Download Pierce chapter 8

Chapter 8 – Bacterial and
Viral Genetic Systems
Bacteria
• Prototrophic
– Wild-type
– Can grow on minimal media
• Contains minimal nutrients – carbon, nitrogen, phosphorous,
vitamins, ions
• Auxotrophic
– Can not produce an essential enzyme or manufacture
essential molecules
– Will only grow on media that contains the “missing”
substance
• Complete media
Culturing bacteria
• Suspension culture
– Liquid media
– Bacteria dies off when
nutrients are used up
or waste buildup
becomes toxic
– Bacteria grow
singularly – no
colonies
Culturing bacteria
• Petri dishes
– Growth media
in agar
– Isolate
individual
colonies
• Each colony
originates from
a single
bacterium
Replica plating
• Gives “carbon
copies” of petri
dish colonies
• Use sterilized
velvet to make a
stamp
– Some bacteria
from each colony is
transferred to
velvet, and then
transferred to new
dishes
Bacterial genome
• Most consist of a single,
circular chromosome
– Some have several
chromosomes, and a few
have linear chromosomes
• Very little “extra” DNA
between genes
• Plasmids
– Small, circular, extrachromosomal DNA
• Usually non-essential
– Replicate independent of
chromosomal DNA
• Have their own origin of
replication
F factor episome
• Episome
– A plasmid that
can replicate
independently
AND also has the
ability to
incorporate into
chromosomes
Gene transfer in bacteria Conjugation
• One bacteria directly
transfers DNA to
another bacterium
• Cytoplasmic
connection forms, and
either entire plasmid or
part of the
chromosome is
transferred from donor
to recipient
• Crossing over may
occur between
homolgous regions
– Creates recombinant
DNA
– Extra DNA is degraded
Gene transfer in bacteria Transformation
• Bacteria takes
up DNA from
surrounding
environment
• Recombination
may occur
Gene transfer in bacteria Transduction
• Viral particle
introduced DNA
from a
bacterium into
a new
bacterium
Conjugation
• Fertility factor/F
factor contains ori
and genes needed
for conjugation
• F+ and F– F+ contains F factor
– Forms a sex pilus –
extension of cell
membrane
• Extends and comes
in contact with Freceptor
– F factor separates,
and one strand is
transferred into F• Double stranded
DNA is created and
F- becomes F+
Conjugation
• Hfr bacteria
– F+ cell that has F factor
incorporated into
chromosome
• As F factor enters recipient,
some chromsome enters –
amount depends on time
length of contact
• Donor DNA made into
double-stranded
– Crossing over can occur
between homologous
regions
– Any DNA not incorporated
is degraded
• Recipient is not usually
converted to F+ since the F
factor is nicked in the middle
Conjugation
• F′ bacteria
– F factor excises out of a chromosome in a Hfr cell
• May remove part of chromosome as well
– F′ plasmid now contains F factor and some genes from
chromosome
• Enters F- bacteria
– Produces merozygotes – partially diploid
Transformation
• Uptake of DNA and
incorporation into
chromosome or plasmid
– Naturally occurring –
dead bacteria
– Artificially introduced
• Competent – cells able to
take up DNA
– CaCl2, heat shock,
electrical fields
• Makes membrane more
permeable to DNA
– DNA does not have to
have bacterial origin
• Transformants – bacteria
that have incorporated
foreign DNA
E. Coli has model organism
• Many strains are
avirulent
• Small and rapid
reproduction
• Easy to culture
• Genome is single
chromosome - haploid
– Small genome
– 4.5 million bp/4,000 genes
• Wild-type are
prototrophic
Viral genetics
• DNA or RNA
(single or double
stranded) as
genetic material
• Can not reproduce
on their own
• Bacteriophages –
viral particles that
infect bacteria
Bacteriophage – lytic cycle
• Virulent phages
• Viral DNA is
injected into host
cell where it
replicated,
transcribed, and
translated into
more phages
• Host cell bursts
open to release
viral particles
Bacteriophages – lysogenic cycle
• Temperate phages
• Phage DNA is
incorporated into
host genome –
prophage
• Passed onto all
progeny cells
• Can be transcribed
and translated
• Can exit from host
genome to enter
lytic cycle
Transduction
• Generalized
– Any gene is transferred
– During lytic cycle, bacterial DNA is degraded
• Some may enter viral protein coat instead of viral genetic material
– Transducing phages
• Can become incorporated into new host’s genome
Transduction
• Specialized
– Few genes are transferred/genes near certain
sites of chromosome
– During lysogenic cycle, prophage enters at
specific sites of host’s genome
– When prophage excises, it may do so
imperfectly and bring some hot DNA with it
• Then introduced to new host
RNA viruses
• Positive strand RNA viruses
– Single strand directly codes for viral proteins
• Negative strand RNA viruses
– Must make complementary RNA strand, which then codes for
proteins
• Retroviruses
– Incorporate into host genome
• Must make DNA from RNA
• Reverse transcriptase
– Makes cDNA from DNA or RNA template
– Enters host genome as a provirus
• Can be transcribed and translated
– Some retroviruses contain oncogenes
• Cause tumors