MCB 130 2nd Exam

PLASMIDS

closed circular or linear DNA molecules that replicate independently of the bacterial chromosome

PLASMIDS

code for functions involved in their own life cycles and for functions which affect the physiology of the host cell

PLASMIDS

pathogenicity in plants and animals

2 kb to 100 kb

Size of plasmids

SUPERCOILED (COVALENTLY CLOSED-CIRCULAR), RELAXED CIRCULAR, SUPERCOILED DENATURED, NICKED OPEN CIRCULAR, LINEARIZED

5 CONFORMATIONS OF PLASMID DNA

SUPERCOILED (COVALENTLY CLOSED-CIRCULAR)

PLASMID CONFORMATION: FULLY INTACT WITH BOTH STRANDS UNCUT

RELAXED CIRCULAR

PLASMID CONFORMATION: FULLY INTACT BUT SUPERCOILS REMOVED

SUPERCOILED DENATURED

PLASMID CONFORMATION: SMALL QUANTITIES FOLLOWING EXCESSIVE ALKALINE LYSIS; BOTH STRANDS ARE UNCUT BUT ARE NOT CORRECTLY PAIRED, RESULTING IN A COMPACTED PLASMID FORM

NICKED OPEN-CIRCULAR

PLASMID CONFORMATION: HAS ONE STRAND CUT

LINEARIZED

PLASMID CONFORMATION: HAS BOTH STRANDS CUT AT ONLY ONE SITE

FERTILITY PLASMID

PLASMID: CARRY ONLY TRA GENES, DO NOT HAVE CHARACTERISTIC BEYOND THE ABILITY TO PROMOTE CONJUGAL TRANSFER OF PLASMIDS

Resistance or R plasmids

PLASMID: carry genes that confer resistance to one or more antibacterial agents (chloramphenicol, ampicillin, and mercury)

Col plasmids

PLASMID: code for colicins, proteins that kill other bacteria

COLICINS

WHAT DO COL PLASMIDS CODE FOR

Degradative plasmids

PLASMID: confer on their bacterial host the ability to metabolize unusual molecules such as toluene and salicylic acid

Virulence plasmids

PLASMID: confer pathogenicity on the bacterial host

ColE1

PLASMID: CODES FOR BACTERIOCIN WHICH KILLS E. COLI

Tol

PLASMID: CONFERS ABILITY TO DEGRADE TOLUENE AND BENZOIC ACID

RK2; Klebsiella aerogenes

PLASMID: CONFERS RESISTANCE TO AMPICILLIN, TETRACYCLIN, AND KANAMYCIN; WHERE IS IT ORGINALLY FOUND

pCG86

Isolated from a piglet with diarrhea; a hybrid plasmid – carries enterotoxin genes (Ent plasmid) and resistance genes (R plasmid)

Host range

Properties of Plasmids: includes the type of bacteria in which the plasmid can replicate

Host range

a fundamental property of a bacterial plasmid and is primarily determined by the plasmid replication system

Narrow

Narrow or Broad host range: ColE1 plasmid type such as pBR322, pET, and pUC

Broad

Narrow or Broad host range: RK2 and RSF 1010 plasmids

Copy number

Property of plasmid: average number of copies per host cell

Low copy

Copy number: plasmids have 15-20 copies per cell

Medium copy

Copy number: plasmids have 20-100 copies per cell

High copy

Copy number: plasmids have 500-700 copies per cell

1

Copy number of F plasmid

4-7

Copy number of RK2 in E. coli

16

Approximate copy number of pBR322

30-50

Approximate copy number of pUC18

20-300

Approximate copy number of pIJ101

Incompatibility (Inc) Groups

Properties of Plasmids: Failure of two plasmids to be stably inherited and maintained, in the absence of a selective pressure, in the same cell line

Incompatibility (Inc) Groups

Properties of Plasmids: Connected with regulation of copy number and segregation

same replication control functions (trans-acting)

similarities between the members of the same Incompatibility (Inc) Groups; each can control replication of the other; one of the two plasmids is lost during random partitioning

conjugative and non-conjugative

Classifications of plasmids according to transferability and mobilizability

Conjugative plasmids

Plasmid Transferability and Mobilizability: possess the ability to promote s*xual conjugation between bacterial cells

Conjugative plasmids

Plasmid Transferability and Mobilizability: can transfer themselves from host to host,

Conjugative plasmids

Plasmid Transferability and Mobilizability: self-transmissible plasmids

Conjugative plasmids

Plasmid Transferability and Mobilizability: contain tra genes and the oriT

Mobilizable plasmids

Plasmid Transferability and Mobilizability: contain only the oriT sequences

Mobilizable plasmids

Plasmid Transferability and Mobilizability: lack the genes encoded in the transfer operon (tra), which encodes most of the functions involved in mating pair formation

Mobilizable plasmids

Plasmid Transferability and Mobilizability: cannot transfer themselves from host to host, but can be mobilized by transmissible plasmids (such as the F plasmid or ColV)

Plasmids or replicons

self-replicating DNA molecules

Plasmids or replicons

code a few proteins required for their own replication, still depend on their hosts for other proteins

Theta and rolling circle replication

2 general mechanisms of plasmid replication

Ø regulating the amount of available primer for the initiation of DNA

replication

Ø regulating the amount of essential replication proteins

Ø regulating the function of essential replication proteins

control of initiation of plasmid replication

1. Regulation by antisense RNA

2. Regulation by binding of replication proteins to repeated 18-22 bp sites called iterons

Two mechanisms to control the initiation of plasmid replication

Plasmid with no par system

when the cell divides, each plasmid has an equal chance of being segregated into one of the daughter cells. at some point, a daughter cell contains no plasmid at all.

