JOURNAL OF STRUCTURAL BIOLOGY 104, 107-111 (1990)

Mechanisms in Microbial Evolution

WERNERARBER

Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Received October 10, 1990

cesses which lead to genetic variation represent im-

Molecular genetic studies with prokaryotic microor- portant biological functions.
ganisms reveal that many different molecular processes
contribute to the formation of spontaneous mutations. MOLECULARMECHANISMSOF
Besides infidelities in DNA replication and the conse- SPONTANEOUSMUTAGENESIS
quences of environmental mutagens, enzyme-mediated Molecular genetic analysis has revealed a multi-
DNA rearrangements bring about important, evolution- tude of mechanisms contributing to the formation of
arily relevant alterations in the genetic information. Par- spontaneous mutations (Table I). Infidelity of the
ticular attention is given in this article to site-specific DNA replication process can be at the origin of nu-
recombination at secondary crossover sites and to the cleotide substitution and other local alterations of
transposition of mobile genetic elements with relaxed DNA sequences. Many environmental mutagens are
target specificity. Besides these diverse processes of ge-
natural, others are man-made, and they affect DNA
nomic mutation the acquisition of genetic information
sequences in various, often quite specific ways. Since
from other organisms plays an uncontested role in micro-
we consider here a mutation to be any alteration in
bial evolution. Enzymes and organelles mediating any of
these mutational processes can be looked at as biological the DNA sequences of a genome, we also count re-
functions acting at the level of populations for the needs combinational DNA rearrangements among the
of biological evolution, rather than to fulfill the needs of spontaneous mutations. To a large extent, DNA re-
individual living organisms. ‘Q Iwo Academic PRSS, IN. arrangements depend on the presence of specific en-
zymes, in particular enzymes for general recombi-
nation, for site-specific recombination, and for trans-
INTRODUCTION position. Finally, the acquisition of genetic
information from other organisms also represents
Biological evolution results from the steady inter- an alteration of the DNA sequences of a genome; it
play between spontaneous mutation and selection, can therefore be counted among the processes of
whereby reproductive and geographical isolation spontaneous mutagenesis. All of these mechanisms
can be counted among the limiting parameters. Se- have been quite well studied with haploid microor-
lection is a function of the living conditions in eco- ganisms, bacteria and their viruses, in which muta-
systems. The living conditions depend both on the tions are rapidly revealed by their phenotypic man-
physicochemical characteristics and on the biologi- ifestation.
cal activities exerted by the organisms present in an The possible consequences of a mutation, i.e., of an
ecosystem. Since these parameters are variable, se- alteration of the nucleotide sequence of the genome,
lection is also variable. A high genetic diversity in- are as follows: (1) A mutation is often lethal, i.e., it
creases the chance that in large populations of or- inhibits further reproduction. (2) A mutation is of-
ganisms some members are able to readily adapt to ten without immediate influence on processes of life.
altered living conditions. While selection tends to Such silent mutations may become relevant for phe-
reduce the genetic diversity, spontaneous mutation notypic manifestation at a later time in combination
counteracts this tendency and it thus represents an with additional mutational changes. (3) Sometimes
important factor for the long-term maintenance of a mutation inhibits life processes only partially, i.e.,
life on the planet under steadily changing living it represents a selective disadvantage. (4) Rather
conditions. rarely, a mutation gives a selective advantage to the
In this contribution, we will focus our attention on organism. These mutations contribute most impor-
the multitude of mechanisms of spontaneous mu- tantly to the evolutionary process in populations.
tagenesis. This will lead us to consider whether pro- We assume that each of the molecular processes

