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5
THE GENETICS
OF BACTERIA
AND THEIR VIRUSES
KEY QUESTIONS
Do bacterial cells ever pair up for any type
of sexual cycle?
Do bacterial genomes ever show
recombination?
If so, in what ways do genomes become
associated to permit recombination?
Does bacterial recombination resemble
eukaryote recombination?
Do the genomes of bacterial viruses ever
show recombination?
Do bacterial and viral genomes interact
physically in any way?
Can bacterial and viral chromosomes be
mapped using recombination?
OUTLINE
5.1 Working with microorganisms
5.2 Bacterial conjugation
5.3 Bacterial transformation
5.4 Bacteriophage genetics
Sexual union of bacteria. Cells of Escherichia coli that have
become attached by pili prior to DNA transfer between donor 5.5 Transduction
and recipient cell types. [Dr. L. Caro/Science Photo Library/Photo 5.6 Physical maps versus linkage maps
Researchers.]
151
44200_05_p151-184 3/5/04 2:59 PM Page 152
Partial genome
Transformation transfer by
DNA uptake
Genome
Genome
Transduction
Figure 5-1 Four ways by which bacterial DNA can be transferred from cell to cell.
44200_05_p151-184 3/5/04 2:59 PM Page 153
5.2 Bacterial conjugation the medium were supplemented with methionine and
biotin; strain B would grow only if it were supplemented
The earliest studies in bacterial genetics revealed the un- with threonine, leucine, and thiamine. Thus, we can des-
expected process of cell conjugation. ignate the strains as
strain A: met bio thr leu thi
Discovery of conjugation
strain B: met bio thr leu thi
Do bacteria possess any processes similar to sexual
reproduction and recombination? The question was Figure 5-4a displays in simplified form the design of
answered by the elegantly simple experimental work of their experiment. Strains A and B were mixed together,
Joshua Lederberg and Edward Tatum, who in 1946 dis- incubated for a while, and then plated on minimal
covered a sexlike process in bacteria. They were study- medium, on which neither auxotroph could grow. A
ing two strains of Escherichia coli with different sets small minority of the cells (1 in 10 7 ) was found to grow
of auxotrophic mutations. Strain A would grow only if as prototrophs and hence must have been wild type,
A + B
Mix
Some
WT progeny
(a)
A B
met bio thr + leu + thi + Mixture met + bio + thr leu thi
Wash cells Wash cells Wash cells Figure 5-4 Lederberg and Tatums demonstration
of genetic recombination between bacterial cells.
Plate ~ 10 8 cells Plate ~ 10 8 cells Plate ~ 10 8 cells (a) The basic concept: two auxotrophic cultures
(A and B) are mixed, yielding prototrophic wild
types (WT). (b) Cells of type A or type B cannot
grow on an unsupplemented (minimal) medium
(MM), because A and B each carry mutations that
cause the inability to synthesize constituents
needed for cell growth. When A and B are mixed
for a few hours and then plated, however, a few
MM MM MM colonies appear on the agar plate. These colonies
No met + bio + thr + leu + thi + No
derive from single cells in which an exchange of
colonies Prototrophic colonies genetic material has occurred; they are therefore
colonies capable of synthesizing all the required
(b) constituents of metabolism.
44200_05_p151-184 3/5/04 2:59 PM Page 156
The F plasmid directs the synthesis of pili, projections part or all of that chromosome into the F cell. The
that initiate contact with a recipient (Figure 5-6) and chromosomal fragment can then engage in recombina-
draw it closer. The F DNA in the donor cell makes a tion with the recipient chromosome. The rare recombi-
single-stranded copy of itself in a peculiar mechanism nants observed by Lederberg and Tatum in F F
called rolling circle replication. The circular plasmid crosses were due to the spontaneous, but rare formation
rolls, and as it turns, it reels out the single-stranded of Hfr cells in the F culture. Cavalli-Sforza isolated ex-
copy like fishing line. This copy passes through a pore amples of these rare cells from F cultures, and found
into the recipient cell, where the other strand is syn- that indeed they now acted as true Hfrs.
thesized, forming a double helix. Hence a copy of F re- Does an Hfr cell die after donating its chromosomal
mains in the donor and another appears in the recipi- material to an F cell? The answer is no. Just like the
ent, as shown in Figure 5-7. Note in the figure that the F plasmid, during conjugation the Hfr chromosome
E. coli genome is depicted as a single circular chromo- replicates and transfers a single strand to the F cell. The
some. (We will examine the evidence for this later.) single-stranded nature of the transferred DNA can be
Most bacterial genomes are circular, a feature quite dif- demonstrated visually using special strains and antibod-
ferent from eukaryotic nuclear chromosomes. We shall ies, as shown in Figure 5-9. The replication of the chro-
see that this feature leads to many idiosyncrasies of mosome ensures a complete chromosome for the donor
bacterial genetics. cell after mating. The transferred strand is converted into
a double helix in the recipient cell, and donor genes may
become incorporated in the recipients chromosome
Hfr strains through crossovers, creating a recombinant cell (Figure
5-10). If there is no recombination, the transferred
An important breakthrough came when Luca Cavalli-
fragments of DNA are simply lost in the course of cell
Sforza discovered a derivative of an F strain with two
division.
unusual properties:
Hfr azi r tonr lac gal str s F azi s tons lac gal str r
Hfr F
and recombination
c+ b+ a+ 0
c b a- Transfer of single-stranded
DNA copy
c+ b+ a+ F
Exconjugant
Exogenote c+ b+ a+
www. ANIMATED ART
Transferred fragment
c b a converted to double helix
Endogenote
Recombinant Lost
c+ b a
Double crossover inserts
c b+ a+ donor DNA
azi r
Hfr or F? When Wollman and Jacob allowed Hfr
Frequency (%) of Hfr genetic
0
O a b c F
0 10 20 30 40 50 60
Time (minutes)
Thus almost none of the F recipients are converted be-
(b) cause the fertility factor is the last element transmitted,
and usually the transmission process will have stopped
F factor
before getting that far.
