Laterally transferred genomic islands in Xanthomonadales related to

RESEARCH LETTER
Laterally transferred genomic islands in Xanthomonadales related
to pathogenicity and primary metabolism
Wanessa C. Lima1, Apuã C.M. Paquola1, Alessandro M. Varani2, Marie-Anne Van Sluys2 &
Carlos F.M. Menck1
1
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil; and 2Department of Botany, Institute of
Biosciences, University of São Paulo, São Paulo, SP, Brazil
Correspondence: Carlos F. M. Menck,
Department of Microbiology, Institute of
Biomedical Sciences, University of São Paulo,
Av. Lineu Prestes, 1374, ICB II, São Paulo, SP,
Brazil. Tel.: 155 11 3091 7357; fax: 155 11
3091 7354; e-mail: [email protected]
Received 10 September 2007; accepted
7 January 2008.
First published online February 2008.
DOI:10.1111/j.1574-6968.2008.01083.x
Editor: Ross Fitzgerald
Keywords
lateral gene transfer; horizontal gene transfer;
Xanthomonas ; genomic islands;
categorization.
Abstract
Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome
evolution, usually associated with increased fitness and/or changes in behavior,
especially if one considers pathogenic vs. non-pathogenic bacterial groups. The
genomes of two phytopathogens, Xanthomonas campestris pv. campestris and
Xanthomonas axonopodis pv. citri, were previously inspected for genome islands
originating from LGT events, and, in this work, potentially early and late LGT
events were identified according to their altered nucleotide composition. The
biological role of the islands was also assessed, and pathogenicity, virulence and
secondary metabolism pathways were functions highly represented, especially in
islands that were found to be recently transferred. However, old islands are
composed of a high proportion of genes related to cell primary metabolic
functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally
inherited by vertical processes.
Introduction
Evolutionary molecular biology started in the 1960s, when
Zuckerkandl & Pauling (1965) noticed that nucleic acid and
protein sequences are sources of information to infer organism evolution. Since then, biologists have tried to reconstruct the evolutionary history of species, based on sequence
similarity of highly conserved genes such as 16S rRNA and
recA (Woese & Fox, 1977; Eisen, 1995). Nevertheless, there
are several examples of genes that do not follow the classical
topology of these phylogenetic trees, and this has been
attributed to the existence of lateral gene transfer (LGT)
events between species (Syvanen, 1994; Gogarten et al.,
1999), contrasting with the vertical process of inheritance
whereby traits are transmitted from parents to offspring
(Eisen, 2000).
Because of the increase in available genomic data, it is
now easier to evaluate the extent by which organisms
increase their genetic diversity through the acquisition of
genes by LGT. Nevertheless, identifying LGT within a given
bacterial genome is not a trivial task. The approaches are
FEMS Microbiol Lett 281 (2008) 87–97
based on the analysis of DNA composition, distinguishing
foreign from indigenous DNA (including GC content, GC
skew, dinucleotide relative abundance and codon usage
bias); analysis of unusual phyletic patterns (as unexpected
ranking of similarities or unusual gene content); unexpected
phylogenetic tree topology; and the presence of mobile
genetic elements (Eisen, 1998; Ragan, 2001a).
The ability to perform comparative analyses on several
genomes at the same time, in order to predict LGT, has
gained precision and is currently becoming the method of
choice for identifying LGT. Currently, several papers assessing the amount of laterally transferred genes in completely
sequenced genomes are available, using different methodologies. The data indicate the overall contribution of LGT
events in shaping microbial genomes, in proportions ranging from virtually none to more than 25% of genes as
resultant from transfers (depending on the organism, its
taxonomy position and its life style) (Ochman et al., 2000;
Merkl, 2004; Beiko et al., 2005; Shi et al., 2005).
Xanthomonadales are gram-negative Gammaproteobacteria
including important plant pathogen species (Vauterin et al.,
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W.C. Lima et al.