107
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108
TABLE I
Diversity of Mechanisms in Spontaneous Mutagenesis
Infidelity of DNA replication
Nucleotide substitution
Small insertions and deletions
Partial duplications
Environmental mutagens (radiation, chemicals, etc.)
Recombinational DNA rearrangements
By general recombination at homologous DNA segments
By site-specific recombination, particularly at secondary
crossover sites
Transpositional recombination
Others (gyrase-mediated and other “illegitimate” processes)
Acquisition of genetic information from other organisms
listed in Table I contributes to the production of
spontaneous mutants for each species with a char-
acteristic efficiency. Thereby, DNA rearrangements
may probably quite often produce lethal mutants,
while nucleotide substitutions may less frequently
lead to lethality. Studies on the cause of spontane-
ous lethal mutations at the molecular level could
clarify the validity of these presumptions. As we will
show below, this is possible with a particular exper-
imental approach. If a relatively high proportion of
spontaneous mutations results in lethality, one has
to postulate that the overall efficiency of spontane-
ous mutagenesis should be significantly lower than
to produce one mutation per genome and generation,
otherwise the survival of the concerned strain might
not be assured.
MUTANTS LETHALLY AFFECTED
BACTERIOPHAGE REPRODUCTION CAN BE
PROPAGATED AND INVESTIGATED IN THE
PROPHAGE CONDITION
In my graduate work done at the University of
Geneva in the 1950s I explored the functional de-
fects resulting from mutations that the bacterio-
phage lambda [Al genome had suffered in its pro-
phage state. The genomes of such mutants prop-
agate perfectly well in the prophage condition.
However, upon induction of the lytic cycle of repro-
duction, any mutation in one of the genes essential
for vegetative growth inhibits the production of in-
fective viral particles. This type of experiment al-
lows one therefore to study lethal mutations. In the
1950s it was already possible to identify functional
defects by analyzing in the electron microscope the
kind of structural products still resulting from the
induction of a defective lysogen. For example, some
of the mutants studied produced only phage heads
but no tails. Other mutants produced both heads and
tails, but still could not assemble infective phage
particles (Arber and Kellenberger, 1958). At the
time when these experiments were done, it was of
course not yet possible to investigate the kind of
WERNER ARBER
sequence alteration that the defective A genome had
suffered. But this has since become possible with the
techniques of molecular genetic analysis. Molecular
studies have, for example, revealed that a majority
of spontaneous mutations carried in populations of
the Pl prophage and affecting the vegetative cycle of
reproduction of phage Pl were due to transposition
of IS elements (Arber et al., 1978; Sengstag and Ar-
ber, 1983).
In the summer of 1956, Jean Weigle brought to
my attention two publications by Morse, Lederberg,
and Lederberg (1956a,b) who had just discovered the
phenomenon of specialized transduction with phage
A. When we observed in the electron microscope in-
duced lysates from bacteria singly lysogenic for the
h gal transducing phage, no phage-like structures
whatsoever could be detected. By genetic comple-
mentation analysis, it was then found that h gal
isolates had long deletions removing a number of
late phage genes essential for tail and head morpho-
genesis (Arber, 1958; Arber et al., 1957). Obviously,
these mutants could be considered as substitutions
in which some of the phage genes were left behind
upon excision of the prophage from the host chromo-
some. Instead, some host genes located adjacent to
the A prophage in the bacterical chromosome had
substituted for the lost A genes (Campbell, 1962).
This exciting finding clearly pointed to the mecha-
nism by which phage A can serve under natural con-
ditions as gene vector in the transfer of host genes
from one bacterial strain to another.
ACQUISITION OF GENETIC INFORMATION IS A
IN WIDELY USED STRATEGY AMONG BACTERIA
Besides bacteriophage-mediated transduction,
other mechanisms of gene transfer among bacteria
include transformation, conjugation and, at least to
some extent, also cell fusion. It is well-known that in
conjugation, a conjugative plasmid serves as natural
gene vector. In contrast, in the transformation pro-
cess, DNA fragments of the donor organism are
taken up by the recipient cell without the interme-
diate of a gene vector. In order to become relevant
for evolution, the DNA taken up by the recipient cell
in any of the transfer processes mentioned must be-
come part of the genome. Depending on the system,
this can be brought about by a recombinational pro-
cess with the chromosome or by the establishment of
an independent replicon.
NATURAL BARRIERS TO GENE ACQUISITION
When the natural gene transfer processes were
studied in bacteria, it soon became evident that
other factors than the one just mentioned, i.e., the
need to ensure propagation, also represent limiting
factors to gene acquisition and thus contribute to
genetic isolation. Surface incompatibility seriously
 MECHANISMS
inhibits the range of genetic exchange in conjuga-
tion as well as in phage-mediated transduction. The
surface composition of recipient cells plays also a
role in the efficiency of DNA uptake in transforma-
tion.
Restriction-modification systems, which are found
in a wide range of bacterial strains, play in my mind
a double role in DNA acquisition under natural con-
ditions. Restriction reduces to a large extent the ef-
ficiency of gene acquisition by any of the gene trans-
fer processes (Arber, 1965). Invading foreign DNA is
recognized as such by restriction enzymes and is
fragmented into relatively small pieces. Although
the resulting DNA fragments become quite rapidly
exonucleolytically degraded, the genetic informa-
tion on a DNA fragment may become integrated
with some low probability into the genome by any of
the recombination pathways exerted in the recipient
cell. Thus restriction-modification systems repre-
sent on the one hand a serious limitation to the ac-
quisition strategy, but they favor on the other hand
acquisition to occur in small steps. The acquisition
of only one or a few genes--or even the information
for one functional domain-can ensure that the re-
cipient cell can better cope with the acquired genetic
information than if a large DNA segment of a donor
genome were acquired. In the latter case, the func-
tional harmony of the recipient organism might of-
ten be disturbed. Functional compatibility indeed
belongs also to the factors limiting gene acquisition.
IN MICROBIAL EVOLUTION 109
cerned tail fiber gene has thus a constant part lo-
cated outside of the invertible DNA segment and a
variable part encoded on the invertible DNA. De-
pending on the orientation of the invertible DNA
segment, Pl phage particles have one or the other
type of tail fibers. Since the biological function of tail
fibers resides in the recognition of host surface ele-
ments, DNA inversion influences the host range of
the phage.
The Cin DNA inversion enzyme occasionally acts
at secondary crossover sites, whereby the inversion
process can bring about novel DNA arrangements
(Iida et al., 1984; Iida and Hiestand-Nauer, 1986).
This is exemplified in Fig. 1. In this experiment car-
ried out by Iida and Hiestand-Nauer (1987) a plas-
mid which carried only one efficiently used crossover
site was constructed. Selection was made for rare
mutational events in which an open reading frame
coding for kanamycin resistance had spontaneously
been provided with a promoter of transcription.
Most often, the expression of kanamycin resistance
could be attributed to DNA inversion occurring be-
tween the consensus-like crossover site gwC* and one
of many different secondary crossover sites. Interest-
ingly, sequence analysis of these secondary cross-
over sites revealed a very wide divergence from the
consensus sequence. Considering the preserved nu-
cleotides still reflecting the consensus, no general
rule could be extrapolated for the criteria to serve as
secondary crossover site. We can thus conclude that