Hfr str s Origin Origin F str r INFERRING INTEGRATION SITES OF F AND CHROMO-
SOME CIRCULARITY Wollman and Jacob went on to
shed more light on how and where the F plasmid
integrates to form an Hfr, and in doing so deduced the cir-
17 min
cularity of the chromosome. They performed interrupted-
mating experiments using different, separately derived Hfr
strains. Significantly, the order of transmission of the alleles
differed from strain to strain, as in the following examples:
Hfr strain
H O thr pro lac pur gal his gly thi F
25 min
1 O thr thi gly his gal pur lac pro F
2 O pro thr thi gly his gal pur lac F
3 O pur lac pro thr thi gly his gal F
AB 312 O thi thr pro lac pur gal his gly F
Figure 5-11 Interrupted-mating conjugation experiments. Each line can be considered a map showing the order of
F streptomycin-resistant cells with mutations in azi, ton, alleles on the chromosome. At first glance, there seems
lac, and gal are incubated for varying times with Hfr cells to be a random shuffling of genes. However, when the
that are sensitive to streptomycin and carry wild-type alleles
identical alleles of the different Hfr maps are lined up,
for these genes. (a) A plot of the frequency of donor alleles
in exconjugants as a function of time after mating. (b) A
the similarity in sequence becomes clear.
schematic view of the transfer of markers (shown in different
colors) over time. [Part a after E. L. Wollman, F. Jacob, and H F thi gly his gal pur lac pro thr O
W. Hayes, Cold Spring Harbor Symp. Quant. Biol. 21, 1956, 141.] (written backwards)
1 O thr thi gly his gal pur lac pro F
2 O pro thr thi gly his gal pur lac F
3 O pur lac pro thr thi gly his gal F
AB 312 F gly his gal pur lac pro thr thi O
(written backwards)
44200_05_p151-184 3/4/04 10:47 AM Page 160
O
Homologous
regions where
pairing can
take place 2
1 2 1
a
a b
1 b c d a 2
Hfr
Transferred last Direction of transfer Transferred first
d c
E. coli d c
chromosome
Figure 5-12 Insertion of the F factor into the E. coli chromosome by crossing-over.
Hypothetical markers 1 and 2 are shown on F to depict the direction of insertion. The origin
(O) is the mobilization point where insertion into the E. coli chromosome occurs; the
pairing region is homologous with a region on the E. coli chromosome; a d are
representative genes in the E. coli chromosome. Fertility genes on F are responsible for the
F phenotype. Pairing regions (hatched) are identical in plasmid and chromosome. They are
derived from mobile elements called insertion sequences (see Chapter 13). In this example,
the Hfr cell created by the insertion of F would transfer its genes in the order a, d, c, b.
The relationship of the sequences to one another is that if F is a ring, then insertion might be by simple cross-
explained if each map is the segment of a circle. This was over between F and the bacterial chromosome (Figure
the first indication that bacterial chromosomes are circular. 5-12). That being the case, any of the linear Hfr chromo-
Furthermore, Allan Campbell proposed a startling hypothe- somes could be generated simply by insertion of F into the
sis that accounted for the different Hfr maps. He proposed ring in the appropriate place and orientation (Figure 5-13).
thi gly his gal pur lac pro thr lac pur gal his gly thi thr pro pro lac pur gal his gly thi thr
H 2 1
thr thr
thi pro thi pro
Figure 5-13 Order of gene transfer. The five E. coli Hfr strains shown each have different
F factor insertion points and orientations. All strains share the same order of genes on
the E. coli chromosome. The orientation of the F factor determines which gene enters the
recipient cell first. The gene closest to the terminus enters last.
44200_05_p151-184 3/4/04 10:47 AM Page 161
Chromosome Plasmid
transfer transfer
F a+
F+ a +
Conjugation and
Insertion of F factor transfer of F factor
F a+ F+ a + F a+
Hfr a +
F a a
Conjugation and
chromosome transfer
F a+ Hfr a + F a+
a+ F+ a +
a F a
F a +/a F a
Recombination No recombination
F a + F a F+ a
Figure 5-14 Conjugation summary. Summary of the various events that take
place in the conjugational cycle of E. coli.
Several hypotheses later supported followed from The E. coli conjugation cycle is summarized in
Campbells proposal. Figure 5-14.
1. One end of the integrated F factor would be the Mapping of bacterial chromosomes
origin, where transfer of the Hfr chromosome begins.
The terminus would be at the other end of F. BROAD-SCALE CHROMOSOME MAPPING USING
TIME OF ENTRY Wollman and Jacob realized that it
2. The orientation in which F is inserted would
would be easy to construct linkage maps from the
determine the order of entry of donor alleles. If the
interrupted-mating results, using as a measure of dis-
circle contains genes A, B, C, and D, then insertion
tance the times at which the donor alleles first appear
between A and D would give the order ABCD or
after mating. The units of map distance in this case are
DCBA, depending on orientation. Check the
different orientations of the insertions in Figure 5-12. minutes. Thus, if b begins to enter the F cell 10 min-
utes after a begins to enter, then a and b are 10 units
How is it possible for F to integrate at different apart. Like eukaryotic maps based on crossovers, these
sites? If F DNA had a region homologous to any of linkage maps were originally purely genetic construc-
several regions on the bacterial chromosome, any one of tions. At the time they were originally devised, there was
these could act as a pairing region at which pairing could no way of testing their physical basis.
be followed by a crossover. These regions of homology
are now known to be mainly segments of transposable FINE-SCALE CHROMOSOME MAPPING BY RECOM-
elements called insertion sequences. For a full explanation BINANT FREQUENCY For an exconjugant to acquire
of these, see Chapter 13. donor genes as a permanent feature of its genome, the
The fertility factor thus exists in two states: donor fragment must recombine with the recipient
chromosome. However, note that time-of-entry mapping
1. The plasmid state: as a free cytoplasmic element F is is not based on recombinant frequency. Indeed the units
easily transferred to F recipients. are minutes, not RF. Nevertheless it is possible to use
2. The integrated state: as a contiguous part of a circular recombinant frequency for a more fine scale type of
chromosome F is transmitted only very late in mapping in bacteria, and this is the method to which we
conjugation. now turn.
44200_05_p151-184 3/4/04 10:47 AM Page 162
Hfr fragment
arg 2 met 2
leu 2 leu 1 arg 2 met 2
2
F chromosome
(b) Insertion of late marker and one early marker
arg 1 met 1
1
leu
(d) Insertion of late and early markers, but not of marker in between
arg 1 met 1
leu 1
Figure 5-16 Mapping by recombination in E. coli. After a cross, selection is made for the
leu marker, which is donated late. The early markers (arg and met) may or may not be
inserted, depending on the site where recombination between the Hfr fragment and the F
chromosome takes place. The frequencies of events diagrammed in parts a and b are used
to obtain the relative sizes of the leu arg and arg met regions. Note that in each case
only the DNA inserted into the F chromosome survives; the other fragment is lost.
