Chapter 7 Microbial Genetics
Plasmids
? Circular genetic elements
that reproduce autonomously
and have an extra-
chromosomal existence.
– 1-1000 KB in size
– Most are circular double-
stranded DNA,some linear ds
DNA
– Transmitted from cell to cell via
conjugation process
– Some can integrated into
chromosome
– Can carry a variety of genes for
production of toxin,resistance
to antibiotics and heavy metals.
Mutations and Mutants
? Phenotype:the observable characteristics of an organism
– designated by a capital letter followed by two small
letters,with either a plus and minus superscript to
indicate the presence or absence of that property,
His-,Glu+ et al
? Genotype,The precise genetic make-up of an organism
– designated by three lower case letters followed by a
capital letter (all in italics) indicating the particular
gene involved,hisC,mutations in the hisC gene would
be designated as hisC1,hisC2 et al
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Replica Plating Technique,Isolation of Mutants
Velveteen (锦绒布）
Auxotroph (营养缺陷型 )
Prototroph (原养型 )
Replica Plating Technique,Isolation of Mutants
? Penicillin-Selection Method
– Penicillin kills only growing cells,If penicillin is added to a
population growing in a medium lacking the growth factor
required by the designed mutant,the parent cells will be killed,
whereas the non-growing mutant cells will be unaffected.
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Molecular
basis of
Mutation
? Point Mutation
– Missense mutation
– Nonsense mutation
– Silent mutation天冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
– Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Mutations involving many base pairs
? Deletions,mutations in which a region of
the DNA has been eliminated
? Insertions,occur when new bases are added
to the DNA,can be a single base or many
bases (called insertion sequences)
? Translocation,large section of
chromosomal DNA is moved to a new
location.
Rates of Mutation
? Spontaneous mutation,10-6,
? Transposition,10-4
? Nonsense mutation,10-6-10-8
? Missense mutation,10-6-10-8
Mutagens
Chemical mutagens:
Base analogs
Chemicals reacting
with DNA
Alkylating agents
Physical mutagens:
UV
Ionizing rdiation
Physical mutagen,Radiation
? Noninonizing (electromagnetic):
– UV,induction of pyrimidine dimers
? Ionizing radiation
– X-ray,cosmic rays,gamma rays; causing water
and other substances to ionize; such as the
generation of hydroxyl radical which reacts with
DNA
SOS regulatory system
Mutations may arise from error prone DNA repair
Other Methods of Creating Mutations
? Biological mutagens
– Transposon mutagenesis,bacteriophage Mu
? Site-directed mutagenesis
– Technique has been developed to make
mutation in a specific DNA site,
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemcals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
The Ames Test
? Histidine auxotrophs of
Salmonella typhimurium
and tryptophan auxotrophs
of Escherichia coli have
been the major tools for the
Ames test.
? Induction of a phage lambda
lysogen has also been used
as an assay of DNA damage
Genetic Recombination
? Homologous or General Recombination
– RecA protein participation
– Homologous DNA sequences have the same or nearly
the same sequence
– New genotypes only arise when two homologous
sequences are genetically distinct
In Prokaryotes,DNA fragments are
introduced into recipients by three means
? Transformation,a process by which free
DNA is inserted directly into a competent
recipient cell
? Transduction,transfer of bacterial DNA
from one bacteria to another within a
temperate or defective virion
? Conjugation (mating),DNA transfer via
actual cell-to-cell contact between the
recipient and the donor cell
Prokaryotic genetic recombination:
1,Transformation
2,Transduction
3,Conjugation
Detection of
Recombination
? Requirement:
reverse mutation
for the selected
characteristic
must be low.
This problem can
often be
overcome by
using double
mutants.
Complementation Test:
cis-tran test
? trans configuration,two
mutations are each on
separate DNA molecules
? cis configuration,Two
mutations were on the
same DNA molecule
? Complementation does not
involve recombination
Transformation
? Competent,cells
able to take up a
molecule of DNA.
