You are on page 1of 10

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 2004531918Review ArticleProphage–chromosome interactionC. Canchaya, G. Fournous and H.

Brüssow

Molecular Microbiology (2004) 53(1), 9–18 doi:10.1111/j.1365-2958.2004.04113.x

MicroReview

The impact of prophages on bacterial chromosomes

Carlos Canchaya,† Ghislain Fournous† and Introduction


Harald Brüssow*
Microbial genomics revealed that, in some bacteria, sub-
Nestlé Research Centre, Nutrition and Health
stantial amounts of the bacterial DNA is in fact prophage
Department/Functional Microbiology Group, CH-1000
DNA (Canchaya et al., 2003; Casjens, 2003). It also
Lausanne 26 Vers-chez-les-Blanc, Switzerland.
became increasingly clear that prophage DNA has played
an important role in the evolution of bacterial pathogenic-
Summary ity (Boyd and Brüssow, 2002). These data have changed
our understanding of phage–bacterium interaction from a
Prophages were automatically localized in se-
simple parasite–host relationship into a two-way co-
quenced bacterial genomes by a simple semantic
evolution of viral and bacterial genomes. Here, we provide
script leading to the identification of 190 prophages
a short overview on the impact of prophage integration on
in 115 investigated genomes. The distribution of
bacterial genome structure and diversification.
prophages with respect to presence or absence in a
given bacterial species, the location and orientation
of the prophages on the replichore was not Methodological problems of prophage identification
homogeneous. In bacterial pathogens, prophages
Prophage identification is not an exact science, but a
are particularly prominent. They frequently encoded
labour-intensive, empirical approach that needs a lot of
virulence genes and were major contributors to the
insight (Casjens, 2003). To keep pace with the increasing
genetic individuality of the strains. However, some
number of sequenced bacterial genomes, a simple script
commensal and free-living bacteria also showed
was written in our laboratory (Fournous, 2003) that trans-
prominent prophage contributions to the bacterial
forms the GenBank file of a bacterial genome into a FASTA
genomes. Lysogens containing multiple sequence-
file of all its open reading frames (ORFs). A BLASTX search
related prophages can experience rearrangements
is then conducted with each individual ORF, and the out-
of the bacterial genome across prophages, leading
put of the significant database matches is searched by a
to prophages with new gene constellations. Transfer
semantic program for its annotations using positive (e.g.
RNA genes are the preferred chromosomal integra-
phage, integrase, tail, capsid, terminase, portal) and neg-
tion sites, and a number of prophages also carry
ative (e.g. macrophage, transposase, transposon, inser-
tRNA genes. Prophage integration into protein cod-
tion) keywords. The hits are plotted for each ORF position
ing sequences can lead to either gene disruption or
of the genome, and a further script transforms this hit list
new proteins. The phage repressor, immunity and
into prophage genomes by performing a neighbour anal-
lysogenic conversion genes are frequently tran-
ysis for further phage-like genes around each individual
scribed from the prophage. The expression of the
hit. An example of the automatic data output is shown for
latter is sometimes integrated into control circuits
Salmonella enterica serovar Typhi strain CT-18. It shows
linking prophages, the lysogenic bacterium and its
all phage hits (ticks on the outer circle) and the prophage
animal host. Prophages are apparently as easily
identification by neighbour analysis (green bars) (Fig. 1A).
acquired as they are lost from the bacterial chromo-
Differences between the automatic and manual annota-
some. Fixation of prophage genes seems to be
tions by Casjens (2003) shown in the second circle were
restricted to those with functions that have been co-
minimal and concern only remotely phage-like elements
opted by the bacterial host.
(Sti5, Sti6; Fig. 1A, centre).
The maps of the candidate prophage elements are
Accepted 27 February, 2004. *For correspondence. E-mail shown in Fig. 1A. Sti1 and Sti4 are clearly lambda-like
harald.bruessow@rdls.nestle.com; Tel. (+41) 21 785 8676; Fax (+41)
21 785 8544. †These authors contributed equally to the database prophages in their genetic organization, but not in their
mining. sequence; the structural genes resemble a Photorhabdus
© 2004 Blackwell Publishing Ltd
10 C. Canchaya, G. Fournous and H. Brüssow
Fig. 1. Prophage distribution.
A. Salmonella enterica serovar Typhi strain
A ori CT18 and its prophages. The two outer circles
represent the circular genome map. Prophages
Sti10
identified by Casjens (2003) are indicated by
Sti9 red boxes. The outermost ring displays the
computer output of our prophage detection pro-
gram (Fournous, 2003). Each tick is a potential
phage hit. Prophages identified by automatic
neighbour analysis in our program are marked
I R1 by a small green box. The gene maps of the
1: candidate prophage elements are displayed
I 2 T R
4: within the circle with their Sti number as identi-
3 4T fier; they are not to scale. The prophage pro-
3: Sti1 teins are coloured according to putative
I 5 R
8: function in the top five maps representing
I 5 6 lambda- (1, 4), Mu- (3) and P2-like (8, 9) proph-
9: ages. The colour code is as follows: red, lysog-
7
Sti8 10:
8 Sti2 eny; violet, lysis; green, head; brown, head-
2: to-tail; blue, tail; mauve, tail fibre; black,
4: 9 1 lysogenic conversion; orange, DNA replication;
5: yellow, transcriptional regulation. In the P4-like
T
6: (10) and prophage remnants (bottom five
R Sti3 maps), phage-related genes are coloured red,
7:
and candidate lysogenic conversion genes are
in black. The following candidate lysogenic con-
version genes were identified: 1, msgA; 2,
Sti4 enterohaemolysin-1; 3, enterohaemolysin-2; 4,
Sti7 type II secretion; 5, restriction–modification; 6,
sopE; 7, protein kinase/phosphatase; 8, pertus-
Sti6 Sti5 sis-like toxin subunits A and B; 9, sspH. Recom-
ter binases are identified with I (integrase), T
(transposase) and R (invertase).
B. Location of prophages from 115 investigated
genomes projected on an idealized replichore
of a bacterial genome (ori: origin, ter: terminus).
C. The ordinate gives the number n of prokary-
ori B C otic genomes, the abscissa the number p of
prophages per genome. The height of the bars
gives the number of genomes showing the indi-
n cated number of prophages.