However, sometimes the exit is not clean, and the plas- exconjugants seemed to carry an F plasmid with a piece
mid carries with it a part of the bacterial chromosome. of the donor chromosome incorporated. The origin of this
An F plasmid carrying bacterial genomic DNA is called F plasmid is shown in Figure 5-17. Note that the faulty
an F (F prime) plasmid. excision occurs because there is another homologous re-
The first evidence of this process came from experi- gion nearby that pairs with the original. The F in our
ments in 1959 by Edward Adelberg and Franois Jacob. example is also called F lac because the piece of host
One of their key observations was of an Hfr in which chromosome that it picked up has the lac gene on it.
the F factor was integrated near the lac locus. Starting F factors have been found carrying many different chro-
with this Hfr lac strain, Jacob and Adelberg found an mosomal genes and have been named accordingly. For ex-
F derivative that in crosses transferred lac to F lac ample, F factors carrying gal or trp are called F gal and
recipients at a very high frequency. (These transferrants F trp, respectively. Because F lac/lac cells are Lac in
could be detected by plating on medium lacking lac- phenotype, we know that lac is dominant over lac.
tose.) Furthermore, these F lac exconjugants occasion- Partial diploids made using F strains are useful for
ally gave rise to F lac daughter cells, at a frequency some aspects of routine bacterial genetics, such as the
of 1 103. Thus, the genotype of these recipients study of dominance or of allele interaction. Some
appeared to be F lac / F lac. In other words the lac F strains can carry very large parts (up to one-quarter)
44200_05_p151-184 3/4/04 10:47 AM Page 164
(a) Insertion F
IS1 lac 1
ton IS2
Integrated F factor
(b)
lac 1
Hfr chromosome
lac 1
(c) Excision
F' lac
1
lac
(d)
lac 2
Figure 5-17 Origin of an F factor. (a) F is inserted in an Hfr strain at a repetitive element
labeled IS1 between the ton and lac alleles. (b) The inserted F factor. (c) Abnormal
outlooping by crossing-over with a different element, IS2, to include the lac locus. (d) The
resulting F lac particle. (e) F lac / lac partial diploid produced by the transfer of the
F lac particle to an F lac recipient. [From G. S. Stent and R. Calendar, Molecular
Genetics, 2d ed. Copyright 1978 by W. H. Freeman and Company.]
Enterococcus Lactococcus
faecium lactis Mycoplasma
Listeria
monocytogenes
29 1 2
3
4
5
28
6
Lactococcus
Enterococcus 27 lactis
faecalis
7
26
25
8
Enterococcus
faecium 24
Listeria
23 Plasmid pk 214
monocytogenes
Staphylococcus 22
9 Streptococcus
aureus
agalactiae
21
Lactobacillus
plantarum
10
Lactococcus
lactis
20 11
12
19 Streptococcus
13
14 pyogenes
Staphylococcus 18
aureus 17 15
16 Escherichia
coli
Enterococcus
faecium Lactococcus
Escherichia Escherichia lactis
coli coli
Staphylococcus
aureus
Figure 5-18 Origins of genes of the Lactococcus lactis plasmid pK214. The genes are from
many different bacteria. [Data from Table 1 in V. Perreten, F. Schwarz, L. Cresta, M. Boeglin,
G. Dasen, and M. Teuber, Nature 389, 1997, 801 802.]
tant to many of these drugs, including penicillin, tetracy- Table 5-2 Genetic Determinants Borne
cline, sulfanilamide, streptomycin, and chloramphenicol. by Plasmids
This resistance to multiple drugs was inherited as a single
genetic package, and it could be transmitted in an infec- Characteristic Plasmid examples
tious manner not only to other sensitive Shigella strains, Fertility F, R1, Col
but also to other related species of bacteria. This talent, Bacteriocin production Col E1
which resembles the mobility of the E. coli F plasmid, is Heavy-metal resistance R6
an extraordinarily useful one for the pathogenic bac- Enterotoxin production Ent
terium because resistance can rapidly spread through a Metabolism of camphor Cam
population. However, its implications for medical science Tumorigenicity in plants T1 (in Agrobacterium
are dire because the bacterial disease suddenly becomes tumefaciens)
resistant to treatment by a large range of drugs.
From the point of view of the geneticist, however, the
mechanism has proved interesting, and useful in genetic been found to carry many different kinds of genes in bac-
engineering. The vectors carrying these multiple resis- teria. Table 5-2 shows some of the characteristics that can
tances proved to be another group of plasmids called R be borne by plasmids. Figure 5-18 shows an example of a
plasmids. They are transferred rapidly on cell conjugation, well-traveled plasmid isolated from the dairy industry.
much like the F plasmid in E. coli. R plasmids become important in designing strains for
In fact, the R plasmids in Shigella proved to be just use in genetic engineering because the plasmids are easily
the first of many similar ones to be discovered. All exist in shuttled between cells, and the R genes can be used to
the plasmid state in the cytoplasm. These elements have keep track of them.
44200_05_p151-184 3/4/04 10:47 AM Page 166
Sheath
End plate
Cell wall
Injected
DNA
Fibers
Uninfected
cell
Phage proteins
synthesized
Mapping phage chromosomes
and genetic using phage crosses
Degraded material replicated;
host host chromosome
chromosome then degraded
Two phage genotypes can be crossed in much the same
way that we cross organisms. A phage cross can be illus-
Figure 5-23 A generalized bacteriophage lytic cycle. [After trated by a cross of T2 phages originally studied by
J. Darnell, H. Lodish, and D. Baltimore, Molecular Cell Biology. Alfred Hershey. The genotypes of the two parental
Copyright 1986 by W. H. Freeman and Company.] strains in Hersheys cross were h r h r. The alle-
les correspond to the following phenotypes:
microscope? In this case, we cannot produce a visible h: can infect two different E. coli strains (which we
colony by plating, but we can produce a visible manifes- can call strains 1 and 2)
tation of a phage by taking advantage of several phage
h : can infect only strain 1
characters.
Lets look at the consequences of a phages infecting r: rapidly lyses cells, thereby producing large plaques
a single bacterial cell. Figure 5-23 shows the sequence of r: slowly lyses cells, producing small plaques
44200_05_p151-184 3/4/04 10:47 AM Page 169
(h r ) (h r )
RF
total plaques
rII gene
parent 1
parent 2
wild type
double
mutant
made use of the fact that rII mutants will not infect a to hydrolytic enzymes. Thus, Lederberg and Zinder had
strain of E. coli called K. Therefore he made the discovered a new type of gene transfer, mediated by a
rII rII cross on another strain and then plated the virus. They were the first to call this process
phage lysate on a lawn of strain K. Only rII recombi- transduction. As a rarity in the lytic cycle, virus particles
nants will form plaques on this lawn. This way of finding sometimes pick up bacterial genes and transfer them
a rare genetic event (in this case a recombinant) is a when they infect another host. Transduction has subse-
selective system: only the desired rare event can produce quently been demonstrated in many bacteria.
a certain visible outcome. Contrast this with screens, To understand the process of transduction we need
systems in which large numbers of individuals are visu- to distinguish two types of phage cycle. Virulent phages
ally scanned to seek the rare needle in the haystack. are those that immediately lyse and kill the host. Tem-
This same approach can be used to map mutant perate phages can remain within the host cell for a pe-
sites within genes for any organism from which large riod without killing it. Their DNA either integrates into
numbers of cells can be obtained, and for which it is the host chromosome to replicate with it or replicates
possible to easily distinguish wild-type and mutant phe- like a plasmid, separately in the cytoplasm. A phage in-
notypes. However, this sort of intragenic mapping has tegrated into the bacterial genome is called a prophage.
been largely superseded by the advent of cheap chemi- A bacterium harboring a quiescent phage is called lyso-
cal methods for DNA sequencing, which identify the genic. Occasionally a lysogenic bacterium lyses sponta-
positions of mutant sites directly. neously. A resident temperate phage confers resistance
to infection by other phages of that type.
