Bacterial Genetics

The science of genetics describes and analyze heredity of physiologic functions that form the properties of organism. These properties are determined by the total of all the genetic information named genome. The basic unit of genetics is gene, a segment of DNA that carries in its nucleotide sequence information for a specific biochemical or physiologic property. A gene is relatively stable but occasionally may undergo a nucleotide change, such a change is called as mutation. Mutations may occur spontaneously or can be induced by a number of physical or chemical agents.

Bacteria may have changes including: Morphological and/or structural changes (L form) Variations of cultural characteristics and biochemical reactions Changes in virulence Variation of antigenicity Changes in drug resistance

These variations of bacteria can be divided into two types: a) Phenotypic variation: non-heritable b) Genotypic variation: heritable (mutation)

Phenotypic variations Normal physiologic responses of bacteria due to the change of bacteria growth environment. The changes are limited, non-hereditary, and revert back to their original state when the conditions are changed back. Flagella of Salmonella spp. are absent due to the presence of 0.1 % phenol in culture medium.

Genotypic variations (Mutation) Stable, heritable changes of bacteria. The changes are due to the mutations in bacterial genomic nucleotide sequences. In this lecture, emphases are given to the content about genotypic variations containing bacterial genome, mutation types and mechanisms.

What are the basic genetic materials in bacteria? Bacterial genome

Bacterial Genome DNA/Genome: the genetic materials relative to bacterial heredity and mutation. A. chromosome B. out of chromosome: a) plasmid b) bacteriophage/phage c) transposable genetic elements

Microbial Genome Features 29% Borrelia burgdorferi G+C content 68% Deinococcus radiodurans single circular chromosome two circular chromosomes Genome organization circular chromosome plus one or more extrachromosomal elements large linear chromosome plus extrachromosomal elements

Bacterial Genomics

Chromosomal DNA Bacterial chromosome consists of a single, circle of double-strand DNA. in average 2 mm long Usually < 5000 Kb) The chromosome carry many genes.

Bacterial Genome DNA/Genome: chromosome out of chromosome: plasmid bacteriophage/phage transposable genetic elements

a). Plasmids Are small，circular/line, extra-chromosomal double-stranded DNA that are capable of autonomous replication. Carry genes associated with specialized functions

The characteristics of plasmids Self-replication Encoding some bacterial properties -F/R/Col/Vi plasmid Not necessary for bacterial viability Transferability

Classification of plasmids Transfer properties Conjugative plasmids (mediate conjugation through sex pilus) Non-conjugative plasmids (can not mediate conjugation because of no gene for encoding sex pilus)

Classification of plasmids Phenotypic effects Fertility plasmid (F factor: carrying a gene that encoding sex pilus protein) Bacteriocinogenic plasmid (carrying genes that encoding bacteriocins that kill other bacteria) Resistance plasmid (R factors: carrying genes that encoding enzymes to destroy antibiotics)

F plasmid

RTF (Resistance Transfer Factor) Structure of R Factors Tn 9 Tn 21 Tn 10 Tn 8 RTF R determinant RTF (Resistance Transfer Factor) Conjugative plasmid Transfer genes R determinant Resistance genes Are often parts of transposons (Tn)

Mode of action of resistance genes a) Modification (detoxification) of antibiotics β-lactamase b) Alteration of the target sites of antibiotics Streptomycin resistance c) Alteration of the uptake ability of antibiotics Tetracycline resistance d) Replacement of sensitive pathway resistance to sulfa drugs

Bacterial Genome DNA/Genome: chromosome out of chromosome: plasmid bacteriophage/phage transposable genetic elements

b). Bacteriophages/phages Phages are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. They are viruses that specially infect bacteria (“bacterial virus”).