ter P

prophage. Sti3 is a Mu-like whereas Sti8 and Sti9 are P2- this typhoid fever-causing Salmonella serovar (see
like prophages. The well-known gene map of these three Fig. 1A legend for annotations). Sti10 resembles a P4-like
phage types allowed the tentative identification of morons prophage and also contains a potential moron (Fig. 1A).
(extra non-phage genes inserted into the genomes of Sti7 represents a tail gene cluster from a P2-like phage
temperate phages) (Juhala et al., 2000). Notably, the flanked by a pin invertase. The presence of recombinases
morons encode important candidate virulence factors for (integrase, transposase, invertase) points to the mobile
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18
Prophage–chromosome interaction 11
character of these DNA elements (Fig. 1A). In contrast, 2002a; Nakagawa et al., 2003; see also the circled proph-
Sti2 and 5 show isolated phage genes that flank pertussis- age in Fig. 2B). This strong bias in phage orientation is
like toxin genes (Sti2) and Salmonella serovar Typhimu- still unexplained, although avoidance of RNA and DNA
rium virulence genes (sspH and msgA) (Sti5). Sti5 and 6 polymerase collision and interference with the normal
were identified by a manual search (Casjens, 2003), but functioning of terminus functions (dif site) have been pro-
not by the automatic program (Fig. 1A). On the basis of posed (Campbell, 2002).
theoretical models, prophages are predicted to decay by
deletion of phage genes resulting in prophage remnants.
Prophages contribute to the genetic individuality of a
In contrast, genes conferring a selective advantage such
bacterial strain
as a moron should be retained (Lawrence et al., 2003).
Sti5 fits this prediction, but its prophage derivation must The role of mobile DNA in the diversification of bacterial
await the discovery of related, but less decayed prophage genomes becomes apparent when the sequenced
elements. genomes from two different strains of the same species
are aligned in a dot plot. For example, the alignment of
two S. agalactiae serotypes showed about a dozen small
Distribution and orientation of prophages
gaps (Fig. 2A). The gaps corresponded nearly exclusively
One hundred and ninety prophages identified by the pro- to mobile DNA; bioinformatic analysis revealed integrative
gram in 115 sequenced prokaryotic genomes were pro- plasmids, transposons and two prophages. Ten gaps
jected on a hypothetical replichore (one half of the showed an atypical nucleotide composition suggesting
chromosome) (Fig. 1B). The position of the prophages is lateral gene transfer, and eight of the genomic islands
given as a percentage of the distance from ori (origin) to were associated with integrases/transposases (Glaser
ter (terminus of replication) regardless of which half (left et al., 2002). The variable genome parts defined by com-
or right) of the chromosome the prophages were identified parative genomics (Glaser et al., 2002), dot plot alignment
on. The representation does not take account of the vari- and microarray hybridization (Tettelin et al., 2002) showed
able length of the different bacterial replichores. Overall, excellent concordance. The relative contribution of proph-
the prophages were relatively evenly distributed, but the ages to the strain-specific DNA varied sometimes for
density of the prophages was greater near ter than near strains from the same species. In some Staphylococcus
ori. Approximately half the sequenced prokaryotic aureus strain comparisons, prophages were the major
genomes do not contain either prophages or well-defined contributors to variability (Fig. 2B) (Kuroda et al., 2001),
remnants (Fig. 1C). This group contains all sequenced whereas in other comparisons, they competed with
Archaea and most intracellular eubacterial pathogens. It genomic islands and transposons for this role (Baba et al.,
has been argued that the latter have undergone recent 2002). An extreme case is presented by Streptococcus
genome contractions in which all non-essential genes for pyogenes in which all major gaps in the alignment of
the intracellular niche have been lost. Prophages have, different M serotypes could be traced to prophage inte-
however, been observed in insect endosymbionts (phage gration events (Smoot et al., 2002a). As all S. pyogenes
APSE-1 and Wolbachia prophages), suggesting that intra- and many S. aureus prophages encode proven or sus-
cellular bacteria can harbour phages and prophages. A pected virulence factors, the prophage-imposed diversity
quarter of all genomes contain one or two prophages. between the strains might be of clinical relevance (Beres
Approximately one-tenth of the genomes contributed the et al., 2002).
majority of the prophage sequences (Fig. 1C). Bacteria The role of prophages in the individuality of the
containing six or more prophages in their genome are strains is not restricted to Gram-positive bacteria, but
mainly pathogens, with the notable exception of Lactococ- can also be seen in Gram-negative bacteria. Two Sal-
cus lactis, an organism used in cheese fermentation and monella Typhi strains could be aligned over the entire
under extreme pressure from bacteriophages. The com- genome when allowing for a large chromosomal inver-
parisons of genomes from the same species suggest a sion across two rRNA gene clusters. From 113 ORFs
stochastic process: prophages might be present or absent specific for one or other strain, 76 were prophage genes
(e.g. Streptococcus agalactiae) (Fig. 2A) or different (Deng et al., 2002). In a Salmonella serovar Typhi and
strains within a species may contain one, two or three Typhimurium comparison, two chromosomal inversions
prophages (e.g. Staphylococcus aureus) (Fig. 2B). and 12 larger alignment gaps were identified. Nine of
Prophages showed a preferred orientation for integra- the gaps can be traced to prophages or prophage rem-
tion: the structural genes pointed mostly in the direction nants. The gaps represent either prophage insertion/
of the majority of the surrounding bacterial genes. When deletion events or prophage replacements at a given
prophages changed position from the right to the left rep- chromosomal position. Escherichia coli can serve as a
lichore, they also changed their orientation (Smoot et al., dramatic example in which half the 1 Mb DNA that dis-
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18
12 C. Canchaya, G. Fournous and H. Brüssow