1995; Van Sluys et al., 2002), although phylogenetic reconstruction of conserved genes (as 16S rRNA gene and recA)
branches them very close to the root of the Beta- and
Gammaproteobacteria subdivision (Martins-Pinheiro et al.,
2004; Lima et al., 2005). Some studies even state that the
extensive transfers among Xanthomonadales and the three
major proteobacterial clades (alpha, beta and gamma) make
it difficult to place them in one of the former groups (Comas
et al., 2006). In Xanthomonas campestris pv. campestris
(XCC) and Xanthomonas axonopodis pv. citri (XAC), estimates of the number of laterally transferred genes vary
between 5% and 20%, depending on the methodology used
(Garcia-Vallve et al., 2003; Merkl, 2004; Nakamura et al.,
2004). The identification, at a genomic scale, of gene clusters
or gene islands laterally transferred was carried out in a few
cases, in general relating them to pathogenicity or virulence
traits. Nakamura et al. (2004) identified four PAIs (pathogenicity islands) in XAC and three in XCC, all of them
containing virulence genes or gene regulators. Mantri &
Williams (2004), searching for islands delivered by sitespecific integrases, identified four islands in each genome
containing transposases, tRNAs, virulence or phage-related
genes, hypothetical genes, peptidases and regulators. More
recently, the genomic repertoire of mobile genetic elements
from six Xanthomonadales species was described, and several
of these elements are located at one of the borders of a
genomic island (Monteiro-Vitorello et al., 2005). Finally,
Chen (2006) identified four islands in XAC and one in XCC,
based on the cumulative GC profile method. In a previous
work, we identified 25% of Xanthomonas genes as potentially acquired through LGT, by unusual ranking of sequence
similarity and clustering analysis (Lima et al., 2005).
In this work, further analysis distinguished potentially
late and early events of gene transfer in Xanthomonas
genome diversification, where recently acquired islands are
bordered by mobile genetic elements, and old islands have
lost most of the LGT identifiers except for the atypical
phylogenetic pattern. Functional categorization of these
clustered genes reveals a bias towards biological roles,
including pathogenicity and virulence traits, transport,
energy metabolism and regulatory functions. The data
reveal a group of potentially transferred genes that are not
usually detected by nucleotide composition methods, and
indicate that LGT may also contribute to build prokaryotic
genomes by replacing genes normally inherited by vertical
processes.
were the GC content bias, the dinucleotide content bias and
the codon usage bias, as described, respectively, in GarciaVallve et al. (2000), Karlin (2001) and Hsiao et al. (2003).
Genes were considered extraneous in terms of (1) the GC
content if their GC content deviated by 4 1.5s (SD) from
the mean value of their genome; (2) the dinucleotide
frequency if the gene deviated 4 2.0s away from the
genome mean; and (3) the codon usage when the gene had
a Mahalanobis distance of 4 2s from the mean genome
value. Genes were defined as possessing an odd nucleotide
composition when displaying at least one of these three
parameters deviant from the genome average.
Analysis of tetranucleotide usage variance was performed
by the implementation of the OLIGOWORDS program (Reva &
Tümmler, 2005; Klockgether et al., 2007). Parameters of
distance (D) and oligonucleotide usage variance (OUV)
were calculated using a sliding window of 8 kbp and a step
size of 2 kbp (both calculations were normalized by monoand dinucleotide frequencies).
Materials and methods
Functional categorization
Nucleotide composition analyses
Each island was inspected for the unusual nucleotide
composition of their genes. The three parameters analyzed
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Phylogenetic analyses
Automatic, large-scale phylogenetic reconstruction was undertaken through the implementation of a modified PYPHY
algorithm (Sicheritz-Ponten & Andersson, 2001). This software performs a BLAST similarity search and keyword search
on the Swiss-Prot and TrEMBL databases, and the sequences
matching predefined criteria are aligned with CLUSTAL software. Distance-based phylogenetic trees (using the NEIGHBOR-JOINING program implemented in the PHYLIP package) are
then generated for all genes in both genomes, whenever the
number of homologs was at least four proteins. Following
this first phylogenetic screening, manual and detailed phylogenetic reconstructions were made to all genes belonging
to some gene cluster of interest, including the trees shown in
this study. In these cases, protein sequences were aligned
with the CLUSTALX program (Thompson et al., 1997), and
regions of the alignments that were ambiguous, hypervariable or containing gaps were excluded from subsequent
analysis (GENEDOC program; Nicholas et al., 1997). Distancebased phylogenetic trees were generated from these alignments using the neighbor-joining algorithm (NEIGHBORJOINING program from PHYLIP package; Felsenstein, 1989).