Composition of Bacteriophage nucleic acid: either DNA or RNA but not both dsDNA, ssRNA, ssDNA Contain unusual or modified bases Encode 3-5 gene products ~ approximate 100 gene products protein: function in infection and protect the nucleic acid

Structure of Bacteriophage different sizes and shapes icosahedral filamentous like a tadpole

Structure of T4 phage Capsid Head DNA Contractile Sheath Tail Tail Fibers Base Plate Head Contractile Sheath Capsid DNA Structure of T4 phage Head consists of DNA surrounded by a protein coat (capsid) Tail is composed of a hollow core surrounded by a contractile sheath with base plate at the end through which are tail fibers.

Interaction between phages and bacteria Phages are wide spread throughout nature and can be found infecting many different genera of bacteria. However, the host range of any specific phage is limit. A given phage can usually infect only a single species of bacteria or closely related species. Based on the pattern of interaction between a given phage and it’s host, phages are divided into two major groups: virulent/Lytic phages and temperate/Lysogenic phages.

Interaction between phages and bacteria Infection with a virulent phage results in phage replication with the production of new phage particles and their subsequent release that causing death and lysis of the host bacteria. Infection with a temperate phage does not necessarily lead to bacterial lysis and death, and the phage may integrate into the bacterial genome without new phase production.

Virulent/Lytic Phages Virulent phages can multiply in bacteria and kill the bacterial cell by lysis at the end of their life cycle. The life cycle of a virulent phage can be divided into four phases: I. adsorption / attachment II. penetration III. biosynthesis / intracellular development IV. maturation and release

I. Adsorption Recognition of host bacterial surface receptors by the tail fibers

II. Penetration The tail sheath contracts, pushing the rigid tail core through the bacterial cell envelope and the phage’s nucleic acid is injected through the hollow core into the bacterial cytoplasm.

III. Biosynthesis Protein synthesis and production of many new copies of new phage DNA or RNA.

IV. Maturation and Release Irreversible combination of phage nucleic acid with it’s protein coat. Induce cell lysis and release of the newly formed phages.

Temperate/Lysogenic Phages Temperate phages are capable to invade host bacteria and inducing lysogenic state without necessarily producing a lethal lytic infection. A temperate phage can either go through the lytic cycle or induce lysogeny by integrating the host DNA in the form of a prophage. Prophage is only a genome of the phage that integrated in genomic DNA of its host bacterium. The bacterial cell harboring a prophage is termed as lysogenic bacterium and this state is called as lysogenization.

Prophage formation II. peneration I. adsorption IV. prophage replicates along with host chromosome III. integrate of phage DNA into host genome

Type Life cycle Virulent phage Lytic Temperate phage Lysogenic lytic

Prophages in lysogenic bacteria will spontaneously proceed through the lytic cycle.

Prophage Lysogenic bacterium Genome of a temperate phage integrating with bacterial genome Lysogenic bacterium A bacterium containing a prophage

The medical significance of phages Phage typing Genetic recombination in bacteria

Bacterial Genome DNA/Genome: chromosome out of chromosome: plasmid bacteriophage/phage transposable genetic elements

Transposable Genetic Elements Definition: segments of DNA that have the capacity to move from one bacterial DNA molecule (bacterial chromosome or plasmid) to another or from one location to another in one DNA molecule. (jumping gene / movable gene)

Properties of transposable genetic elements “Random” movement: move with no any regularity. Transposase: coded by the transposable genetic elements and mediates transposition. Not capable of self replication: usually replicated as a part of some other replicon (plasmid or chromosome). Site-specific recombination: dependent on the inserted sites, but does not require homology between the recombining molecules. Transposition may be accompanied by replication: In some cases, one copy remains of the element at the original site and the other is moves to a new site.

Types of Transposable Genetic Elements I. Insertion sequences (IS) II. Transposons (Tn)

Insertion sequences (IS) Definition: a type of transposable Genetic Elements that carry no other genes except the genes involving in transposition (transposase coding genes). Structure: a small DNA that has repeated sequences at its ends, which are involved in transposition. In the middle between the terminal repeated sequences there is a transposase coding gene (usually one and occasionally more). Function: introduction of an insertion sequence into a bacterial gene will result in the inactivation of the gene. Transposase ABCDEFG GFEDCBA

Importance i) Mutation - The introduction of an insertion sequence into a bacterial gene will result in the inactivation of the gene. ii) Plasmid insertion into chromosomes - The sites at which plasmids insert into the bacterial chromosome are at or near insertion sequence in the chromosome.