A Fig. 2. Prophages contribute to the individuality of bacterial strains.


A. Dot plot comparison of the DNA sequences from Streptococcus
2000000 agalactiae strains 2603V/R (vertical) and NEM316 (horizontal). The
SA2 red boxes identify mobile DNA elements characterized by compara-
tive genomics (Glaser et al., 2002) and microarray analysis (Tettelin
et al., 2002). The gaps in the alignment are located by thin horizontal
and vertical lines. The numbers are bp positions. The two prophages
are annotated with SA1 and SA2. The dot plot was done with the
DOTTUP program (http://www.emboss.org/), using the direct and
2603V/R

reverse sequence; word size was 15, output format was POSTSCRIPT,
Tn916
the program was run in the direct and reverse direction and the figures
were combined.
B. Dot plot of the DNA sequence alignment from the Staphylococcus
SA1 aureus strains MSSA476 and NCTC 8325. Prophages are annotated
as red boxes. A prophage that changed the replichore and the orien-
tation is circled in red. The MSSA476 sequencing data were produced
by the Microbial Sequencing group at the Sanger Institute and can
be obtained from ftp://ftp.sanger.uk/pub/sa/ (NCBI accession number
NC_002953). The NCTC8325 sequencing data were produced by the
Staphylococcus aureus Genome Sequencing Project of J. Iandolo at
Streptooccus agalactiae NEM316 the University of Oklahoma Health Sciences Center (NCBI accession
number NC_002954).
B C. Microarray analysis in the gut commensal Lactobacillus johnsonii.
Outer circle: eight L. johnsonii strains were hybridized against the
2500000
reference strain NCC533. Inner circle: eight different Lactobacillus
species were hybridized against the NCC533 strain (ring 1): L. gas-
seri ATCC19992 and DSM20234 (2, 3), L. helveticus CNRZ303 (4),
L. crispatus DSM20584 (5), L. gallinarum ATCC33199 (6), L. amylo-
2

vorus DSM20531 (7), L. acidophilus ATCC4356 (8), L. reuteri


DSM20016 (9) and L. plantarum ATCC14917 (10). Red, blue and
MSSA476

black segments indicate regions that do or not do cross-hybridize with


NCC533 and that are not represented on the microarray respectively.
The outermost thin circle locates prophages and prophage remnants
(orange boxes), integrase (red) and transposase (green) genes. For
1

details, see Ventura et al. (2003a).

tinguish E. coli O157:H7 from K-12 are accounted for by


prophage DNA (Ohnishi et al., 2001). Interestingly,
SCC
prophage comparisons between strains sharing a very
1 23 recent common ancestor (E. coli O157 Sakai and
Staphylococcus aureus NCTC 8325 EDL933 or the two M3 S. pyogenes strains) already
showed modular exchange reactions (Makino et al.,
C 1999) (Fig. 3B, two tail fibre genes in P5 versus 315.2
ori
comparison), suggesting that prophages are a highly
dynamic part of the bacterial genomes.
Even in the comparisons of some closely related sister
species such as Listeria innocua and L. monocytogenes
(Glaser et al., 2001) or Lactobacillus johnsonii and L. gas-
seri (Pridmore et al., 2004; T. R. Klaenhammer and F.
Desiere, unpublished), prophages account for a substan-
tial part of the major alignment gaps.