Bootstrap assessment of tree topology (one thousand replicates) was performed with the SEQBOOT program (PHYLIP).
The functional, biological role of each gene within the
islands was assigned through the Xanthomonas genome
project homepage (da Silva et al., 2002) and the Comprehensive Microbial Resource in TIGR (Peterson et al., 2001).
FEMS Microbiol Lett 281 (2008) 87–97
89
LGT islands in Xanthomonas
Table 1. Analysis of unusual nucleotide composition of Xanthomonas
islands
Number of
genes
% of
atypical genes
Number of mobile
genetic elements
Island
XAC
XCC
XAC
XCC
XAC
XCC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
A1
A2
A3
A4
A5
C1
C2
C3
C4
C5
70
20
24
19
28
18
43
39
34
17
19
13
22
18
10
30
13
13
18
77
17
23
34
19
16
30
25
24
16
108
25
17
36
32
14
–
–
–
–
–
42
21
23
17
51
24
30
33
39
20
22
13
13
19
10
31
13
14
18
32
23
27
27
24
15
11
30
22
16
94
–
–
–
–
–
57
50
19
59
36
81
60
29
56
64
50
33
44
44
35
21
38
55
56
20
37
15
23
44
77
47
56
44
32
37
60
40
37
19
48
96
29
53
97
50
–
–
–
–
–
62
43
43
82
69
92
40
39
59
40
18
15
46
63
20
39
15
29
61
78
57
37
37
46
60
45
30
41
56
59
–
–
–
–
–
95
58
95
75
81
3
3
0
0
1
2
2
0
0
0
0
0
0
0
0
5
0
1
2
25
0
0
2
2
0
7
1
0
0
12
14
0
2
4
0
–
–
–
–
–
0
2
0
0
4
2
0
0
1
0
1
0
0
0
0
5
0
1
1
6
0
2
0
0
0
0
2
0
0
13
–
–
–
–
–
19
23
9
29
14
The percentages consider the number of genes within each island
displaying one or more of the following atypical nucleotide composition
features: GC content, codon usage and dinucleotide frequency.
Results
Detection of unusual features in laterally
transferred islands
Xanthomonas genomes comprise an elevated number of
genes with the highest similarity to genes from phylogenetiFEMS Microbiol Lett 281 (2008) 87–97
cally distant organisms, and in most cases the genes are
grouped into islands instead of simply being scattered
throughout the genome. In a previous report, we identified
40 islands whose best matches (through BLAST searches) were
to distantly related organisms (non-Gammaproteobacteria)
in both XAC and XCC, including 30 islands shared by both
genomes (islands 01–30, in Table 1) and five exclusive to
either one (identified in Table 1 as A1–A5 when belonging to
XAC, or C1–C5 when belonging to XCC) (Lima et al., 2005;
the genomic coordinates for all islands are provided as
supplementary material). Genomes, publically available,
from other Xanthomonas species were also assessed, and, as
expected, almost all islands are also present, with the
exception of those classified as exclusive. XAC-exclusive
islands were not found in the other six Xanthomonas
genomes, while XCC-exclusive islands were found in other
X. campestris strains (data not shown).
In this study, genes within the islands were further
investigated with respect to nucleotide composition and
phylogenetic position. A gene is considered to have unusual
nucleotide composition if it differs significantly from the
genome average in at least one of the following characteristics: GC content, codon usage and dinucleotide frequency.
As shown in Table 1, the islands have different proportions
of atypical genes, ranging from 15% to 97%. The exclusive
islands were among the islands with the highest proportion
of atypical genes, and the presence of an elevated number of
mobile genetic elements within these islands strengthens the
hypothesis of recent events of lateral transmission originating from such exclusive islands.