MESSAGE Recombination between phage chromosomes Only temperate phages can transduce. There are
can be studied by bringing the parental chromosomes two kinds of transduction: generalized and specialized.
together in one host cell through mixed infection. Progeny Generalized transducing phages can carry any part of
phages can be examined for parental versus recombinant the bacterial chromosome, whereas specialized trans-
genotypes.
ducing phages carry only certain specific parts.
a+
a+ b+
b+
a+
Donor bacterium b+
b+
Phages carrying
donor genes
a+
a+
a+ a a+
a
Figure 5-27 The mechanism of generalized transduction. In reality, only a very small
minority of phage progeny (1 in 10,000) carry donor genes.
a single piece of DNA. For example, suppose that we that is also arg is measured. Strains transduced to both
wanted to find the linkage between met and arg in met and arg are called cotransductants. The greater the
E. coli. We could grow phage P1 on a donor met arg cotransduction frequency, the closer two genetic markers
strain, and then allow P1 phages from lysis of this strain must be (the opposite of most mapping correlations).
to infect a met arg strain. First, one donor allele is se- Linkage values are usually expressed as cotransduction
lected, say, met. Then, the percentage of met colonies frequencies (Figure 5-28).
2.8 (77), 13
42, 40 (70), 46
68, 74
70
21, 16
2.0
particles that now carry a nearby gene and leave behind 5.6 Physical maps
some phage genes [see Figure 5-31b(ii)]. The resulting
phage genome is defective because of the genes left behind, versus linkage maps
but it has also gained a bacterial gene gal or bio. These
Some very detailed chromosomal maps for bacteria have
phages are referred to as dgal (-defective gal) or dbio.
been obtained by combining the mapping techniques of
The abnormal DNA carrying nearby genes can be packaged
interrupted mating, recombination mapping, transforma-
into phage heads and can infect other bacteria. In the pres-
tion, and transduction. Today, new genetic markers are
ence of a second, normal phage particle in a double infec-
typically mapped first into a segment of about 10 to 15
tion, the dgal can integrate into the chromosome at the
map minutes by using interrupted mating. This method
attachment site (Figure 5-31c). In this manner, the gal
allows the selection of markers to be used for mapping by
genes in this case are transduced into the second host.
P1 cotransduction or by recombination.
By 1963, the E. coli map (Figure 5-32) already de-
MESSAGE Transduction occurs when newly forming
tailed the positions of approximately 100 genes. After 27
phages acquire host genes and transfer them to other
bacterial cells. Generalized transduction can transfer any host years of further refinement, the 1990 map depicted the
gene. It occurs when phage packaging accidentally incorporates positions of more than 1400 genes. Figure 5-33 shows a
bacterial DNA instead of phage DNA. Specialized transduction 5-minute section of the 1990 map (which is adjusted to
is due to faulty outlooping of the prophage from the bacterial a scale of 100 minutes). The complexity of these maps
chromosome, so the new phage includes both phage and illustrates the power and sophistication of genetic analy-
bacterial genes. The transducing phage can transfer only sis. How well do these maps correspond to physical real-
specific host genes.
ity? In 1997, the DNA sequence of the entire E. coli
D
A
B
I
C
thrA,D
pdxA
uvrA
malB
pyrA
(lex)
metA
*purH
(ace M
ara
aceE
*purthl
pgl
A
leu
aceF
azi
Y
*su yc
pan *
Z
A,D
ftsA
D
P
p
O
*c H
arg B
80 0
arg gC
1
ar E
la c
79
la
pr cl
ph C
ar pc
*
o
oA
2 *
g
*serB
(mutT)
*thyR
C
p rts
(trpR)
oR
(tp p)
78 10
(gua pil
B
*valS
tonA
* d)
ph n *
hsp
(ra etF
(ast)
A
D
argF
m etB K
lo in *
,B
pyr
C)
m lp
m
pE
pro
11
B
77 su s*
fdp
g
*p
rh
rn pG*
oB
P
u
a
*a
rA
11.5 su A*
pr
B A
m
tfr glt
pA
C 76 0 / 90 15
ilv c
O 85 5 s x su L*
A rbs 74 t l sup ,B*
D pho mb rE
(da S tolA
E rA) 80 10 pu G K
me 16 aro T
*tna tE nicA
R E
*tna
A gal ) O
73
B
(chlB
75 15 (mglR
C bgl
) (phr)
A pyrE 17
xyl gltH* 2)
(att434.8
(gad) glyS aroA* att l
72
*gltC 70 20 pyrD chlA
pyrC bioA
mtl 18
urvB*
purB
(cat) 24 supC,O
71
65 25 supF*
aroE tdk*
(chlC
67 *spcA ) gaIU
argR 25 att f
asd *linA pab 80
60 30 B tonB
D yA A
glp *er sp
a
aro
H trp B Figure 5-32 The 1963
glpR 66 G aro *
arg np pp D 26 cys
B
C genetic map of E. coli.