DNA–DNA microarray analysis

Microarray analysis demonstrated that, in Salmonella


serovar Typhimurium strains, genetic differences were
essentially limited to prophage content (Porwollik et al.,
2002; Chan et al., 2003). The same was true for S.
pyogenes (Smoot et al., 2002b). Vibrio cholerae strains
recovered as far back as 1910 demonstrated only 1%
difference between the strains (Dziejman et al., 2002).
Lactobacillus johnsonii The prepandemic clinical isolates and an environmen-
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18
Prophage–chromosome interaction 13
tal non-toxigenic O1 El Tor isolate lacked the CTX Prophages as target regions for chromosomal
prophage encoding the cholera toxin CT, identifying rearrangement
this prophage as a major determinant of genetic vari-
ability. Microarray analysis showed that lateral gene It is not rare that different prophages from the same
transfer has played a fundamental role in the diversifi- lysogen share DNA sequence identity. As these regions
cation of S. aureus: up to 22% of the genome com- are targets for homologous recombination, it was pre-
prised variable genetic material. Prophages comprised dicted that prophages mediate rearrangements of bacte-
17% of the total amount of 250 kb mobile and variable rial chromosomes (Brüssow and Hendrix, 2002). Several
DNA (Fitzgerald et al., 2001). As prophages from bac- genome alignments support this prediction. For example,
terial pathogens frequently encode virulence factors, a Japanese S. pyogenes M3 strain differed from an Amer-
one could suspect that the analysis of pathogenic bac- ican M3 isolate mainly by two sequential DNA inversions
teria overestimates the contribution of prophages to (Nakagawa et al., 2003). One inversion occurred across
the genetic individuality of a given strain. However, two prophages (Fig. 3A). Genomics analysis suggested
similar data were obtained with the gut commensal that the cross-over point was located in the lysis modules.
Lactobacillus johnsonii. Hybridization of eight molecu- As the lysogenic conversion genes from S. pyogenes
larly distinct strains of L. johnsonii revealed that the prophages are encoded downstream of the lysis genes,
prophage DNA contributed approximately half the the cross-over results in a reshuffling of virulence genes
strain-specific DNA of the reference strain (Fig. 2C) between prophages (Fig. 3B). This might allow a wider
(Ventura et al., 2003a). When lactobacilli belonging to horizontal spread of these genes as they become associ-
seven different Lactobacillus species were included in ated with phages with potential new host ranges and
that analysis, genes related to the prophages of the belonging to new immunity groups. This flexibility extends
reference strain were not detected. This observation the possibilities of conversion gene permutation in
demonstrates that mobile DNA is not necessarily polylysogenic hosts such as S. pyogenes. A spectacular
widely distributed across species borders. case of apparently prophage-mediated recombinations is

A B
M3 315
1'

2'

3'
4'
5'
6'

1'

B 6

ssa
2'
1
2
3
4

ssa
6
com M3 SSI-1 com

Fig. 3. Genome rearrangements in Streptococcus pyogenes.


A. The US S. pyogenes serotype M3 strain MGAS315 (top) was aligned with the Japanese S. pyogenes serotype M3 strain SSI-1 (bottom) using
the Artemis comparison tool. MGAS315 shows the likely original constellation, SSI-1 experienced a first inversion across the duplicated com
genes and a second inversion across prophages P5 and P6. The position of the prophages is indicated by the red boxes.
B. Alignment of the prophages P5 and P6 with the corresponding prophages 315.1 and 315.2 locating the site of recombination within the lysis
cassette. The shading connects regions of high DNA sequence identity between the compared phages. The modular structure of the prophages
is colour-coded (key in Fig. 1).

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18


14 C. Canchaya, G. Fournous and H. Brüssow
presented by the Gram-negative plant pathogen Xylella somal site to the left of Xt1 (Fig. 4B and C) and, finally,
fastidiosa. The pathovars 9a5c and Temecula shared DNA an inversion across the prophages Xt8 and Xt2 sharing
sequence identity essentially over the entire genome, but highly related integrase genes (Fig. 4C), leading to nearly
showed a very complex alignment pattern, suggesting perfectly aligned genomes (Fig. 4D). A genome inversion
three successive chromosomal inversion events (Van between the two sequenced O157 E. coli strains can also
Sluys et al., 2003). Genomics analysis suggested the fol- be traced to a recombination between prophages 993O
lowing sequence of events (Fig. 4): inversion across and 933P. However, prophages are not a privileged
prophage elements XfP1 and XfP2 sharing extensive recombination site: two Yersinia pestis genomes differ by
DNA sequence over their entire length (Canchaya et al., a large number of small genome inversions and contain
2003) (Fig. 4A and B), followed by an illegitimate recom- numerous prophage remnants, but only three inversions
bination between prophage remnant Xt10 and a chromo- are flanked at one side by a prophage sequence (Deng

B
A
Xylella fastidiosa Temecula ? Xt1 Xt10

2500000

XfP2
9.a.5.c
XfP1

2500000

observed
C D
reconstructed
Xt8 Xt2

Fig. 4. Genome rearrangements in Xylella fastidiosa.


A. Dot plot comparison of X. fastidiosa pathovar Temecula (horizontal) and pathovar 9a5c (vertical). The position of the prophages is indicated
by red boxes. The chromosome inversion between prophages XfP1 and XfP2 leads to the dot plot shown in (B). An inversion across prophage
Xt10 and an unknown chromosomal site leads to the dot plot depicted in (C). A final inversion between prophages Xt8 and Xt2 results in the
near-perfect alignment of the two genomes shown in (D).

