chromosome- a dense structure inside cells that physically carries hereditary informationfrom one generation to the next.

Bacterial Cell - contains only one chromosome, consisting of a single molecule of double-stranded deoxyribonucleic acid (DNA) in the form of closed circle.

*Procaryotic chromosome is:1. Naked (lacking the nuclear membrane found in eucaryotic cell)2. is twisted, coiled and packaged into a highly compact form (because

bacterial chromosome has length of about 1200 times of the entire cell)

-in addition to its chromosome, a bacterial cell contains one or more PLASMIDS (double-stranded DNA molecules that are much smaller than the chromosome and can replicate independently of the chromosome).

-most are circular but linear plasmids have beenfound in few bacteria(e.g. Spirochete that causeLyme disease)-have been used extensively in genetic engineeringtechniques.

DNA REPLICATION - process that copies the nucleotide sequence of a double-stranded parent DNA into twodouble-stranded daughter molecules

Figure 5.14. Origin of replication in E. coli Replication initiates at a unique site on the E. coli chromosome, designated the origin (ori)

Component Function

Initiator protein Binds to origin and separates strands of DNA to initiate replication

DNA helicase Unwinds DNA at replication fork

Single-strand-binding proteins Attach to SS-DNA and prevent 20 structures from forming

DNA gyrase Moves ahead of the replication fork , making and resealing breaks in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork

DNA primase Synthesizes a short RNA primer to provide a 3’-OH group for the attachment of DNA nucleotides

DNA polymerase III Elongates a new nucleotide strand from the 3’-OH group provided by the primer

DNA polymerase I Removes RNA primers and replaces them with DNA

Table 4.1 Components required for replication in bacterial cells

Component Function

DNA ligase Joins Okazaki fragments by sealing nicks in the sugar-phosphate of newly synthesized DNA

Transcription Elongation in Eucaryotes Is Tightly Coupled To RNA Processing

Figure 6-21. Summary of the steps leading from gene to protein in eucaryotes and bacteria. The final level of a protein in the cell depends on the efficiency of each step and on the rates of degradation of the RNA and protein molecules. (A) In eucaryotic cells the RNA molecule produced by transcription alone (sometimes referred to as the primary transcript) would contain both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm.

Variability in Microorganisms-associated with its genotype and its phenotype

Genotype - represents the inheritable total potential of a cell

Phenotype- represents the portion of the genetic potential that is actually

expressed by the cell under a given set of condition.(eg. Maybe particular color or size of bacterial colony or presence of bacterial

capsules which may or may not formed by certain bacteria depending on their environment)

Phenotypic changes-both the genotype and the environment influence the phenotypeof

an organismeg. Bacteria of the genus Azomonas form large, gummy colonies

when grown with the sugar sucrose and smaller, nongummy colonies in the absence of this sugar

Genotypic changes-although some phenotypic changes are the result of Environmental

influences, others are the result of changes in the DNA.These can occur as the result of:

1. mutation – a change in the nucleotide sequence of a gene or

2. recombination –a process that leads to new combinations of genes on a chromosome

Mutation and Recombination

• Mutation is a heritable change in DNA sequence that can lead to a change in phenotype. By definition, a mutant differs from its parental strain in genotype, the nucleotide sequence of the genome.

• Selectable mutations are those that give the mutant a growth advantage under certain environmental conditions and are especially useful in genetic research. If selection is not possible, mutants must be identified by screening.

• Although screening is always more tedious than selection, methods are available for screening large numbers of colonies in certain types of mutations. For instance, nutritionally defective mutants can be detected by the technique of replica plating (Figure 8.2).

Molecular Basis of Mutation

• Mutations, which can be either spontaneous or induced, arise because of changes in the base sequence of the nucleic acid of an organism's genome.

Mutations can be classified into various types based upon the kinds of changes they produce in a gene. Two common types are:

1. Point mutations –results from the substitution of 1 nucleotide for another in a gene

a. Neutral mutationeg. AAU to AAC still codes for

asparagine b. missense mutation

eg. AAU become AAG asparagine to lysine

c. nonsense mutationeg. UAU to UAA premature halting

2. Frameshift mutation –addition or loss of one or more nucleotides in a gene

a. insertionb. deletion

• A point mutation, which results from a change in a single base pair, can lead to a single amino acid change in a polypeptide or to no change at all, depending on the particular codon involved (Figure 8.3).

• Deletions and insertions cause more dramatic changes in the DNA, including frameshift mutations, and often result in complete loss of gene function (Figure 8.4).

Figure 8-4. Different types of mutations

• Table 10.1 lists various kinds of mutants.

Mutation Rates• Different types of mutations can occur at

different frequencies. For a typical bacterium, mutation rates of 10–7 to 10–11 per base pair are generally seen.

• Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes.

Mutagenesis

• Mutagens are chemical, physical, or biological agents that increase the mutation rate. Mutagens can alter DNA in many different ways, but such alterations are not mutations unless they can be inherited.

• Table 8.2 gives an overview of some of the major chemical and physical mutagens and their modes of action.