? Uptake of DNA
? Integration of
transforming
DNA
Other methods for introducing
DNA into bacterial cells
? Transfection,transformed DNA is extracted
from a bacterial virus
? Artificially induced competence,e.g treat E,
coli with high concentration of Ca ions,and
then stored the cells at low T,the E,coli will
become competent at low efficiency
? Electroporation,pulsed electrical fields
generate pores in the cell membranes,
allowing DNA molecules to enter the cells,
Transformation (transfection) of
eukaryotic cells
? Transfection,introducing DNA into
mammalian cells
– phagocytosis in animal cells
– Yeast,spheroplasts added with Ca ions plus
polyethylene glycol
– Electroporation
– Particle gun,or gens gun
Transduction
1,Generalized transduction
2,Specialized transduction
Transduction
? Generalized transduction,host genes
derived from virtually any portion of the
host genome become part of the DNA of
the mature virus genome.
? Specialized transduction,occurs only in
some temperate viruses,s apecific group of
host genes is integrated directly into virus
genome-usually replacing some of the virus
genes-and is transferred to the recipient
during lysogenization
Specialized
Transduction
? Under rare
conditions,the
phage genome is
excised incorrectly.
? Lambda dgal
(defective galactose)
under the assistance
of helper,the
defective phage can
be replicated and
can transduce the
galactose genes.
Phage conversion
? A prophage is immune to further infection by the same
type of phage.
– Change in structure of a polysaccharide on the cell surface of
Salmonella anatum upon lysogenization with e15,
– Conversion of nontoxin producing strains of Corynebacterium
diphtheriae to toxin producing (pathogenic) strains.
? Information for production of these new materials is
apparently an integral part of the phage genome and
is automatically and exclusively transferred upon
infection by the phage and lysogenization.
Plasmids
? Circular genetic elements
that reproduce autonomously
and have an extra-
chromosomal existence.
– 1-1000 KB in size
– Most are circular double-
stranded DNA,some linear ds
DNA
– Transmitted from cell to cell via
conjugation process
– Some can integrated into
chromosome
– Can carry a variety of genes for
production of toxin,resistance
to antibiotics and heavy metals.
Mutations and Mutants
? Phenotype:the observable characteristics of an organism
– designated by a capital letter followed by two small
letters,with either a plus and minus superscript to
indicate the presence or absence of that property,
His-,Glu+ et al
? Genotype,The precise genetic make-up of an organism
– designated by three lower case letters followed by a
capital letter (all in italics) indicating the particular
gene involved,hisC,mutations in the hisC gene would
be designated as hisC1,hisC2 et al
Isolation of Mutants
? Pigmented and
nonpigmented
mutants
? Antibiotic-resistant
mutants within the
inhibition zone
? Genetic marker,
such as the
expression of b-
galactosidase that
produces the blue
color
Replica Plating Technique,Isolation of Mutants
Velveteen (锦绒布）
Auxotroph (营养缺陷型 )
Prototroph (原养型 )
Replica Plating Technique,Isolation of Mutants
? Penicillin-Selection Method
– Penicillin kills only growing cells,If penicillin is added to a
population growing in a medium lacking the growth factor
required by the designed mutant,the parent cells will be killed,
whereas the non-growing mutant cells will be unaffected.
Mutants that are detectable
? Non-motile
? Non-capsulated
? Rough colony
? Nutritional
? Sugar fermentation
? Drug resistant
? Virus resistant
? Temperature sensitive
? Pigmentless
? Cold sensitive
Molecular
basis of
Mutation
? Point Mutation
– Missense mutation
– Nonsense mutation
– Silent mutation天冬
酰
胺
酪
氨
酸
Frameshift mutations
? Occur in protein-encoding gene that including
the reading frame
? If occurs in promoter region,it is not frameshift
Back Mutations or Reversions
? Point Mutations are reversible
? Revertant
– wild type phenotype that was lost in the
mutant is restored.
? Same site revertants
? True revertants
? Second site mutation
– Suppressor mutation,restore the wild type
phenotype by mutation somewhere else.
Mutations involving many base pairs
? Deletions,mutations in which a region of
the DNA has been eliminated
? Insertions,occur when new bases are added
to the DNA,can be a single base or many
bases (called insertion sequences)
? Translocation,large section of
chromosomal DNA is moved to a new
location.