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18


Prophage–chromosome interaction 15
et al., 2002). In other bacteria, rDNA and duplicated bac- tional protein. For example, lambdoid phage 21 inserts
terial genes were used for genome rearrangements (com within the isocitrate dehydrogenase gene and introduces
genes in the case of Fig. 3A). an alternative 165 bp 3¢ end for that gene (Campbell et al.,
1992). In rare cases, the phage recombination site attP is
located within the phage integrase gene, and prophage
Integration sites
integration leads to an altered int gene, which apparently
Many prophages from both Gram-negative and Gram- stabilizes the lysogen (Magrini et al., 1999), or the bacte-
positive bacteria integrate into tRNA genes in a preferred rial attB site complements the int gene (Bruttin et al.,
orientation (Campbell, 1992; 2002). Mostly, but not always 1997).
(Ventura et al., 2003a), the attP site of the phage recon-
stitutes the tRNA upon integration. Interestingly, an
Transcription of prophage genomes
increasing number of prophages are described that carry
tRNA genes (Fig. 5D). The phage-encoded tRNA differed In the lambdoid phages, it is known that most genes in
from the chromosomal tRNA and were transcribed in the the integrated prophages are not transcribed, and the bulk
lysogen (Ventura et al., 2004). Such constellations might of the prophage genes are thus ‘passive genetic cargo’ to
alleviate the consequences of prophage integration the lysogen. However, this statement does not apply to all
events into tRNA genes that do not reconstitute the orig- lambda prophage genes: lambda genes bor and lom are
inal tRNA gene. However, integration into tRNA genes is expressed from the lysogen and confer serum resistance
far from being universal. Integration into intergenic regions to the E. coli lysogen during in vivo growth (Barondess
and into ORFs has also been described (Canchaya et al., and Beckwith, 1990). Two lambdoid prophages from E.
2002). The prophage attachment site frequently faithfully coli O157 encode Shiga-like toxins (Stx), the major viru-
complements the coding sequence of the protein. How- lence factor of this important food pathogen. Their expres-
ever, cases of inactivation of the protein-encoding function sion is tightly controlled. Expression is achieved during
have also been described, a well-known case being the prophage induction and when iron, sensed by the Fur
negative lysogenic conversion of the lipase gene by an S. transcriptional regulator, becomes growth limiting (Wag-
aureus phage (Lee and Iandolo, 1986). In addition, inte- ner et al., 2002). Stx induces bleeding into the gut, and
gration into an ORF can also lead to an altered but func- iron thus becomes available from decaying blood cells. Stx

ori
A 124
2356 irp6A
2162
2159 pi3

adhA

B DT
539

625

SPIF
pi2
C
speC
spd1
mf2-
1734

hmuO pi1
D
894
mf4

mf2

922
1520

1061
dtxR
1296

Fig. 5. Transcription control of prophage genes.


A. Genome map of the Corynebacterium diphtheriae strain NCTC13129 (Cerdeno-Tarraga et al., 2003). The transcriptional regulator gene dtxR
is shown together with all genes regulated by DtxR (arrows) including the DT toxin encoded by the corynephage F shown in (B). The transcribed
lysogenic conversion genes from a Streptococcus pyogenes prophage are shown in (C). They are under the control of SPIF released from
pharyngeal cells (Broudy et al., 2001). Map of Lactobacillus plantarum prophage Lp1 in (D). The modular structure of the prophages is indicated
by a colour code (key in Fig. 1). A similar modular structure is proposed for the corynephage despite the phylogenetic distance separating the
bacterial hosts. The horizontal arrows next to the prophage maps indicate the prophage transcripts. In (B) and (C), only the right prophage end
was investigated for transcription.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18


16 C. Canchaya, G. Fournous and H. Brüssow
has no physiological export system, but E. coli O157 has by web-like phylogenies as a result of the rampant effect
‘learned’ to avoid this suicidal production by charging of horizontal gene transfer (HGT) in bacteria (Doolittle,
bystander intestinal E. coli strains with its synthesis via 1999). Other researchers claim that the role of HGT for
infection with the induced prophage (Gamage et al., the evolution of bacterial genomes is overstated (Kurland
2003). In a number of Gram-positive pathogens, important et al., 2003). The sequencing of multiple strains from
virulence factors are encoded between the phage lysin the same bacterial species demonstrated that HGT
gene and attR, the right attachment site (Beres et al., accounted for the majority of the intraspecies genome
2002). Only a low transcription level of these genes was differences. Prophages are the major contributors to