Importance iii) Phase Variation - In Salmonella there are two genes which code for two antigenically different flagellar antigens. The expression of these genes is regulated by an insertion sequences. In one orientation one of the genes is active while in the other orientation the other flagellar gene is active. Thus, Salmonella can change their flagella in response to the immune systems' attack. Phase variation is not unique to Salmonella flagellar antigens. It is also seen with other bacterial surface antigens. Also the mechanism of phase variation may differ in different species of bacteria (e.g. Neisseria; transformation).

Transposons (Tn) Definition: a type of transposable Genetic Elements that carry other genes and insertion sequences (IS). Structure: the extra genes are located between the terminal repeated sequences. Function: Since transposons can jump from one DNA molecule to another and frequently carry antibiotic resistance genes, these transposons participate the development of drug resistance in bacteria.

Importance: Many antibiotic resistance genes are located on transposons. Since transposons can jump from one DNA molecule to another, these antibiotic resistance transposons are a major factor in the development of plasmids which can confer multiple drug resistance on a bacterium harboring such a plasmid. These multiple drug resistance plasmids have become a major medical problem because the indiscriminate use of antibiotics have provided a selective advantage for bacteria harboring these plasmids.

Mutation types Self Mutations: low frequency Spontaneous mutation: Mutations for a given gene spontaneously occur with a certain frequency (from 10-8-10-6) in a population derived from a single bacterium. Induced mutation: Some chemical agents and radiation can induce bacterial mutation. Gene transfer and recombination: high frequency one bacterium uptake exogenous DNA segment from another bacterium or phage (Gene transfer) and then the DNA segment is incorporated into DNA of itself (recombination).

Terms about Bacterial Gene Transfer Donor: a bacterium to offer DNA segment (but not entire chromosome) to other bacteria. Recipient: a bacterium to receive DNA segment offered by other bacteria.　 Bacterial genes are usually transferred among members of the same species but occasionally transferred to other species.

Major mechanisms and modes of Bacterial Gene Transfer Gene mutation Gene transfer and recombination Transformation Conjugation Transduction Lysogenic conversion Protoplast fusion

Types of mutation Base substitution Frame shift Insertion sequences

Types of Mutations Normal DNA

Base Substitution Mutation C Missense Mutation

Base Substitution Mutation Nonsense Mutation

Frame Shift Mutation ATG CAT GCA TGC ATT TCC TGC TTA AAA 1. Addition Mutation AAT GCA TGC ATG CAT TTT CCT GCT TAA Reading Frame is Shifted 2. Deletion Mutation (A)TGC ATG CAT GCA TTT CCT GCT TAA

What can cause mutation? Chemicals: nitrous acid; alkylating agents 5-bromouracil benzpyrene Radiation: X-rays and Ultraviolet light Viruses/ phage

Gene transfer and recombination a relatively small fragment of a donor genome to a recipient cell Recombination: Exogenous DNA integrated into the chromosome

Transformation Definition: a bacterial recipient uptake naked chromosomal DNA segment offered by bacterial donor in environment and then the DNA segment recombined with the recipient’s chromosomal DNA .

Factors affecting transformation DNA size: Double stranded DNA segment with at least 500 kbp works best. Competence of the recipient: Only the bacteria in a particular time during their growth cycle called as competent stage can take up DNA by transformation, while the non-competent bacteria can not.

Steps in transformation Uptake of DNA Recombination

Significance for transformation Transformation occurs in nature and it can lead to increased virulence ( e.g. Streptococcus pneumoniae) and drug resistance. In addition transformation is widely used in recombinant DNA technology.

