Microbial Genetics

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Microbial Genetics

İ. Çağatay Acuner

M.D., Clinical Microbiologist, Associate Professor

Department of Microbiology

Faculty of Medicine, Yeditepe University

Learning Objectives

Explain basics of nucleic acid function in bacteria

(replication of DNA, chromosome replication, DNA repair, gene expression, gene organization)

Describe sources of mutation and variation in bacteria

Describe basic features of bacteriophages and plasmids

Describe mechanisms of gene transfer in bacteria

Explain the importance of genomic plasticity in bacteria

References and Recommended Further Readings

1.

2.

3.

Medical Microbiology, 7th Ed. Murray P.R., Rosenthal K.S., Pfaller M.A.

2013, Mosby.

Mim’s Medical Microbiology, 5th Ed. Goering R.V., et al. 2012, Mosby.

Sherris Medical Microbiology, 5th Ed. Ryan K.J., et al. 2010, McGraw Hill.

Bacterial Genome

Bacterial genome

is the total collection of genes carried by a bacterium

on chromosome

on extrachromosomal genetic elements

Genes

are sequences of nucleotides that have a

biologic function

Examples;

protein-structural genes

(cistrons, which are coding genes),

ribosomal ribonucleic acid ( RNA) genes

,

recognition and binding sites for other molecules (

promoters and operators

)

Each genome contains many operons, which are made up of genes

Eukaryotes have two distinct copies of each chromosome; diploid

Bacteria

have only

one copy of their chromosomes ; haploid

Because bacteria have only one chromosome , alteration of a gene (mutation) will have a

more obvious effect

on the cell

The

structure

of the bacterial chromosome is

maintained by

polyamines;

spermine and spermidine (not by histones)

Bacteria may also contain

extrachromosomal genetic elements

;

plasmids

bacteriophages

(bacterial viruses) these elements are

independent of the bacterial chromosome

• in most cases can

be transmitted from one cell to another

Escherichia coli genome

Comparative genomes (genomics) of Escherichia coli strains

Transcription

Transcription ;

The information carried in the genetic memory of the DNA is

messenger RNA (mRNA)

transcribed into

for subsequent translation into protein a useful

RNA synthesis

is performed by a

DNA-dependent RNA polymerase

sigma factor;

recognizes a particular sequence of nucleotides in the DNA (the promoter)

binds tightly to this site

»

promoter sequences occur just before the start of the DNA that actually encodes a protein

» sigma factors bind to these promoters to provide a docking site for the RNA polymerase

»

Some bacteria encode several sigma factors to allow transcription of a group of genes under

special conditions, such as heat shock, starvation, special nitrogen metabolism, or sporulation

polymerase binds to the appropriate site on the DNA

sequential addition of ribonucleotides occur complementary to the sequence in the DNA

• entire gene or group of genes (operon) is transcribed

RNA polymerase dissociates from the DNA

Other RNA types that are also transcribed from the DNA;

transfer RNA (tRNA)

– which is used in protein synthesis

ribosomal RNA (rRNA

) a component of the ribosomes

DNA codon table

Transcription

Transcription ;

Promoters and operators control the expression of a gene by;

• influencing which sequences will be transcribed into messenger RNA (mRNA)

Operons;

groups of;

– one or more

»

structural genes ; expressed from a particular promoter

»

ending at a transcriptional terminator

Thus all the genes

coding for the enzymes of a particular pathway;

»

can be coordinately regulated

Operons with many structural genes

polycistronic

;

Example;

E. coli

lac operon ; includes all the genes necessary for lactose metabolism

, and

the control mechanisms

;

»

turning off ( in the presence of glucose ) or

»

turning on ( in the presence of galactose or an inducer )

lac operon includes;

– a repressor sequence,

– a promoter sequence, and

structural genes for the β-galactosidase enzyme

a permease, and

an acetylase

E. coli lac operon

Translation

Translation

;

Translation is the process by which the language of the

genetic code

,

in the form of mRNA,

• is converted (translated) into

the protein product a sequence of amino acids

;

Translation

Translation ;

Codon ;

a set of three nucleotides

– each correspond to either;

»

» an amino acid or a stop or start information

64 different codon combinations

– encoding;

»

20 amino acids

»

start (5’UTR →fMet≈AUG in prokaryotes) and termination -stop- (UAA,

UGA, UAG) codons

‘Degeneracy’ of the genetic code ;

some of the amino acids are encoded by more than one triplet codon

this feature protects the cell from the effects of minor mutations in the DNA or mRNA

Anticodon;

each tRNA molecule;

– contains a three-nucleotide sequence complementary to one of the codon

sequences

»