observed in broth growth, whereas contact with pharyn- genome diversification in some species; in others, proph-
geal cells induced the expression of these virulence ages compete with integrative plasmids, transposons or
genes in S. pyogenes prophages (Broudy et al., 2001) pathogenicity islands (which themselves show links to
(Fig. 5C). The expression of the diphtheria toxin in prophages) for this role. Still other sequenced bacteria
Corynebacterium diphtheriae is regulated via the DtxR lack prophages as a result of sampling bias (e.g. Strepto-
transcriptional regulator that binds in an iron-dependent coccus pneumoniae) or perhaps a genuine absence of
way to operators of many bacterial genes (Fig. 5A), phages (e.g. Helicobacter pylori ).
including the diphtheria toxin gene encoded by a cory- Prophages played an important and, in some cases, a
nephage (Fig. 5B). DtxR and Fur are the master regula- decisive role in the emergence of bacterial pathogens. V.
tors of large iron regulons, and prophage virulence gene cholerae, E. coli O157 and C. diphtheriae are examples
expression thus comes under the control of the host bac- in which the disease-specifying toxins are encoded by
terium. In contrast, constitutive transcription of moron-like prophages. S. aureus, S. pyogenes and Salmonella are
extra phage genes was seen in commensals and free- examples of pathogens in which many disease-modifying
living bacteria (Ventura et al., 2003b). The morons were factors are encoded by multiple prophages and each indi-
located in the vicinity of the left and right phage attach- vidual prophage contributes only an incremental virulence
ment sites (Fig. 5D). Interestingly, morons from pathogens increase.
and commensals sharing the same habitat (e.g. S. pyo- With respect to the impact of HGT on bacterial evolu-
genes and Lactobacillus plantarum both isolated from the tion, we believe that two processes must be distinguished.
oral cavity) also shared sequence-related putative In the field of medical microbiology and epidemiology, we
lysogenic conversion genes in their prophages (e.g. mf- observe events that occur across time frames that rarely
type DNases; Fig. 5C and D). exceed 100 years. Obviously, vertical modes of bacterial
In S. pyogenes and S. aureus, the prophage moron genome evolution are not very efficient over these short
transcription is increased by prophage induction via an time periods. Therefore, the emergence of new pathogens
increase in copy number (Broudy et al., 2002; Sumby and is likely to rely on the acquisition of lateral DNA (or loss
Waldor, 2003). In these cases, it is not clear whether these of DNA or a combination of both). In this context, phages
genes are of ecological benefit to the phage, the lysogen are an ideal carrier for horizontal DNA and thus a likely
or both (Broudy and Fischetti, 2003). Microarray analysis motor for short-term bacterial diversification. But does this
in a number of systems revealed that prophage genes process influence the structure of bacterial genomes over
figured prominently under the upregulated genes of the evolutionary time periods? Over this time scale, it is not
lysogen under stress conditions. This was observed in S. the acquisition, but the fixation, of prophage sequences
pyogenes growing in phagocytes (Voyich et al., 2003), that counts (Lawrence and Roth, 1999). In our survey, we
recovered from mice (Kazmi et al., 2001) and human have seen little, if any, evidence that prophages were fixed
patients or experiencing growth temperature changes into the chromosome of a bacterial species. Prophages
(Smoot et al., 2001). are apparently acquired as easily as they are lost from the
A prominent upregulation of prophage gene expression chromosome. A site occupied by a prophage in one strain
was also seen in Gram-negative lysogens upon infection might be empty in another strain or occupied by another
of animals (Dozois et al., 2003) or when changed from prophage. Within the confines of a bacterial species, a few
planktonic to biofilm growth (Pseudomonas aeruginosa; cases of closely related, although not identical, prophages
Whiteley et al., 2001). were reported that occupied the same chromosomal site
in different bacteria (Casjens, 2003). The Neisseria proph-
ages may be the clearest examples of long residence
Outlook
times during prophage decay. However, this case might
There is currently a lively discussion about the relative be an exception as it relates to Mu-like phages that very
contribution of vertical and horizontal elements of evolu- rarely excise precisely once they are inserted.
tion in bacteria. Some researchers argue that the tree-like However, fixing of entire prophages would not be
representations of Darwinian evolution should be replaced expected as it makes no evolutionary sense to bacteria.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18