However, it is worth noting that several islands potentially
originated by LGT events show low levels of deviation in
nucleotidic parameters. In such cases, the concomitant
findings of atypical phylogenetic reconstruction and atypical
similarity ranking of genes, and in most cases the association
with mobile genetic elements (MGEs), support their origin
via lateral gene transfer events. These islands may be the
result of an early LGT event and, due to the amelioration
process, the nucleotide composition became similar to the
host’s genome.
A second approach to assess the deviation on the nucleotide composition was also employed, and involved the
analysis of the tetranucleotide usage variance in each island,
compared with the median values of the whole host chromosome (Reva & Tümmler, 2005; Klockgether et al., 2007).
It is important to note that, differently from the approaches
presented before, the tetranucleotide parameters (denoted
here by OUV) were calculated based on the entire island
(using a sliding window of 2 Kbp) and not individual genes.
In general, the islands with higher levels of genes with
atypical nucleotide composition also present tetranucleotide
signatures distinct from that of the host chromosome and
those with less atypical genes present OUV values in
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90
W.C. Lima et al.
Fig. 1. Tetranucleotide usage variance of two
more-deviant (a) and two less-deviant (b) islands
in XAC and XCC genomes. The horizontal lines
correspond to the limits of the 95% confidence
interval of OUV in a randomly generated
sequence with the same length and
tetranucleotide content as the host genome.
accordance with the genome average. In Fig. 1, this analysis
is illustrated for one island more deviant and one less
deviant of each genome (and in the Supplementary Fig. S1,
the variance of tetranucleotide frequency is shown for the
three other islands, in each genome, with the highest
frequencies of genes with atypical nucleotide composition).
Clearly, the results indicate that the islands denoted as more
deviant present regions with strong heterogeneity of tetranucleotide frequencies, but for most of the islands these
values are consistently different from the median OUV
values for the two Xanthomonas genomes. These data are in
agreement with a potential recent acquisition of these
islands by LGT.
To support the best-BLAST-match analysis, first used to
detect the laterally transferred islands, a large-scale phylogenetic analysis was conducted in both XAC and XCC
genomes. As expected, to the genes where it was possible to
generate a tree (at least four homologs found in GenBank),
most of them displayed an atypical phylogenetic behavior
(data not shown). A gene is considered to have atypical
phylogenetic reconstruction if it branches outside the Beta/
Gammaproteobacteria group. Both groups (beta and gamma) were considered as typical branching to Xanthomonas
genomes by the close relationship they present in phylogenetic trees (Figure S2 shows a phylogenetic tree for the 16S
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rRNA gene, depicting the position of the Xanthomonadales
group inside the Bacteria clade).
Functional categorization of genes within
islands
The classification of genes within the islands into functional
categories was assessed, and the proportion of category
distribution is presented in Fig. 2, compared with the
average found in the entire genome. As expected for
laterally transferred genes, the functional categories that
have higher proportions of genes within the islands
are those related to pathogenicity and virulence as well
as MGEs.
The distribution of functional categories was also assessed
discriminating islands with the highest as well as the lowest
levels of deviation in nucleotide composition (the seven
more-or-less deviants in each genome). As shown in Fig. 3a,
islands with highly divergent genes (potentially recent LGT
events) display a clear predominance of genes related to
MGE, besides pathogenicity and virulence. In fact, for
these islands the other functional categories are barely
represented, except as regards cell structure. On the
contrary, islands with lower levels of atypical nucleotide
composition, although also carrying genes related to
FEMS Microbiol Lett 281 (2008) 87–97
91
LGT islands in Xanthomonas
(a)
Frequency (%)
*
*
*
(b)
*
Frequency (%)
*
*
*
Fig. 2. Distribution of functional categories
within islands (light bars) compared with genes
in the whole genome (dark bars), in XAC (a) and
XCC (b). Categories were defined by the original
description (http://cancer.lbi.ic.unicamp.br/
xanthomonas/]).
pathogenicity and virulence, have an increased number of
genes related to intermediary metabolism and cellular
processes (Fig. 3b).
