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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
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
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
B
A
Xylella fastidiosa Temecula ? Xt1 Xt10
2500000
XfP2
9.a.5.c
XfP1
2500000
observed
C D
reconstructed
Xt8 Xt2
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