Gene movement in bacteria

Phages can mediate

transfer of DNA to new

bacterial cell. Donor for

the DNA dies in lyticinfection. In some

bacteria, naked DNA

from dead donor can be

taken up into recipient

cell.

Fig. 10.11 top

Transformation with naked DNA

• Griffith mixed deadpathogenic smoothStrep. pneumoniae cellswith live rough cells inmouse infection.

• S.pneumoniae cellsfrom dead mice weresmooth.

• Avery, MacLeod, &McCarty showed the“transforming substance”from dead smoothS.pneumoniae cellswas nuclease-sensitive

Transformation

Natural transformation (upperscheme) occurs in variety ofeubacteria and archaea;uptake of linear DNAfragments, which mustrecombine with host DNA

Molecular biology and cloninghave led to development ofartificial means to make cellstake up autonomouslyreplicating dsDNAs orplasmids (lower scheme)

Bacteria that undergo

natural transformation

Gram positive: Streptococcus pneumoniae

Bacillus subtilis

Gram negative: Haemophilus influenza

Neisseria gonorrheae

Acinetobacter calcoaceticus

Bacteria that undergo

artificial transformation

Chemically induced: Escherichia coli

Salmonella typhimurium

Pseudomonas aeruginosa

(Salt + cold temps; dsDNA taken up by cells)

Electroporation: Many species of eubacteria and archaea

(and some eukaryotes)

(Pulsed electrical fields; dsDNA)

Natural

transformation

Fig. 10.14

Three stages:

a) Acquisition of

competence

b) DNA uptake c) Recombination

•Stage a varies by species

•Stage b varies between

Gram+ vs. Gram -•Stage c is the same in

all Bacteria

Natural transformation : Gram +

DNA uptake:

1) nonspecific

linear dsDNA

(10-20 Kb) bindsto 10-50 sites/cell

2) DNA breaks

to 6-8 Kb pieces

3) nuclease

converts tossDNA during

transport across

membrane

(100 nt/sec)

Fig. 10.14

Free nucleotides

released in medium

Natural transformationDNA uptake stage:

•In G–, dsDNA

becomes nuclease

resistant as it passesthrough OM into

some protected

compartment via OM

“secretin” protein

•In G+/–, passageinto cytoplasm via

conserved cyto.

membrane channel

Transformation

in Haemophilus influenzae

Specific 9-11 bp sequence in dsDNA required for binding

to competent cell (5’ AAGTGCGGT 3’).

CompetenceSpecific

DNA binding

Seq. specific receptors?

Uptake

Translocation

3’

Recombination Transformed

genome

Transformasome

TransformasomesDNA-binding sites

Cell envelope

Specific DNA sequences

recognized in transformation

• H. influenzae: 5’ AAGTGCGGT 3’ sequence ispresent 1465 times in genome-every 4Kb (vs. 8-9predicted frequency, if random)

• Neisseria: 5’GCCGTCTGAA 3’sequence is present1910 times in genome (vs. 4 predicted)

• Acinetobacter also seems to prefer to take up specificDNA, but basis of selectivity is unknown

Specific DNA may allow repair of essential functions orallow testing of options to ! fitness

No sequence motifs for DNA binding for Gram +

Gene movement in bacteria, part III

Conjugation allows DNA movement from live

donor to recipient, dependent on cell-cell contact.

The proteins that enable this movement are

usually encoded by genes on plasmids.Fig 10.19

Plasmids

Fundamental characteristics for each plasmid:

• Size/Copy number-from 3 to 200 Kb and from 1 to 100/cell

• Host range-bacterial strains where the plasmid willreplicate

• Incompatibility group-refers to whether 2 plasmidscan be maintained in same cell; based oncompatibility of replication and segregationsystems.

Representative bacterial plasmids

Conjugal; drug

resistance; phage

sensitivity

Broad:

Gram neg.

(Ec/Pseud)

60 / 4RP4

Causes plant tumorAgro/Rhizo200 / 1Ti

Mobilizable; colicinsNarrow9/30ColEI

Conjugal; phage

sensitivity; drug &Hg resistance

Enterics89 / 1R100

Conjugal; phagesensitivity

Narrow

(E. coli)

100 / 1F

TraitsHost rangeSize (Kb)

/ CN

Plasmid

F (fertility) plasmid

• Conjugalability due totransfer (tragenes)

• 99kb plasmidcontainsseveral IS/Tnelements

• Rep/Parfunctions

Fig 10.18

F plasmid and R100

• Can mediate their transfer from donor to recipient by

conjugation and are sensitive to particular phages

due to conjugal pili

• Have similar yet distinct Inc functions and arecompatible with each other

! !