SITE-SPECIFIC RECOMBINATION AT SECONDARY

CROSSOVER SITES IS OF IMMINENT
EVOLUTIONARYRELEVANCE

It is well known that in the lysogenization process

the phage A genome becomes integrated most fre-
quently into a specific chromosomal integration site. ‘$I\’ ‘;7.8_; :. J?‘TLG.,‘ ;: (- -: &(: dA&
With lower frequencies, however, secondary inte- ;i1 <t\*cJ391 “a! c?.cct :t gq !S :&.I‘El :c f;gaqr i .)
tc yu*Q3’x ciZajzr g&t t.g; s. -‘. :l.:ccm;:r ,?aggc~ c.1
gration sites can be used (Weisberg and Landy, ii :-lx*Q1x’) i‘g~ct,Jst~ .cag & c:c;dil:~I.t~gc: i. I
1983). Upon the already discussed excision of the A d, gl\*Q3R7 TTggcTy: cli‘q:: yi (-:;‘!‘i~!.c~t :gyg~ ( >)
c/ !2lu*@Xh Tmdt gmgq TVt ‘&gc~:bcl,jg&t. ( I
prophage from the chromosome, the cutting out pro- f, glu*~3xs :3qCctTCtBtq a a~gTl’c~~c;c’lJtc (.‘I
cess is again most often highly site-specific. The pro- 61 git*Q3X3 .:ctct-~tt.cg:J .bJ :x~a3’:r!~igct: ( .! j
duction of A gal transducing phage due to excision at !I , i!iY”Q3XXB ,:~~macJqtq&3 7 !*A.’ ~.‘--T.~J”l::.i~~i i ‘I
I/ gl\‘cJ30.? ! d 5 ~r 5 I a.vLx~-
-C~ 1 !&? _~I ‘I1 1 1<I i t :: I’i: (P!
other sites occurs about 10 000 times less frequently h, y,‘()“‘1 ^ <-i-JyL;‘- -~._.&
- 2, I. ? *~_~~ ~r;-: ,’ ..-. -. 411“_:. I i !
than the excision at the junctions between chromo- FIG. 1. Secondary crossover sites occasionally used by the
some and prophage. The illegitimate recombination DNA inversion enzyme Cin. Their locations on plasmid pSHI383
leading to the formation of A gal is likely due to the are shown above and their nucleotide sequences below. The data
interaction of site-specific recombinases at second- are taken from Iida and Hiestand-Nauer (1987). On plasmid
pSHI383 the tin. gene is under the control of the lacUV5 pro-
ary crossover sites.
moter. The intact km reading frame does not provide resistance
This phenomenon is also seen in site-specific DNA to kanamycin, since this gene has no promoter. However, either
inversion. The Gin recombinase is encoded by a gene the lacUV5 or the PI promoter can control the expression of km
of the bacteriophage Pl (Iida, 1984). This site- after spontaneous DNA inversion has occurred between the gir*
specific recombinase usually brings about the inver- crossover site and one of the secondary crossover sites labeled a to
f and g to k, respectively. In the DNA sequences the underlined
sion of the nucleotide sequences carried between two capital letters correspond to the consensus sequence for crossover
crossover sites, one of which is located in the reading sites used with high efficiency by Cin recombinase and its rela-
frame for a Pl tail fiber protein. This inversion pro- tives. Numbers in parentheses indicate how many times each
cess is repeated every few generations. The con- particular sequence was independently used in DNA inversion.
110
DNA inversion systems can bring about a wide va-
riety of DNA rearrangements. Those studied in Fig.
1 represent operon fusions. The already discussed
DNA inversion occurring in wild type Pl DNA
brings about a gene fusion. In analogy, gene fusions
are also expected to result from recombination at
secondary crossover sites. It is thus clear that site-
specific inversion represents a potent means for mi-
crobial populations to produce a large number of dif-
ferent DNA arrangements without any loss of nu-
cleotides. Under natural selective pressure any such
new DNA sequence will be tried out for its func-
tional relevance. Since the production of a novel
DNA sequence by this process is quite a rare event,
it does not matter to the cell population that newly
arranged genomes might often lead to lethality. The
possibility that a small number of rearranged ge-
nomes might represent a selective advantage leads
me to postulate that DNA inversion systems might
primarily have the function to provide a wide vari-
ety of new DNA arrangements in large microbial
populations. In this sense, the DNA inversion sys-