Rates of Mutation
? Spontaneous mutation,10-6,
? Transposition,10-4
? Nonsense mutation,10-6-10-8
? Missense mutation,10-6-10-8
Mutagens
Chemical mutagens:
Base analogs
Chemicals reacting
with DNA
Alkylating agents
Physical mutagens:
UV
Ionizing rdiation
Physical mutagen,Radiation
? Noninonizing (electromagnetic):
– UV,induction of pyrimidine dimers
? Ionizing radiation
– X-ray,cosmic rays,gamma rays; causing water
and other substances to ionize; such as the
generation of hydroxyl radical which reacts with
DNA
SOS regulatory system
Mutations may arise from error prone DNA repair
Other Methods of Creating Mutations
? Biological mutagens
– Transposon mutagenesis,bacteriophage Mu
? Site-directed mutagenesis
– Technique has been developed to make
mutation in a specific DNA site,
Mutagenesis and Carcinogenesis
The Ames Test
? Bacteria can be used as screening agents for
the potential mutagenicity of chemicals
– As it has been found that many mutagenic chemcals are
also carcinogenic,capable of causing cancer in animals and
humans
? The fact that a compound is mutagenic in a bacterial
system serves as a warning of possible danger
? Dr,Bruce Ames at the UC Berkeley has developed
the Ames Test technique for the screening of
carcinogenic compounds.
The Ames Test
? Histidine auxotrophs of
Salmonella typhimurium
and tryptophan auxotrophs
of Escherichia coli have
been the major tools for the
Ames test.
? Induction of a phage lambda
lysogen has also been used
as an assay of DNA damage
Genetic Recombination
? Homologous or General Recombination
– RecA protein participation
– Homologous DNA sequences have the same or nearly
the same sequence
– New genotypes only arise when two homologous
sequences are genetically distinct
In Prokaryotes,DNA fragments are
introduced into recipients by three means
? Transformation,a process by which free
DNA is inserted directly into a competent
recipient cell
? Transduction,transfer of bacterial DNA
from one bacteria to another within a
temperate or defective virion
? Conjugation (mating),DNA transfer via
actual cell-to-cell contact between the
recipient and the donor cell
Prokaryotic genetic recombination:
1,Transformation
2,Transduction
3,Conjugation
Detection of
Recombination
? Requirement:
reverse mutation
for the selected
characteristic
must be low.
This problem can
often be
overcome by
using double
mutants.
Complementation Test:
cis-tran test
? trans configuration,two
mutations are each on
separate DNA molecules
? cis configuration,Two
mutations were on the
same DNA molecule
? Complementation does not
involve recombination
Transformation
? Competent,cells
able to take up a
molecule of DNA.
? Uptake of DNA
? Integration of
transforming
DNA
Other methods for introducing
DNA into bacterial cells
? Transfection,transformed DNA is extracted
from a bacterial virus
? Artificially induced competence,e.g treat E,
coli with high concentration of Ca ions,and
then stored the cells at low T,the E,coli will
become competent at low efficiency
? Electroporation,pulsed electrical fields
generate pores in the cell membranes,
allowing DNA molecules to enter the cells,
Transformation (transfection) of
eukaryotic cells
? Transfection,introducing DNA into
mammalian cells
– phagocytosis in animal cells
– Yeast,spheroplasts added with Ca ions plus
polyethylene glycol
– Electroporation
– Particle gun,or gens gun
Transduction
1,Generalized transduction
2,Specialized transduction
Transduction
? Generalized transduction,host genes
derived from virtually any portion of the
host genome become part of the DNA of
the mature virus genome.
? Specialized transduction,occurs only in
some temperate viruses,s apecific group of
host genes is integrated directly into virus
genome-usually replacing some of the virus
genes-and is transferred to the recipient
during lysogenization
Specialized
Transduction
? Under rare
conditions,the
phage genome is
excised incorrectly.
? Lambda dgal
(defective galactose)
under the assistance
of helper,the
defective phage can
be replicated and
can transduce the
galactose genes.
Phage conversion
? A prophage is immune to further infection by the same
type of phage.
– Change in structure of a polysaccharide on the cell surface of
Salmonella anatum upon lysogenization with e15,
– Conversion of nontoxin producing strains of Corynebacterium
diphtheriae to toxin producing (pathogenic) strains.
? Information for production of these new materials is
apparently an integral part of the phage genome and
is automatically and exclusively transferred upon
infection by the phage and lysogenization.