F plasmid! oriC of

chromosome R100 (or RP4) plasmid

Low copy number plasmids seem to localize to particular spots during division.

Plasmid transfer via F

conjugation

Fig. 10.22a

DNA transfer inF conjugation

• oriT is nicked,5’"3’ ssDNA

transfer

• DNA pol III, etcreplicates

(leading indonor)

• Religation oforiT at end

Fig. 10.22b

Mobilizable plasmids

Conjugal plasmids encode proteins for an apparatus thatallows DNA movement.Mobilizable plasmids (like ColEI) can exploit the apparatusencoded by a co-resident conjugal plasmid. Usually amoblizable plasmid-specific DNA-processing function (stillusing ssDNA transfer process). ColE1 cannot move byitself!

= conjugal plasmid

Without a conjugal plasmid,

ColE1 cannot mobilize from here!

F plasmid can integrate into

E. coli chromosome

Fig. 10.23

Process forms

Hfr…

IS/Tns can allow

plasmids to recombinewith host chromosomes

F plasmid and E. coli Hfrs

Fig. 10.24

When plasmids recombinewith chromosome,conjugation functionscan lead to transfer ofchromosomal DNA (nextto integrated plasmid)

to recipient via conjugativeapparatus.

Directionality of processleads to rare transfer of tra genes

Conjugal plasmids can mediatetwo distinct DNA transfer events

Fig 10.11Conjugal or mobilizable

DNA must recombine with

recipient; recipient rarely becomes

a donor; 10-100 kb transferred

In both cases, ssDNA

transferred

Outcomes from exchange of genetic info

Gene substitution:transduction, naturaltransformation, orHfr conjugation

Gene addition:phages forminglysogens, plasmidconjugation &mobilization, orartificialtransformation withplasmids.

Gene movement: the bacterium

fights back…

While many mechanisms to move DNA fromone cell to another exist, the bacterial cell isnot necessarily a “passive” recipient. Someincoming DNA can obviously have negativeimpact on cell (Phage infection/sensitivity).

Bacteria have developed one important strategyto combat the flow of DNA into a cell:Restriction-Modification (R-M) systems

Discovery of R-M systemsWork from several phage groups (50’s-60’s):

# infects both B and K strains of E. coli, but…

• # preps grown on E. coli B strain with 10000xlower titer on K strain than on B

• # preps grown on E. coli K strain with 10000xlower titer on B strain than on K

• Discovered that reduction in “efficiency” ofinfection due to strain-specific nucleases

• Demonstrated that the R-M enzymes actindiscrimantly on dsDNA in cell; normal hostDNA is protected due to its modification

Discovery of R-M systems

• Upon entering E. coli K,DNA from # grown on

B strain could either bedegraded (restricted) ormodified

• If modified, subsequentinfections of phage in Kstrains would not besubject to K-specificrestriction

K cell

Grown on B

Recognition sequence for

E. coli Type II R-M enzyme

See Fig. 7.21

The EcoRI restriction enzyme makes staggered cuts on

both strands of DNA, leaving ss “sticky ends”. The

modifying enzyme adds -CH3 to 1 base of each strand in

recognition sequence and prevents cleavage. R-M systemswidespread in Bacteria and Archaea (rare in euks).

Three types of R-M systems

NoNoYesATP-dependent

YesNoYesJoint Nuclease/

Methylase?

24-26 bp on

3’ side

(closeby)

Between G

and A (in

sequence)

ca 1 Kb

away

(distant)

Cleavagesite

AGACCGAATTCTGAN8TGCTRecognitionsite

EcoPIEcoRIEcoBExample

Type IIIType IIType I

More on R-M systems

Because they have separate R/M enzymes and they

cleave in recognition sequence, the restriction

endonucleases of Type II systems are useful for

molecular biology.

Restriction enzymes recognizing different sequences

have been isolated from wide variety of bacteria.

Type II systems most common but Type I systems

widely distributed; Type III systems rare

Phages and plasmids have developed ways tocircumvent the protection hosts get from RM systems