Conjugation Donor Recipient Definition: Gene transfer from a donor to a recipient by direct physical contact between two bacterial cells. Donor: the bacterium (F+) has fertility plasmid called as F factor. The F factor offers the bacterium an ability to produce a sex pilus. Recipient: the bacterium (F- ) that lack of F factor.

only one strand of DNA is transferred

Physiological States of F Factor (I) According to the different patterns and characteristics of gene transfer, conjugation can divided into three types. I. Autonomous (F+): the F factor is autonomous and carries only those genes necessary for its replication and for DNA transfer (no chromosomal genes of bacterial donor). So in this type of conjugation, there is low transfer level of bacterial donor’s genes. In crosses of the F+ and F- bacteria, the F- bacterium becomes F+ and the F+ bacterium remains F+.

Model of Autonomous Conjugation by F+ The F+ bacterium transfers extra chain of F+ factor and then the completed F+ factors in the two bacteria is synthesized by rolling circle replication.

Physiological States of F Factor (II) II. Integrated (Hfr): The F factor has integrated on bacterial chromosome, and only bacterial DNA is transferred with a high frequency (usually the F factor is not transferred). In crosses of the Hfr and F- bacteria, the F- bacterium rarely becomes Hfr but obtaining DNA segment from donor bacterium, and the Hfr bacterium remains Hfr.

Model of Integrated Conjugation Hfr

Physiological States of F Factor (III) III. Autonomous (F’): In this pattern, the F factor is autonomous but it now carries some of bacterial chromosomal genes (F’), because this F factor is a excised integrated F factor with host’s chromosomal sequences at its two sides. In crosses of the F' and F- bacteria, the F- bacterium becomes F' and the F' bacterium remains F'.

Model of autonomous Conjugation by F’ Hfr F-

Significance for conjugation In most of Gram negative bacteria, conjugation is the major way of bacterial gene transfer, which frequently result in multiple antibiotic resistance. In some of Gram positive bacteria, conjugation is also an active way of bacterial gene transfer. Multiple antibiotic resistance genes in a Gram positive bacterium can be obtained by conjugation or by transduction.

Transduction Definition: a chromosomal DNA segment of bacterial donor transferred to a bacterial recipient by way of a bacteriophage, and then the DNA segment recombinate with the recipient’s chromosomal DNA .

Types of Transduction Generalized Transduction: is the transduction in which potentially any genes of the bacterial donor can be transferred to the recipient. Specialized transduction: is the transduction that only certain bacterial genes can be transferred to the recipient.

Generalized Transduction Virulent phages that mediate generalized transduction generally breakdown host DNA into smaller pieces. Occasionally, one of the host DNA pieces is randomly packaged into the phage particle. Thus, any genes of bacteria can be potentially transferred. When the bacterial DNA contained phage infect a bacterial recipient, donor DNA enters the recipient. In the recipient, the event of a recombination of donor DNA and recipient DNA can occur.

Specialized Transduction As the introduction above, sometimes prophage, like virulent phage, can spontaneously entry the lytic cycle. During the excision of prophage, occasionally some of the host DNA segments at either sides of prophage gene sequence is excised with the phage DNA. After a bacterial recipient is infected with this phage and then forms its lysogenization, the recipient’s genomic DNA contains the donor DNA.

Significance for Transduction Transduction occurs in nature and it can lead to increased virulence and drug resistance of recipient bacteria.

Lysogenic conversion Definition: a bacterial recipient is infected with bacteriophage from a bacterial donor, and the genes of phage itself, but not genes of the bacterial donor, recombined with the recipient’s chromosomal DNA. As an example, Corynebacterium diphtheriae will produces diphtheria toxin after it is infected by the β- phage, because the gene encoding the toxin is carried by the phage.

Summary The genetic materials of bacteria. Concepts of Transformation, Transduction, Conjugation and Lysogenic conversion Four forms of genetic recombination in bacteria The Significance of bacterial mutation (changes of bacterial virulence, drug resistance, antigenicity and so on).