Prophage–chromosome interaction 17
The mechanism would no doubt be characterized by fix- pharyngeal cells induces a phage-encoded extracellular
ation of particular phage genes, the function of which has DNase. Infect Immun 70: 2805–2811.
been co-opted by the host. Prophage genes without selec- Brüssow, H., and Hendrix, R.W. (2002) Phage genomics:
small is beautiful. Cell 108: 13–16.
tive value to the host are therefore likely to be deleted.
Bruttin, A., Foley, S., and Brüssow, H. (1997) The site-
Owing to the lack of surrounding prophage genes, com- specific integration system of the temperate Streptococcus
pletely fixed prophage genes are thus difficult to detect. thermophilus bacteriophage phiSfi21. Virology 237: 148–
In the case of bacterial pathogens, a conspicuous obser- 158.
vation is virulence genes flanked by isolated phage-like Campbell, A.M. (1992) Chromosomal insertion sites for
genes. Examples are Shiga toxin genes in Shigella dys- phages and plasmids. J Bacteriol 174: 7495–7499.
enteriae (McDonough and Butterton, 1999), sopE2 in Sal- Campbell, A.M. (2002) Preferential orientation of natural
lambdoid prophages and bacterial chromosome organiza-
monella Typhimurium, sspH and pertussis-like toxin genes
tion. Theor Popul Biol 61: 503–507.
in Salmonella Typhi (Fig. 1A). As related genes are found Campbell, A., Schneider, S.J., and Song, B. (1992) Lamb-
as lysogenic conversion genes in well-established proph- doid phages as elements of bacterial genomes (integrase/
ages (Stx prophages in E. coli O157, SopE prophages in phage21/Escherichia coli K-12/icd gene). Genetica 86:
S. Typhimurium), we probably have here examples of fixed 259–267.
prophage genes. As these genes are likely to extend the Canchaya, C., Desiere, F., McShan, W.M., Ferretti, J.J.,
ecological range of their bacterial hosts, prophages might Parkhill, J., and Brüssow, H. (2002) Genome analysis of
an inducible prophage and prophage remnants integrated
have a greater impact on bacterial genomes than is indi-
in the Streptococcus pyogenes strain SF370. Virology 302:
cated by the presence of clear-cut prophage DNA 245–258.
sequences in the sequenced bacterial genomes. Canchaya, C., Proux, C., Fournous, G., Bruttin, A., and Brüs-
sow, H. (2003) Prophage genomics. Microbiol Mol Biol Rev
67: 238–276.
Acknowledgements Casjens, S. (2003) Prophages and bacterial genomics: what
have we learned so far? Mol Microbiol 49: 277–300.
We thank Sherwood Casjens, Chris Blake, Anne Constable
Cerdeno-Tarraga, A.M., Efstratiou, A., Dover, L.G., Holden,
and Anne Bruttin for critical reading of the manuscript, and
M.T., Pallen, M., Bentley, S.D., et al. (2003) The complete
the Swiss National Science foundation for financial support
genome sequence and analysis of Corynebacterium diph-
to Carlos Canchaya (research grant 5002-057832).
theriae NCTC13129. Nucleic Acids Res 31: 6516–6523.
Chan, K., Baker, S., Kim, C.C., Detweiler, C.S., Dougan, G.,
References and Falkow, S. (2003) Genomic comparison of Salmonella
enterica serovars and Salmonella bongori by use of an S.
Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K., enterica serovar typhimurium DNA microarray. J Bacteriol
Oguchi, A., et al. (2002) Genome and virulence determi- 185: 553–563.
nants of high virulence community-acquired MRSA. Lancet Deng, W., Burland, V., Plunkett, G., III, Boutin, A., Mayhew,
359: 1819–1827. G.F., Liss, P., et al. (2002) Genome sequence of Yersinia
Barondess, J.J., and Beckwith, J. (1990) A bacterial virulence pestis KIM. J Bacteriol 184: 4601–4611.
determinant encoded by lysogenic coliphage lambda. Doolittle, W.F. (1999) Phylogenetic classification and the uni-
Nature 346: 871–874. versal tree. Science 284: 2124–2129.
Beres, S.B., Sylva, G.L., Barbian, K.D., Lei, B., Hoff, J.S., Dozois, C.M., Daigle, F., and Curtiss, R. (2003) Identification
Mammarella, N.D., et al. (2002) Genome sequence of a of pathogen-specific and conserved genes expressed in
serotype M3 strain of group A Streptococcus: phage- vivo by an avian pathogenic Escherichia coli strain. Proc
encoded toxins, the high-virulence phenotype, and clone Natl Acad Sci USA 100: 247–252.
emergence. Proc Natl Acad Sci USA 99: 10078–10083. Dziejman, M., Balon, E., Boyd, D., Fraser, C.M., Heidelberg,
Boyd, E.F., and Brüssow, H. (2002) Common themes among J.F., and Mekalanos, J.J. (2002) Comparative genomic
bacteriophage-encoded virulence factors and diversity analysis of Vibrio cholerae: genes that correlate with chol-
among the bacteriophages involved. Trends Microbiol 10: era endemic and pandemic disease. Proc Natl Acad Sci
521–529. USA 99: 1556–1561.
Broudy, T.B., and Fischetti, V.A. (2003) In vivo lysogenic Fitzgerald, J.R., Sturdevant, D.E., Mackie, S.M., Gill, S.R.,
conversion of Tox(–) Streptococcus pyogenes to Tox(+) and Musser, J.M. (2001) Evolutionary genomics of Staphy-
with lysogenic streptococci or free phage. Infect Immun 71: lococcus aureus: insights into the origin of methicillin-
3782–3786. resistant strains and the toxic shock syndrome epidemic.
Broudy, T.B., Pancholi, V., and Fischetti, V.A. (2001) Proc Natl Acad Sci USA 98: 8821–8826.
Induction of lysogenic bacteriophage and phage- Fournous, G. (2003) Automatisation de la recherche et de
associated toxin from group A streptococci during cocul- l’analyse de prophages dans les génomes bactériens. Tra-
ture with human pharyngeal cells. Infect Immun 69: vail de DEA, Université Henri Poincaré, Nancy, France.
1440–1443. Gamage, S.D., Strasser, J.E., Chalk, C.L., and Weiss, A.A.
Broudy, T.B., Pancholi, V., and Fischetti, V.A. (2002) The in (2003) Nonpathogenic Escherichia coli can contribute to
vitro interaction of Streptococcus pyogenes with human the production of Shiga toxin. Infect Immun 71: 3107–3115.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18