» allows base pairing

binds to the codon sequence on the mRNA

Amino acid ;

attached to the opposite end of the tRNA

corresponds to the particular codon-anticodon pair

Control of Gene Expression

Bacteria have developed;

mechanisms to adapt quickly and efficiently to ;

changes and triggers from the environment

adaptation mechanisms allow them to;

coordinate and regulate the expression of genes

for ;

»

multicomponent structures or

»

the enzymes of one or more metabolic pathways

– many bacterial

genes are controlled at

;

multiple levels

and

by multiple methods

a coordinated change in the expression of many genes occurs through use of a

different sigma factor for the RNA polymerase (e.g. sporulation)

this would change the specificity of the RNA polymerase and allow

mRNA synthesis for the necessary genes while ignoring unnecessary genes

– bacteria might produce

more than six different sigma factors

provide global regulation in response to; to;

– stress,

– shock, starvation,

coordinate production of complicated structures (e.g. flagella)

Control of Gene Expression

Simple triggers can turn on or turn off the transcription of a single gene or a group of genes;

temperature,

osmolarity,

pH,

nutrient availability, or the concentration of specific small molecules

• such as oxygen or iron

,

The expression of the components of

– also

virulence mechanisms

coordinately regulated from an operon

;

Salmonella invasion genes within a pathogenicity island are turned on by high osmolarity and low oxygen, conditions present in the gastrointestinal tract

E. coli senses its exit from the gut of a host by;

a drop in temperature,

» and inactivates its adherence genes

Low iron levels;

– can activate expression of hemolysin in E. coli or

– diphtheria toxin from Corynebacterium diphtheriae, potentially to kill cells and provide iron

Quorum sensing

• for; virulence factors of S. aureus

• biofilm production by Pseudomonas spp.

Replication of DNA

Replication of the bacterial genome ;

linked to the growth rate of the cell

initiated at a specific sequence in the chromosome called

requires many enzymes;

Helicase

;

OriC

– to unwind the DNA at the origin to expose the DNA,

Primase

; to synthesize primers to start the process, and

DNA-dependent DNA polymerase/s

;

– that synthesize a copy of the DNA,

» but only if there is a

direction primer sequence

to add to and only in the

5' to 3'

– new DNA is synthesized

semiconservatively; using both strands of the parental DNA as templates

– new DNA synthesis;

• occurs at

growing forks

• proceeds

bidirectionally

leading strand

is copied continuously in the

5' to 3' direction lagging strand fragments)

must be synthesized

as many pieces of DNA using RNA primers (Okazaki

Replication of DNA

lagging-strand

available

– then the

DNA must be extended in the

5' to 3' direction pieces are ligated together by the enzyme DNA ligase

as its template becomes

to maintain the high degree of accuracy required for replication,

– the

DNA polymerases possess "proofreading" functions

,

• which allow the enzyme to confirm that the appropriate nucleotide was inserted and

to correct any errors that were made

during log-phase growth in rich medium, many initiations of chromosomal replication may occur before cell division

– this process produces a

series of nested bubbles

with its pair of growth forks of new DNA synthesis of new daughter chromosomes, each

– the polymerase moves down the DNA strand, incorporating the appropriate

(complementary) nucleotide at each position

replication is complete

when;

the two replication forks meet 180 degrees from the origin

the process of DNA replication

puts great torsional strain on the chromosomal circle

of DNA;

this strain is relieved by topoisomerases (e.g., gyrase), which supercoil the DNA

Bacterial DNA replication

New DNA synthesis occurs at growing forks and proceeds bidirectionally.

DNA synthesis progresses in the 5' to 3' direction continuously (leading strand) or in pieces (lagging strand).

Assuming it takes 40 minutes to complete one round of replication, and assuming new initiation every 20 minutes, initiation of DNA synthesis precedes cell division.

Multiple growing forks may be initiated in a cell before complete septum formation and cell division.

The daughter cells are "born pregnant."

Bacterial cell division

Replication requires extension of the cell wall and replication of the chromosome and septum formation.

Membrane attachment of the DNA pulls each daughter strand into a new cell.