lA p ) 55 35 D
ma m s* pyr Units are minutes, based
laS an F E
B (a *fda 36
aro ) et
C * O
on interrupted-mating
ph sB)
oB 50 40
(bi m rgP A
eS
(ft S
65 a er 45
* mo experiments, timed from
ar d)
G s
ys tB
c rel
g
(e f)
*c 37
*m ysC
A an arbitrarily located
*
m
d
(zw
(da utS
ab )
ot
(da pA)
*p
A
(so
*pr )
D 64
pB
(mg
rg
uv
A
(tolC
m)
(a rA
glpT
rC
purC
st 56
purF
*ctr
38
*nicB
supN
aroC
dsdA
dsdC
)
lP)
p S
)
(tr
su
48
ra
sh
(re T
pD
50
*a
his
)
iA
cB
)
gnd
galR
lysA
G. S. Stent, Molecular
thyA
argA
gua
fuc
purG*
glyA
tyrA
aroF*
pheA
uraP*
mraA,B*
dapC*
cdsA*
gInD*
optA*
dapD
sefA*
polC
hlpA
IpxA
IpxB
rpsB
murG
murC
murE
firA
murF
erivA
mutT
secA
orf
orf
orf
polB
frsQ
tsf
ftsA
ftsZ
ddl
ftsl
orf
rpsT
lspA
ileS
ACBD
orf
KJ DABC DBCA IH DE
ABC AB
panBCD
acrC*
proS*
pyrH*
tadE*
popC*
sefA*
metD
mafB*
garB*
serR*
guaC
nadC
mrcB
pcnB
aceE
prlD*
dadB
fruR*
aceF
ftsM*
brnS*
ssyD
chlG*
aroP
(rimG ) rimF*
apaH
gprA*
dapB
mafA
pdxA
ksgA
kefC
(popD) gprB
spe
Irs*
hpt
fhu
Ipd
folA
ant*
ara
leu
dna
orf
ilvJ
tolJ
car
ilv
orf
thr
(envN )
(tdi )
(toll )
0 1 2 3 4 5
Figure 5-33 Linear scale drawing of a 5-minute section of the 100-minute 1990 E. coli
linkage map. The parentheses and asterisks indicate markers for which the exact location
was unknown at the time of publication. Arrows above genes and groups of genes indicate
the direction of transcription. [From B. J. Bachmann, Linkage Map of Escherichia coli K-12,
Edition 8, Microbiological Reviews 54, 1990, 130 197.]
genome was completed, allowing us to compare the ial and plasmid genomes coincided with the publication
exact position of genes on the genetic map with the cor- and popularization of J. R. R. Tolkiens The Lord of the
responding position of the respective coding sequence Rings. Consequently a review of bacterial genetics at
on the linear DNA sequence. Figure 5-34 makes this that time led off with the following quotation from the
comparison for a segment of both maps. Clearly, the trilogy:
genetic map is a close match to the physical map.
As an aside in closing, it is interesting that many of One Ring to rule them all, One Ring to find them,
the historical experiments revealing circularity of bacter- One Ring to bring them all and in the darkness bind them.
(a)
cysC cysH eno relA argA recC mutH
60 ptr thyA 61
(b)
mutS rpoS pcm cysC iap cysH eno relA barA syd sdaC exo gcvA mltA argA ptr recC thyA ptsP mutH aas galR araE glyU
Figure 5-34 Correlation of the genetic and physical maps. (a) Markers on the 1990 genetic
map in the region near 60 and 61 minutes. (b) The exact positions of every gene, based
on the complete sequence of the E. coli genome. (Not every gene is named in this figure,
for simplicity.) The elongated boxes are genes and putative genes. Each color represents a
different type of function. For example, red denotes regulatory functions, and dark blue
denotes functions in DNA replication, recombination, and repair. The correspondence
of the order of genes on both maps is indicated.
Does bacterial recombination resemble eukaryote Do bacterial and viral genomes interact physically in
recombination? any way?
Yes, in the sense that A B can be produced from A b and Yes, temperate phages can pick up bacterial genome
a B. No, in the sense that recombination always takes fragments during lysis and transfer them to other bacte-
place in a partial diploid (merozygote) as opposed to a ria when they reinfect. Some phages pick up only one
true diploid. Hence the recombination is formally a specific region; others can pick up any segment that can
double-crossover event, needed to maintain the circular be stuffed into a phage head.
bacterial chromosome intact.
Can bacterial and viral chromosomes be mapped
using recombination?
Do the genomes of bacterial viruses ever show Yes, all bacterial merozygotes can be used for recombi-
recombination? nant frequency-based mapping. There are also some un-
Yes, if a bacterial host is infected simultaneously by usual methods, for example, mapping by time of entry
two phage types, recombinant phage are found in the during conjugation, and frequency of cotransduction by
lysate. phage. Virus chromosomes can be mapped, too.
SUMMARY
Advances in microbial genetics within the past 50 years transformation to occur, DNA must be taken into a
have provided the foundation for recent advances in recipient cell, and recombination between a recipient
molecular biology (discussed in subsequent chapters). chromosome and the incorporated DNA must then take
Early in this period, gene transfer and recombination place.
were found to take place between different strains of Bacteria can be infected by bacteriophages. In one
bacteria. In bacteria, however, genetic material is passed method of infection, the phage chromosome may enter
in only one direction from a donor cell (F or Hfr) to the bacterial cell and, using the bacterial metabolic ma-
a recipient cell (F). Donor ability is determined by the chinery, produce progeny phage that burst the host bac-
presence in the cell of a fertility factor (F), a type of terium. The new phages can then infect other cells. If
plasmid. two phages of different genotypes infect the same host,
On occasion, the F factor present in the free state in recombination between their chromosomes can take
F cells can integrate into the E. coli chromosome and place in this lytic process.
form an Hfr cell. When this occurs, gene transfer and In another mode of infection, lysogeny, the injected
subsequent recombination take place. Furthermore, be- phage lies dormant in the bacterial cell. In many cases,
cause the F factor can insert at different places on the this dormant phage (the prophage) incorporates into the
host chromosome, investigators were able to show that host chromosome and replicates with it. Either sponta-
the E. coli chromosome is a single circle, or ring. Inter- neously or under appropriate stimulation, the prophage
ruption of the transfer at different times has provided can arise from its latency and can lyse the bacterial host
geneticists with a new method for constructing a linkage cell.
map of the single chromosome of E. coli and other simi- Phages can carry bacterial genes from a donor to a
lar bacteria. recipient. In generalized transduction, random host
Genetic traits can also be transferred from one bacte- DNA is incorporated alone into the phage head during
rial cell to another in the form of pieces of DNA taken lysis. In specialized transduction, faulty excision of the
into the cell from the extracellular environment. This prophage from a unique chromosomal locus results in
process of transformation in bacterial cells was the first the inclusion of specific host genes as well as phage
demonstration that DNA is the genetic material. For DNA in the phage head.