• There are several classes of chemical mutagens, one being the nucleotide base analogs (Figure 8.5).

• Several forms of radiation are highly mutagenic (Figure 8.6).

• Some DNA damage can lead to cell death if not repaired. A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage and initiates a number of DNA repair processes, both error-prone and high-fidelity (Figure 8.7).

Mutagenesis and Carcinogenesis: The Ames Test

• The Ames test employs a sensitive bacterial assay system for detecting chemical mutagens in the environment.

Recombination•DNA rearrangements are caused by a set of mechanisms that are collectively called genetic recombination.

• Two broad classes:1. general recombination2. site-specific recombination.

General recombination (also known as homologous recombination)-genetic exchange takes place between a pair of homologous DNA sequences

The breaking and rejoining of two homologous DNA double helices creates two DNA molecules that have “crossed over.” In meiosis, this process causes each chromosome in a germ cell to contain a mixture of maternally and paternally inherited genes.

Homologous recombination arises when closely related DNA sequences from two distinct genetic elements are combined in a single element (Figure 10.9)

• Recombination is an important evolutionary process, and cells have specific mechanisms for ensuring that recombination takes place.

In bacteria, gene transfer that can lead to recombination may occur in any of three different ways:

1. transformation- simplest type of gene transfer; a recipient cell acquires genes from “free floating” DNA molecules in the

surrounding medium 2. transduction –gene transfer in which a virus serves as vehicle for carrying DNA from a donor bacteriumto a recipient

bacterium.3. conjugation- a process of gene transfer that requires

cell-to-cell contact and thus differs from transformation and trasnduction.

TRANSFORMATION

TRANSDUCTION

CONJUGATION

PART II Genetic Exchange in Prokaryotes

• The discovery of transformation was one of the seminal events in biology because it led to experiments demonstrating that DNA is the genetic material (Figure 8.13).

• Certain prokaryotes exhibit competence, a state in which cells are able to take up free DNA released by other bacteria.

• Incorporation of donor DNA into a recipient cell requires the activity of single-stranded binding protein, RecA protein, and several other enzymes. Only competent cells are transformable (Figure 8.14).

Transduction• Transduction involves the transfer of host genes from

one bacterium to another by bacterial viruses.

• In generalized transduction (Figure 8.15), defective virus particles incorporate fragments of the cell's chromosomal DNA randomly, but the efficiency is low.

• In specialized transduction (Figure 8.16), the DNA of a temperate virus excises incorrectly and takes adjacent host genes along with it; transducing efficiency in this case may be very high.

Plasmids: General Principles

• Plasmids are small circular or linear DNA molecules that carry any of a variety of unessential genes. Although a cell can contain more than one plasmid, they cannot be closely related genetically.

• Figure 10.18 shows a genetic map of the F (fertility) plasmid, a very well characterized plasmid of Escherichia coli.

• Lateral transfer in the process of conjugation can transfer plasmids (Figure 8.19).

Types of Plasmids and Their Biological Significance

• The genetic information that plasmids carry is not essential for cell function under all conditions but may confer a selective growth advantage under certain conditions.

• Examples include antibiotic resistance (Figure 8.20), enzymes for degradation of unusual organic compounds, and special metabolic pathways. Virulence factors of many pathogenic bacteria are often plasmid-encoded.

Types of plasmids

1. Conjugative plasmids: transmitted during conjugation, carry a variety of information

2. R plasmids: resistance plasmids; protect against environmental factors, MDR (multiple drug resistance) plasmid

3. Hfr plasmids: promotes genomic recombination4. Col-plasmids: codes for proteins that kill other

microbes5. Degradative plasmids: contain sequencing that

allows host to digest uncommon substances (ex: toluene, salicylic acid)

6. Virulence plasmids: codes for altering the microbe into a pathogen

• Table 10.3 lists some phenotypes that plasmids confer on prokaryotes.

Conjugation: Essential Features

• Conjugation is a mechanism of DNA transfer in prokaryotes that requires cell-to-cell contact.

• Genes carried by certain plasmids (such as the F plasmid) control conjugation, and the process involves transfer of the plasmid from a donor cell to a recipient cell (Figure 8.22). Plasmid DNA transfer involves replication via the rolling circle mechanism.

The Bacterial Chromosome

Genetic Map of the Escherichia coli Chromosome

• The Escherichia coli chromosome has been mapped usingconjugation, transduction, molecular cloning, and sequencing (Figure 8.42).

• E. coli has been a useful model organism, and a considerable amount of information has been obtained from it, not only about gene structure but also about gene function and regulation.

Regulation of Gene ExpressionOperon – in bacteria, the genes that code for the enzymes of a metabolic pathway are usually arranged in a consecutive manner to form a functional unit.

*most transcriptional control mechanisms for operons involve either enzyme induction or end-product repression

1. Induction –form of control of gene transcription, with the gene transcribed only when appropriate substrate for the protein is present

-used mainly to control the synthesis of proteins that are used totransport and breakdown nutrients.

2. End-product repression – transcription of an operon for a synthetic pathway is often regulated by its end product, and not by the initial substrate of thepathway.

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