Mechanisms of Genetic Transfer between Cells

The

exchange of genetic material between bacterial cells

may occur by;

(1 ) conjugation

;

which is the mating or quasisexual exchange of genetic information

bacterium (the donor) to another bacterium (the recipient)

from one

(2)

transformation ;

• which results in acquisition of new genetic markers by the incorporation of

exogenous or foreign DNA from the environment

(3

) transduction ;

• which is the transfer of genetic information from one bacterium to another

bacteriophage by a

(4?) Once inside a cell,

a transposon

can jump between different DNA molecules (e.g., plasmid to plasmid or plasmid to chromosome)

Transformation

Bacteria

take up fragments of naked DNA

and incorporate them into their genomes

First mechanism of genetic transfer to be discovered in bacteria

(1928, Griffith, +15 years Avery, MacLeod, and McCarty);

• observed that pneumococcus virulence was related to the presence of a surrounding polysaccharide capsule

– and that extracts of encapsulated bacteria producing smooth colonies could transmit this trait to nonencapsulated bacteria, normally appearing with rough edges

Gram-positive and gram-negative bacteria can take up and stably maintain exogenous DNA

Certain species are naturally capable of taking up exogenous DNA;

competent;

Haemophilus influenzae, Streptococcus pneumoniae, Bacillus species, and Neisseria species

Most bacteria do not exhibit a natural ability for DNA uptake

Conjugation

Conjugation;

one-way transfer of DNA from a donor (or male) cell to a recipient (or female) cell through the sex pilus (or adhesin)

the mating type (sex) of the cell depends on;

the presence (male) or absence (female) of a conjugative plasmid

;

such as the

F plasmid

of E. coli

»

F plasmid is defined as conjugative because;

carries all the genes necessary for its own transfer

• including the ability to make sex pili initiate DNA synthesis at the transfer origin (OriT) of the plasmid

transfers itself

converting recipients into F+ male cells

usually occurs between members of the same or related species

,

– but also may occur

between prokaryotes and cells from plants, animals, and fungi

Examples;

E. coli, bacteroides, enterococci, streptococci, streptomycetes, and clostridia

Large conjugative plasmids specify ;

colicin

antibiotic resistance (by adhesins)

Conjugation

Conjugation;

Hfr (high frequency recombination) cell;

If a fragment of

chromosomal DNA

has been incorporated into the plasmid;

it is designated an F prime (F') plasmid

» when it transfers into the recipient cell,

• it carries that fragment with it and converts it into an F' male

if the F’ plasmid sequence (F’) is integrated into the bacterial chromosome,

the cell is designated an Hfr (high frequency recombination) cell

DNA that is transferred

by conjugation is

a single-stranded molecule

recircularized and its complementary strand synthesized in the recipient cell

Integration of an F plasmid into the bacterial chromosome generates an Hfr cell

Conjugation results in transfer of;

a part of the plasmid sequence, and

some portion of the bacterial chromosomal DNA

– because of the fragile connection between the mating pairs,

usually aborted before being completed;

» such that

the transfer is only the chromosomal sequences adjacent to the integrated

F are transferred

Transduction

Genetic transfer by transduction;

– is mediated by;

bacterial viruses (bacteriophages);

pick up fragments of DNA

package them into bacteriophage particles

DNA is;

»

delivered to infected cells

»

becomes incorporated into the bacterial genomes

Transduction can be classified as;

specialized;

• if the phages in question transfer particular genes (usually those adjacent to their integration sites in the genome)

generalized;

• if the selection of the sequences is random because of accidental packaging of host

DNA into the phage capsid

Underlying common mechanism: Recombination

Incorporation of extrachromosomal (foreign) DNA into the chromosome

recombination.

There are two types of recombination:

homologous

nonhomologous

Homologous (legitimate) recombination

;

– occurs

between closely related DNA sequences

– generally

substitutes one sequence for another

requires a set of enzymes

produced (in E. coli) by the

rec genes

Nonhomologous (illegitimate) recombination;

– occurs

between dissimilar DNA sequences

generally produces ;

insertions or deletions or

both

– requires

specialized (sometimes site-specific) recombination enzymes

,

• such as those produced by;

many transposons

lysogenic bacteriophages

occurs

by

Plasmid

The pBR322 plasmid is one of the plasmids used for cloning DNA.

This plasmid encodes resistance to ampicillin (Amp) and tetracycline (Tet) and an origin of replication (ori).

The multiple cloning site in the pGEM plasmid provides different restriction enzyme cleavage sites for insertion of DNA within the βgalactosidase gene (lacZ).

The insert is flanked by bacteriophage promoters to allow directional messenger RNA expression of the cloned sequence.

Transposon

A, The insertion sequences code only for a transposase (tnp) and possess inverted repeats (15 to 40 base pairs) at each end.

B, The composite transposons contain a central region coding for antibiotic resistances or toxins flanked by two insertion sequences (IS), which can be either directly repeated or reversed.

C, Tn3, a member of the TnA transposon family. The central region encodes three genes-a transposase (tnpA), a resolvase

(tnpR), and a β-lactamase-conferring resistance to ampicillin. A resolution site (Res site) is used during the replicative transposition process. This central region is flanked on both ends by direct repeats of 38 base pairs.

D, Phage-associated transposon is exemplified by the bacteriophage mu.

Mechanisms of bacterial gene transfer

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