KEY TERMS
attachment site (p. 000) conjugation (p. 000) endogenote (p. 000)
auxotrophic (p. 000) cotransductants (p. 000) exconjugants (p. 000)
bacteriophages (p. 000) donor (p. 000) exogenote (p. 000)
clone (p. 000) double infection (p. 000) F plasmid (p. 000)
colony (p. 000) double transformation (p. 000) fertility factor (F) (p. 000)
44200_05_p151-184 3/4/04 10:48 AM Page 177
generalized transduction (p. 000) phage recombination (p. 000) screen (p. 000)
genetic markers (p. 000) plaque (p. 000) selective system (p. 000)
Hfr (p. 000) plasmid (p. 000) specialized transduction (p. 000)
interrupted mating (p. 000) plating (p. 000) temperate phage (p. 000)
lysis (p. 000) prokaryotes (p. 000) terminus (p. 000)
lysogenic (p. 000) prophage (p. 000) transduction (p. 000)
merozygote (p. 000) prototrophic (p. 000) transformation (p.000)
minimal medium (p. 000) R plasmids (p. 000) unselected markers (p. 000)
mixed infection (p. 000) recipient (p. 000) virulent phages (p. 000)
origin (O) (p. 000) resistant mutants (p. 000) viruses (p. 000)
phage (p. 000) rolling circle replication (p. 000) zygotic induction (p. 000)
SOLVED PROBLEMS
1. Suppose that a cell were unable to carry out general- zymes. Therefore, a rec recipient can still inherit genetic
ized recombination (rec). How would this cell be- markers by specialized transduction.
have as a recipient in generalized and in specialized
transduction? First compare each type of transduc- 2. In E. coli, four Hfr strains donate the following ge-
tion and then determine the effect of the rec muta- netic markers, shown in the order donated:
tion on the inheritance of genes by each process.
Strain 1: Q W D M T
Solution Strain 2: A X P T M
Strain 3: B N C A X
Generalized transduction entails the incorporation of
Strain 4: B Q W D M
chromosomal fragments into phage heads, which then
infect recipient strains. Fragments of the chromosome
All these Hfr strains are derived from the same F
are incorporated randomly into phage heads, so any
strain. What is the order of these markers on the cir-
marker on the bacterial host chromosome can be trans-
cular chromosome of the original F?
duced to another strain by generalized transduction. In
contrast, specialized transduction entails the integra-
tion of the phage at a specific point on the chromo- Solution
some and the rare incorporation of chromosomal mark- A two-step approach works well: (1) determine the un-
ers near the integration site into the phage genome. derlying principle and (2) draw a diagram. Here the
Therefore, only those markers that are near the specific principle is clearly that each Hfr strain donates genetic
integration site of the phage on the host chromosome markers from a fixed point on the circular chromo-
can be transduced. some and that the earliest markers are donated with
Markers are inherited by different routes in general- the highest frequency. Because not all markers are do-
ized and specialized transduction. A generalized trans- nated by each Hfr, only the early markers must be do-
ducing phage injects a fragment of the donor chro- nated for each Hfr. Each strain allows us to draw the
mosome into the recipient. This fragment must be incor- following circles:
porated into the recipients chromosome by recombina-
tion, with the use of the recipient recombination sys- B Q
Q
tem. Therefore, a rec recipient will not be able to W W
incorporate fragments of DNA and cannot inherit mark- D B D
ers by generalized transduction. On the other hand, the M M N M
C
major route for the inheritance of markers by special- T
A
XP
T A
X
ized transduction is by integration of the specialized
transducing particle into the host chromosome at the Strain 1 Strain 2 Strain 3 Strain 4
specific phage integration site. This integration, which
sometimes requires an additional wild-type (helper) From this information, we can consolidate each circle
phage, is mediated by a phage-specific enzyme system into one circular linkage map of the order Q, W, D, M, T,
that is independent of the normal recombination en- P, X, A, C, N, B, Q.
44200_05_p151-184 3/4/04 10:48 AM Page 178
3. In an Hfr F cross, leu enters as the first marker, rather than two. For instance, if the order were met, thi,
but the order of the other markers is unknown. If the pur, then met thi pur recombinants would be very
Hfr is wild-type and the F is auxotrophic for each rare. On the other hand, if the order were met, pur, thi,
marker in question, what is the order of the markers then the four-crossover class would be met pur thi.
in a cross where leu recombinants are selected if 27 From the information given in the table, it is clear that
percent are ile, 13 percent are mal, 82 percent are the met pur thi class is the four-crossover class and
thr, and 1 percent are trp? therefore that the gene order met, pur, thi is correct.
Solution c. Refer to the following diagram:
Recall that spontaneous breakage creates a natural gradi- met 15.4 m.u. pur 1.8 m.u. thi
ent of transfer, which makes it less and less likely for a re- Hfr
cipient to receive later and later markers. Because we
met pur thi
have selected for the earliest marker in this cross, the fre-
quency of recombinants is a function of the order of entry
for each marker. Therefore, we can immediately deter- F
mine the order of the genetic markers simply by looking
To compute the distance between met and pur, we
at the percentage of recombinants for any marker among
compute the percentage of met pur thi, which is
the leu recombinants. Because the inheritance of thr is
52/388 15.4 m.u. The distance between pur and thi is,
the highest, this must be the first marker to enter after
similarly, 6/338 1.8 m.u.
leu. The complete order is leu, thr, ile, mal, trp.
5. Compare the mechanism of transfer and inheritance
4. A cross is made between an Hfr that is met thi of the lac genes in crosses with Hfr, F, and F-lac
pur and an F that is met thi pur. Interrupted- strains. How would an F cell that cannot undergo
mating studies show that met enters the recipient normal homologous recombination (rec) behave in
last, so met recombinants are selected on a crosses with each of these three strains? Would the
medium containing supplements that satisfy only cell be able to inherit the lac gene?
the pur and thi requirements. These recombinants
are tested for the presence of the thi and pur al- Solution
leles. The following numbers of individuals are Each of these three strains donates genes by conjugation.
found for each genotype: In the Hfr and F strains, the lac genes on the host chro-
mosome are donated. In the Hfr strain, the F factor is in-
met thi pur 280 tegrated into the chromosome in every cell, so efficient
met thi pur 0 donation of chromosomal markers can occur, particularly
met thi pur 6 if the marker is near the integration site of F and is do-
met thi pur 52 nated early. The F cell population contains a small per-
a. Why was methionine (Met) left out of the selec- centage of Hfr cells, in which F is integrated into the
tion medium? chromosome. These cells are responsible for the gene
transfer displayed by cultures of F cells. In the Hfr- and
b. What is the gene order? F-mediated gene transfer, inheritance requires the incor-
c. What are the map distances in recombination units? poration of a transferred fragment by recombination (re-
Solution call that two crossovers are needed) into the F chromo-
some. Therefore, an F strain that cannot undergo
a. Methionine was left out of the medium to allow recombination cannot inherit donor chromosomal mark-
selection for met recombinants, because met is the last ers even though they are transferred by Hfr strains or Hfr
marker to enter the recipient. The selection for met cells in F strains. The fragment cannot be incorporated
ensures that all the loci that we are considering in the into the chromosome by recombination. Because these
cross will have already entered each recombinant that fragments do not possess the ability to replicate within the
we analyze. F cell, they are rapidly diluted out during cell division.
b. Here it is helpful to diagram the possible gene orders. Unlike Hfr cells, F cells transfer genes carried on the
Because we know that met enters the recipient last, F factor, a process that does not require chromosome
there are only two possible gene orders if the first transfer. In this case, the lac genes are linked to the F
marker enters on the right: met, thi, pur or met, pur, thi. and are transferred with the F at a high efficiency. In the
How can we distinguish between these two orders? For- F cell, no recombination is required, because the F lac
tunately, one of the four possible classes of recombinants strain can replicate and be maintained in the dividing F
requires two additional crossovers. Each possible order cell population. Therefore, the lac genes are inherited
predicts a different class that arises by four crossovers even in a rec strain.