F+

a plasmid independent of the bacterial genome; contains only F factor DNA

Hfr (High frequency of recombination)

F factor integrated into the bacterial genome

F’

F factor exists as an extrachromosomal element but contains some sections of the chromosomal DNA

F-

strain with no F factor

Cloning vector

a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA.

1. sequences that permit the propagation of itself in bacteria (or in yeast for YACs)

2. cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes

3. method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; usually accomplished by selectable markers for drug resistance

Three features of cloning vectors

Mobile Genetic Elements (MGEs)

referred to as transposable elements; genetic materials that can move around within a genome or across different genomes

Insertion sequences

MGE: encode gene products required for transposase synthesis and regulation

Composite transposons

MGE: flanked by IS elements on the left and right ends

Non-composite transposons

MGE: lack flanking IS elements

Insertion Sequences

small and compact mobile genetic elements

Insertion Sequences

MGE: typically between 0.75 and 2.0 kb in size

Insertion Sequences (IS)

MGE: termini tend to be 10 to 40 base pairs in length with perfect/nearly perfect repeats

Insertion Sequences (IS)

encode one large ORF (300-400 aa) involved in the transposition event; usually present in a small number of copies in the genome

Gene inactivation

most common effect of transposition of IS

(a) inactivation of gene product by IS insertion within target gene

(b) inactivation of gene expression by IS insertion that disrupts or deletes a target host gene promoter

how does IS inactivate genes

Transposons

mobile genetic elements containing additional genes unrelated to transposition functions

Class 1 (compound or composite Tns)

Class 2 (complex Tns)

Two general classes of transposons:

Class 1 Transposons

Transposon class: encode drug resistance genes flanked by copies of an IS in a direct or indirect repeat

IS sequence

supplies transposition function in class 1 transposons

Class 2 Transposons

Transposon class: do not contain IS elements

Class 1 Transposons

Transposon class: ends of the transposon are capable of transposing separately

Class 2 Transposons

Transposon class: always transposed as a unit

Class 2 Transposons

flanked by short (30-40 bp) indirect repeats with the genes for drug resistance and transposition encoded in the middle

Replicative Transposition

transposon is duplicated; donor and target DNA are fused, another copy of Tn is generated

Non-replicative Transposition

"cut-and-paste" mechanism

Phage Mu

a transposable phage, named for its “mutator" effects when integrated into the host DNA

Class 2 transposon

Class of Phage Mu

A and B genes

Phage Mu genes: encode transposase

A protein

Phage Mu protein: required for all transposition events

B protein

Phage Mu protein: only required for replicative transposition events

c gene

Phage Mu genes: regulates expression of the transposase genes.

INTEGRONS

a mobile genetic element with the ability to capture genes by site- specific recombination

intI

Integron: encoding a site-specific recombinase belonging to the integrase family

attI

Integron: adjacent recombination site, recognized by the integrase, receptor site for the cassettes

promoter (Pc)

located upstream of the integration site, necessary for efficient transcription and expression of cassette-encoded gene present in the integron

Gene Cassette

mobile element that includes a gene (most commonly an antibiotic- resistance gene) and an integrase-specific recombination site (member of a family of sites known as 59-base elements); can exist either free in a circularized form or integrated at the attI site (only when integrated is a cassette formally part of an integron)

Vertical gene transfer or vertical evolution

movement of genetic material by descent where information travels through the generations as the cell divides

Vertical gene transfer or vertical evolution

strictly a matter of Darwinian evolution driven by principles of natural selection

Asexual Mode

Most common and important in bacterial growth cycle (e.g. fission)

Genetic recombination

exchange of genes between 2 DNA molecules to form combination of genes in a chromosome

Mutation

change in the base sequence of a cell/organism which may occur naturally or through induction by agents

Donor (“male”) and Recipient (“female”)

Two functional/mating types of bacteria

Lateral or horizontal gene transfer (HGT)

movement of genes between cells that are not direct descendants of one another

Lateral or horizontal gene transfer (HGT)

the movement of genetic material between bacteria other than by descent

Conjugation

physical contact between bacterial cells is needed for genetic recombination to take place. (Bernard Davis)

William Hayes

discovered that the transfer of genetic material in E. coli is not reciprocal. One cell acts as the donor, and the other cell acts as the recipient

William Hayes

suggested that donor ability is itself a hereditary state imposed by a fertility factor (F)

1. Requires cell-cell contact

2. Occurs via a conjugal/membrane pore

3. DNA transfer occurs in one direction - from donor to recipient not vice versa

4. Does not require protein synthesis in donor cell

5. Requires energy in donor cell - primarily ATP

Characteristics of Conjugation

F- encoded (sex) pilus

a hollow cylinder with an exterior diameter of 8 nm and interior diameter of 2 nm; up to 20 um in length