18 C. Canchaya, G. Fournous and H. Brüssow
Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, revealed by microarray analysis. Proc Natl Acad Sci USA
A., Baquero, F., et al. (2001) Comparative genomics of 99: 8956–8961.
Listeria species. Science 294: 849–852. Pridmore, D., Berger, B., Desiere, F., Vilanova, D., Barretto,
Glaser, P., Rusniok, C., Buchrieser, C., Chevalier, F., C., Pittet, A.C., et al. (2004) The genome sequence of the
Frangeul, L., Msadek, T., et al. (2002) Genome sequence probiotic intestinal bacterium Lactobacillus johnsonii NCC
of Streptococcus agalactiae, a pathogen causing invasive 533. Proc Natl Acad Sci USA 101: 2512–2517.
neonatal disease. Mol Microbiol 45: 1499–1513. Smoot, L.M., Smoot, J.C., Graham, M.R., Somerville, G.A.,
Juhala, R.J., Ford, M.E., Duda, R.L., Youlton, A., Hatfull, G.F., Sturdevant, D.E., Migliaccio, C.A., et al. (2001) Global dif-
and Hendrix, R.W. (2000) Genomic sequences of bacte- ferential gene expression in response to growth tempera-
riophages HK97 and HK022: pervasive genetic mosaicism ture alteration in group A Streptococcus. Proc Natl Acad
in the lambdoid bacteriophages. J Mol Biol 299: 27–51. Sci USA 98: 10416–10421.
Kazmi, S.U., Kansal, R., Aziz, R.K., Hooshdaran, M., Norrby- Smoot, J.C., Barbian, K.D., Van Gompel, J.J., Smoot, L.M.,
Teglund, A., Low, D.E., et al. (2001) Reciprocal, temporal Chaussee, M.S., Sylva, G.L., et al. (2002a) Genome
expression of SpeA and SpeB by invasive M1T1 group a sequence and comparative microarray analysis of serotype
streptococcal isolates in vivo. Infect Immun 69: 4988– M18 group A Streptococcus strains associated with acute
4995. rheumatic fever outbreaks. Proc Natl Acad Sci USA 99:
Kurland, C.G., Canback, B., and Berg, O.G. (2003) Horizon- 4668–4673.
tal gene transfer: a critical view. Proc Natl Acad Sci USA Smoot, L.M., McCormick, J.K., Smoot, J.C., Hoe, N.P.,
100: 9658–9662. Strickland, I., Cole, R.L., et al. (2002b) Characterization of
Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., two novel pyrogenic toxin superantigens made by an acute
Kobayashi, I., et al. (2001) Whole genome sequencing of rheumatic fever clone of Streptococcus pyogenes associ-
methicillin-resistant Staphylococcus aureus. Lancet 357: ated with multiple disease outbreaks. Infect Immun 70:
1225–1240. 7095–7104.
Lawrence, J.G., and Roth, J.R. (1999) Genomic flux: Sumby, P., and Waldor, M.K. (2003) Transcription of the toxin
genomic evolution by gene loss and acquisition. In Orga- genes present within the Staphylococcal phage phiSa3ms
nization of the Prokaryotic Genome. Charlebois, R.L. (ed.). is intimately linked with the phage’s life cycle. J Bacteriol
Washington, DC: American Society for Microbiology Press, 185: 6841–6851.
pp. 263–289. Tettelin, H., Masignani, V., Cieslewicz, M.J., Eisen, J.A.,
Lawrence, J.G., Hendrix, R.W., and Casjens, S. (2003) Peterson, S., Wessels, M.R., et al. (2002) Complete
Where are the pseudogenes in bacterial genomes. Trends genome sequence and comparative genomic analysis of
Microbiol 9: 535–540. an emerging human pathogen, serotype V Streptococcus
Lee, C.Y., and Iandolo, J.J. (1986) Lysogenic conversion of agalactiae. Proc Natl Acad Sci USA 99: 12391–12396.
staphylococcal lipase is caused by insertion of the bacte- Van Sluys, M.A., de Oliveira, M.C., Monteiro-Vitorello, C.B.,
riophage L54a genome into the lipase structural gene. J Miyaki, C.Y., Furlan, L.R., Camargo, L.E., et al. (2003)
Bacteriol 166: 385–391. Comparative analyses of the complete genome sequences
McDonough, M.A., and Butterton, J.R. (1999) Spontaneous of Pierce’s disease and citrus variegated chlorosis strains
tandem amplification and deletion of the shiga toxin of Xylella fastidiosa. J Bacteriol 185: 1018–1026.
operon in Shigella dysenteriae 1. Mol Microbiol 34: Ventura, M., Canchaya, C., Pridmore, D., Berger, B., and
1058–1069. Brüssow, H. (2003a) Integration and distribution of Lacto-
Magrini, V., Storms, M.L., and Youderian, P. (1999) Site- bacillus johnsonii prophages. J Bacteriol 185: 4603–4608.
specific recombination of temperate Myxococcus xanthus Ventura, M., Canchaya, C., Kleerebezem, M., de Vos, W.M.,
phage Mx8: regulation of integrase activity by reversible, Siezen, R.J., and Brüssow, H. (2003b) The prophage
covalent modification. J Bacteriol 181: 4062–4070. sequences of Lactobacillus plantarum strain WCFS1. Virol-
Makino, K., Yokoyama, K., Kubota, Y., Yutsudo, C.H., Kimura, ogy 316: 245–255.
S., Kurokawa, K., et al. (1999) Complete nucleotide Ventura, M., Canchaya, C., Pridmore, R.D., and Brüssow, H.
sequence of the prophage VT2-Sakai carrying the vero- (2004) The Prophages of Lactobacillus johnsonii NCC 533:
toxin 2 genes of the enterohemorrhagic Escherichia coli comparative genomics and transcription analysis. Virology
O157:H7 derived from the Sakai outbreak. Genes Genet 320: 229–242.
Syst 74: 227–239. Voyich, J.M., Sturdevant, D.E., Braughton, K.R., Kobayashi,
Nakagawa, I., Kurokawa, K., Yamashita, A., Nakata, M., S.D., Lei, B., Virtaneva, K., et al. (2003) Genome-wide
Tomiyasu, Y., Okahashi, N., et al. (2003) Genome protective response used by group A Streptococcus to
sequence of an M3 strain of Streptococcus pyogenes evade destruction by human polymorphonuclear leuko-
reveals a large-scale genomic rearrangement in invasive cytes. Proc Natl Acad Sci USA 100: 1996–2001.
strains and new insights into phage evolution. Genome Wagner, P.L., Livny, J., Neely, M.N., Acheson, D.W., Fried-
Res 13: 1042–1055. man, D.I., and Waldor, M.K. (2002) Bacteriophage control
Ohnishi, M., Kurokawa, K., and Hayashi, T. (2001) of Shiga toxin 1 production and release by Escherichia coli.
Diversification of Escherichia coli genomes: are bacte- Mol Microbiol 44: 957–970.
riophages the major contributors? Trends Microbiol 9: 481– Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R.,
485. Teitzel, G.M., Lory, S., and Greenberg, E.P. (2001) Gene
Porwollik, S., Wong, R.M., and McClelland, M. (2002) Evolu- expression in Pseudomonas aeruginosa biofilms. Nature
tionary genomics of Salmonella: gene acquisitions 413: 860–864.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 9–18

You might also like