44200_05_p151-184 3/4/04 10:48 AM Page 179
Problems 179
PROBLEMS
BASIC PROBLEMS following numbers of individuals are found for each
1. Describe the state of the F factor in an Hfr, F , and genotype:
F strain. arg bio leu 320
arg bio leu 8
2. How does a culture of F cells transfer markers from arg bio leu 0
the host chromosome to a recipient? arg bio leu 48
3. With respect to gene transfer and integration of the
transferred gene into the recipient genome compare a. What is the gene order?
the following: b. What are the map distances in recombination
a. Hfr crosses by conjugation and generalized trans- percentages?
duction. 9. Linkage maps in an Hfr bacterial strain are calcu-
b. F derivatives such as F lac and specialized trans- lated in units of minutes (the number of minutes
duction. between genes indicates the length of time it takes
4. Why is generalized transduction able to transfer any for the second gene to follow the first in conjuga-
gene, but specialized transduction is restricted to tion). In making such maps, microbial geneticists as-
only a small set? sume that the bacterial chromosome is transferred
from Hfr to F at a constant rate. Thus, two genes
5. A microbial geneticist isolates a new mutation in separated by 10 minutes near the origin end are
E. coli and wishes to map its chromosomal location. assumed to be the same physical distance apart as
She uses interrupted-mating experiments with Hfr two genes separated by 10 minutes near the F-
strains and generalized-transduction experiments attachment end. Suggest a critical experiment to
with phage P1. Explain why each technique, by test the validity of this assumption.
itself, is insufficient for accurate mapping.
10. A particular Hfr strain normally transmits the pro
6. In E. coli, four Hfr strains donate the following marker as the last one in conjugation. In a cross of
markers, shown in the order donated: this strain with an F strain, some pro recombi-
nants are recovered early in the mating process.
Strain 1: M Z X W C
When these pro cells are mixed with F cells, the
Strain 2: L A N C W
majority of the F cells are converted into pro cells
Strain 3: A L B R U
that also carry the F factor. Explain these results.
Strain 4: Z M U R B
11. F strains in E. coli are derived from Hfr strains. In
All these Hfr strains are derived from the same F
some cases, these F strains show a high rate of inte-
strain. What is the order of these markers on the cir-
gration back into the bacterial chromosome of a sec-
cular chromosome of the original F?
ond strain. Furthermore, the site of integration is
7. You are given two strains of E. coli. The Hfr strain is often the same site that the sex factor occupied in
arg ala glu pro leu T s; the F strain is arg ala the original Hfr strain (before production of the F
glu pro leu T r. The markers are all nutritional ex- strains). Explain these results.
cept T, which determines sensitivity or resistance to
phage T1. The order of entry is as given, with arg 12. You have two E. coli strains, F str r ala and Hfr str s
entering the recipient first and T s last. You find that ala, in which the F factor is inserted close to ala.
the F strain dies when exposed to penicillin (pens), Devise a screening test to detect strains carrying F ala.
but the Hfr strain does not (penr). How would you 13. Five Hfr strains A through E are derived from a sin-
locate the locus for pen on the bacterial chromo- gle F strain of E. coli. The following chart shows the
some with respect to arg, ala, glu, pro, and leu? For- entry times of the first five markers into an F strain
mulate your answer in logical, well-explained steps when each is used in an interrupted-conjugation
and draw explicit diagrams where possible. experiment:
8. A cross is made between two E. coli strains: Hfr A B C D E
arg bio leu F arg bio leu. Interrupted-
mal (1) ade (13) pro (3) pro (10) his (7)
www.
mating studies show that arg enters the recipient str s (11) his (28) met (29) gal (16) gal (17)
last, so arg recombinants are selected on a medium ser (16) gal (38) xyl (32) his (26) pro (23)
ade (36) pro (44) mal (37) ade (41) met (49)
containing bio and leu only. These recombinants are
his (51) met (70) str s (47) ser (61) xyl (52)
tested for the presence of bio and leu. The
44200_05_p151-184 3/4/04 10:48 AM Page 180
a. Draw a map of the F strain, indicating the (Problem 16 is reprinted with the permission of Macmillan
positions of all genes and their distances apart in Publishing Co., Inc., from Monroe W. Strickberger, Genetics.
minutes. Copyright 1968 by Monroe W. Strickberger.)
b. Show the insertion point and orientation of the F 17. With the use of P22 as a generalized transducing
plasmid in each Hfr strain. phage grown on a pur pro his bacterial donor, a
c. In the use of each of these Hfr strains, state recipient strain of genotype pur pro his is in-
which gene you would select to obtain the highest fected and incubated. Afterward, transductants for
proportion of Hfr exconjugants. pur, pro, and his are selected individually in ex-
periments I, II, and III, respectively.
14. Streptococcus pneumoniae cells of genotype str s mtl
are transformed by donor DNA of genotype str r a. What media are used for these selection experi-
mtl and (in a separate experiment) by a mixture of ments?
two DNAs with genotypes str r mtl and str s mtl. b. The transductants are examined for the presence of
The accompanying table shows the results. unselected donor markers, with the following results:
a. What does the first line of the table tell you? What is the order of the bacterial genes?
Why? c. Which two genes are closest together?
b. What does the second line of the table tell you? d. On the basis of the order that you proposed in
Why? part c, explain the relative proportions of genotypes
observed in experiment II.
15. Recall that in Chapter 4 we considered the possibil-
(Problem 17 is from D. Freifelder, Molecular Biology and
ity that a crossover event may affect the likelihood
Biochemistry. Copyright 1978 by W. H. Freeman and
of another crossover. In the bacteriophage T4, gene
Company, New York.)
a is 1.0 m.u. from gene b, which is 0.2 m.u. from
gene c. The gene order is a, b, c. In a recombination 18. Although most -mediated gal transductants are
experiment, you recover five double crossovers be- inducible lysogens, a small percentage of these trans-
tween a and c from 100,000 progeny viruses. Is it ductants in fact are not lysogens (that is, they con-
correct to conclude that interference is negative? tain no integrated ). Control experiments show
Explain your answer. that these transductants are not produced by muta-
tion. What is the likely origin of these types?