tem can be considered to function according to the
principle of a variety generator by its action on a
very large number of secondary crossover sites.
SEVERAL ENZYMES ACT ON DNA AS
VARIETY GENERATORS
Interestingly, other enzymes interacting with the
DNA also function according to the variety genera-
tor principle. Among these are type I restriction en-
zymes. These cut long DNA molecules almost ran-
domly, although the distinction between foreign and
own DNA is made at specific nucleotide sequences
(Arber, 1974). DNA rearrangement by transposition
also follows the variety generator principle. Indeed,
different transposition systems show a more or less
relaxed target specificity, so that a wide variety of
different DNA arrangements can result upon trans-
position. The mobile genetic element IS2, for exam-
ple, displays a regional target specificity, but within
the so-called hot target regions many different inte-
gration sites are used, and IS2 can also transpose to
various sequences outside of hot target regions
(Sengstag and Arber, 1983, 1987). Other IS ele-
ments, such as IS30, insert with high preference into
a particular site on the target DNA molecule. How-
ever, with lower efficiency IS30 can also transpose
elsewhere and at a variety of different sites (Caspers
et al., 1984).
SPECIFIC BIOLOGICAL FUNCTIONS PROMOTE
MICROBIAL EVOLUTION
With regard to the evolutionary process, one can
defend the idea that evolution is the result of accu-
mulated errors. In contrast, one may also postulate
WERNER ARBER
that living organisms express specific biological
functions which promote evolutionary changes. I
personally favor the latter interpretation. Indeed, I
consider biological evolution an absolutely impor-
tant process for the long-term maintenance of life on
this planet. I therefore conclude that some of the
enzymes that promote a wide variety of DNA rear-
rangements may indeed perform biological func-
tions that promote evolution and that these enzymes
had been selected for by these functions carried out
at the population level. One may also consider
whether promoters of lateral gene transfer, such as
viruses and plasmids, might have their main func-
tional role in the evolutionary process.
This conceptional point of view leads to the follow-
ing conclusion: One can subdivide biological func-
tions into two categories. On the one hand, many
biological functions meet the needs of individual
lives. They must act at the right time and with the
right efficiency in each single member of a popula-
tion. On the other hand, factors promoting genetic
diversity exert their action only at the population
level. Of course, each individual member of a popu-
lation will carry the genes for the evolutionarily rel-
evant processes. But only a small proportion of in-
dividuals should experience their activities. Recom-
bination enzymes bringing about novel DNA
rearrangements in only a small proportion of a pop-
ulation are good examples for such biological func-
tions. Of course, the proposed classification is some-
what arbitrary, and it is clear that some gene prod-
ucts may serve both purposes and fulfill the needs of
individual lives as well as those of biological evolu-
tion in large populations. DNA ligase might be a
good example for such biological functions. Still one
may wonder at what level the selective value might
be. In the case of DNA inversion systems, I person-
ally consider the selective value of activities gener-
ating sequence varieties as more important than
that of the more readily seen turning back and forth
of a particular DNA segment located between two
consensus crossover sites.
The author expresses his deep gratitude to Eduard Kellen-
berger for originally stimulating research on defective prophages
and for his continued interest and appreciated encouragement
throughout the decades of research on the many facets of micro-
bial evolution.
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