16. You have infected E. coli cells with two strains of T4
virus. One strain is minute (m), rapid-lysis (r), and 19. An ade arg cys his leu pro bacterial strain is
turbid (tu); the other is wild-type for all three mark- known to be lysogenic for a newly discovered phage,
ers. The lytic products of this infection are plated but the site of the prophage is not known. The bac-
and classified. The resulting 10,342 plaques were terial map is
distributed among eight genotypes, as follows: arg
his
m r tu 3467 m11 520 cys
111 3729 1 r tu 474 leu
mr1 853 1r 172 ade
m 1 tu 162 1 tu 965 pro
a. Determine the linkage distances between m and The lysogenic strain is used as a source of the phage,
r, between r and tu, and between m and tu. and the phages are added to a bacterial strain of geno-
type ade arg cys his leu pro. After a short incu-
b. What linkage order would you suggest for the
bation, samples of these bacteria are plated on six
three genes?
different media, with the supplementations indicated
c. What is the coefficient of coincidence (see Chap- in the table below. The table also shows whether
ter 4) in this cross, and what does it signify? colonies were observed on the various media.
44200_05_p151-184 3/4/04 10:48 AM Page 181
Problems 181
5. Define the terms prototroph and auxotroph. all possible combinations and (after incubation) are
6. Which cultures in this experiment are prototrophic plated to determine the frequency of a b recom-
and which are auxotrophic? binants. The following results are obtained, where
M many recombinants, L low numbers of re-
7. Given some strains of unknown genotype regarding combinants, and 0 no recombinants:
thiamine and proline, how would you test their
genotypes? Give precise experimental details, 1 2 3 4
including equipment.
5 0 M M 0
8. What kinds of chemicals are proline and thiamine? 6 0 M M 0
Does this matter in this experiment? 7 L 0 0 M
9. Draw a schematic diagram showing the full set of 8 0 L L 0
manipulations performed in the experiment.
10. Why do you think the experiment was done? On the basis of these results, assign a sex type (ei-
ther Hfr, F, or F) to each strain.
11. How was it established that pro enters after thi?
Give precise experimental steps. 26. An Hfr strain of genotype a b c d str s is mated
with a female strain of genotype a b c d str r. At
12. In what way does an interrupted-mating experiment
various times, the culture is shaken vigorously to
differ from the experiment described in this
separate mating pairs. The cells are then plated on
problem?
agar of the following three types, where nutrient A
13. What is an exconjugant? How do you think that allows the growth of a cells; nutrient B, of b cells;
exconjugants were obtained? (This might involve nutrient C, of c cells; and nutrient D, of d cells (a
genes not described in this problem.) plus indicates the presence of, and a minus the ab-
14. When the pro gene is said to enter after thi, does it sence of, streptomycin or a nutrient):
mean the pro allele, the pro allele, either, or both?
Agar type Str A B C D
15. What is fully supplemented medium in the
context of this question? 1
2
16. Some exconjugants did not grow on minimal
3
medium. On what medium would they grow?
17. State the types of crossovers that are involved in
Hfr F recombination. How do these crossovers a. What donor genes are being selected on each
differ from crossovers in eukaryotes? type of agar?
b. The table below shows the number of colonies
18. What is a recombination unit in the context of the
on each type of agar for samples taken at various
present analysis? How does it differ from the map
times after the strains are mixed. Use this informa-
units used in eukaryote genetics?
tion to determine the order of genes a, b, and c.
24. A generalized transduction experiment uses a metE
pyrD strain as donor and metE pyrD as recipient. Number of colonies
MetE transductants are selected and then tested for Time
on agar of type
the pyrD allele. The following numbers were of sampling
obtained: (minutes) 1 2 3
0 0 0 0
metE pyrD 857 5 0 0 0
metE pyrD 1 7.5 100 0 0
10 200 0 0
Do these results suggest that these loci are closely 12.5 300 0 75
linked? What other explanations are there for the 15 400 0 150
lone double? 17.5 400 50 225
20 400 100 250
CHALLENGING PROBLEMS 25 400 100 250
25. Four E. coli strains of genotype a b are labeled 1,
2, 3, and 4. Four strains of genotype a b are la- c. From each of the 25-minute plates, 100 colonies
beled 5, 6, 7, and 8. The two genotypes are mixed in are picked and transferred to a dish containing agar
44200_05_p151-184 3/4/04 10:48 AM Page 183
Problems 183
with all the nutrients except D. The numbers of b. What is the probable order of the three tightly
colonies that grow on this medium are 89 for the linked genes?
sample from agar type 1, 51 for the sample from agar (Problem 28 is from Franklin Stahl, The Mechanics of In-
type 2, and 8 for the sample from agar type 3. Using heritance, 2d ed. Copyright 1969, Prentice Hall, Engle-
these data, fit gene d into the sequence of a, b, and c. wood Cliffs, NJ. Reprinted by permission.)
d. At what sampling time would you expect
colonies to first appear on agar containing C and 29. You have two strains of that can lysogenize E. coli;
streptomycin but no A or B? the following figure shows their linkage maps:
(Problem 26 is from D. Freifelder, Molecular Biology
and Biochemistry. Copyright 1978 by W. H. Freeman and Strain X Strain Y
Company.)
c b c b
27. In the cross Hfr aro arg ery str F aro arg
r s
a. One of the genes is obviously quite distant from 31. A generalized transducing phage is used to transduce
the other three, which appear to be tightly (closely) an a b c d e recipient strain of E. coli with an
linked. Which is the distant gene? a b c d e donor. The recipient culture is plated
44200_05_p151-184 3/4/04 10:48 AM Page 184
on various media with the results shown in the table termed tonB that confers resistance to the virulent
below. (Note that a indicates a requirement for A as phage T1:
a nutrient, and so forth.) What can you conclude
about the linkage and order of the genes? att80
tonB
Presence () or They used an F lac plasmid that could not repli-
Compounds added absence () cate at high temperatures in a strain carrying a dele-
to minimal medium of colonies tion of the lac genes. By forcing the cell to remain
lac at high temperatures, the researchers could
CDE select strains in which the plasmid had integrated
BDE into the chromosome, thereby allowing the F lac to
BCE be maintained at high temperatures. By combining
BCD this selection with a simultaneous selection for resis-
ADE tance to T1 phage infection, they found that the
ACE only survivors were cells in which the F lac had in-
ACD tegrated into the tonB locus, as shown in the figure
ABE below.
ABD
ABC F
tonB att80
lac
32. In 1965, Jon Beckwith and Ethan Signer devised a This placed the lac region near the integration site
method of obtaining specialized transducing phages for phage 80. Describe the subsequent steps that
carrying the lac region. They knew that the integra- the researchers must have followed to isolate the
tion site, designated att80, for the temperate phage specialized transducing particles of phage 80 that
80 (a relative of phage ) was located near a gene carried the lac region.