It is worth noting the presence of entire clusters of genes
related to metabolic pathways, among islands with a low
percentage of genes with discrepant nucleotide composition
(Table 2). Notable examples are islands involved in the
metabolism of NAD, arginine and cysteine and in energetic
metabolism, including tricarboxylic acid cycle (Lima &
Menck, 2008). Phylogenetic reconstructions for such genes
confirm these islands as the result of transfer from distantly
related organisms, considering closely related organisms
from the Beta/Gammaproteobacteria group (Fig. S2). In
some of these important metabolic pathways, we found
genes branching close to homologs of the Eukarya and
Archaea clades, as well as other Bacteria groups distantly
related, such as Actinobacteria, Firmicutes and Bacteroidetes,
instead of branching with closely related homologs from
proteobacteria. These results support the origin of such
genes from LGT events, despite the fact that these genes are
related to primary metabolic functions in the cell.
One clear example of this ‘core metabolism gene transfer’
refers to the island bearing the genes related to cystein
FEMS Microbiol Lett 281 (2008) 87–97
biosynthesis. Five genes (cysNC, cysD, cysJ, cysI and cysH),
responsible for the assimilation and activation of inorganic
sulfate to organic sulfide, are clustered, and phylogenetic
reconstructions branch them with non-Gammaproteobacteria
groups. This is illustrated by Fig. 4, as CysI, which encodes
the beta subunit of NADPH sulfite reductase, groups with
Firmicutes and Fungi, in a separate branch from the other
Proteobacteria.
Concerning recently acquired islands, seven are related to
virulence and pathogenicity, which may provide selective
traits advantageous to these plant pathogens (Table 2).
These islands carry genes related to type II, III and IV
secretion systems, xanthan gum production and host
cell wall degradation, and some of them have been described
previously (Van Sluys et al., 2002; Lima et al., 2005).
The majority are associated to MGEs (phages, transposons
or tRNA), and have discrepant nucleotide composition
(Table 1).
Phylogenetic trees of these genes also reveal an atypical
pattern. For example, the island carrying the xanthan gum
operon (composed of 14 genes) has strongest similarity to
Bacillus species, although the genes from this gram-positive
bacterium were not annotated as being involved in xanthan
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92
W.C. Lima et al.
XAC
(a)
XCC
*
*
*
*
*
*
*
(b)
*
*
*
*
XAC
XCC
**
*
*
**
*
*
**
Fig. 3. Distribution of functional categorization of island genes according to atypical nucleotide composition, showing the highest (a) and the lowest
(b) levels of deviation from the nucleotide composition. Genes within islands (light bars) were compared with genes in the whole genome (dark bars), in
XAC and XCC. For columns marked with one asterisk, the difference in relation to the total genome is statistically significant (P o 0.01, determined for
each pair with a w2 test). In the columns marked with two asterisks, a P o 0.05 was considered. The following islands were considered as less deviant in
XAC (03, 11, 15, 17, 18, 29, A2) and in XCC (11, 12, 15, 17, 18, 27) and as more deviant in XAC (01, 02, 05, 20, 26, A1, A4) and XCC (04, 06, 20, C1,
C3, C4, C5).
Table 2. Islands identified in XAC and XCC related to pathogenicity and central metabolic pathways
Island
Function and/or prominent genes
Comments
5
8
13
16
18
19
20
Type III secretory system, effector proteins and host cell wall degradation genes (b-galactosidases)
Type II secretory system (operon 1) and host cell wall degradation genes (b-galactosidases)
NAD metabolism
Energetic metabolism (TCA cycle)
Arginine biosynthesis
Xanthan gum production genes
Type IV secretory system, fimbriae production and phage genes
tRNA-flanked, presence of transposase
22
23
24
25
30
Host cell wall degradation genes (glucosidases, mannosidases, b-galactosidase, rhamnosidase)
Cysteine metabolism
Host cell wall degradation genes (esterase, amylase, rhamnogalacturonase and cellulases)
Type II secretory system (operon 2) and proteases
Host cell wall degradation genes (xylosidases, glucosidases, xylanases, b-galactosidase)
production. Trees for two genes belonging to this system
(gumF presented in Fig. 5a and gumH in Fig. 5b) reveal that
both genes appear only in distantly related groups, such as
Archaea, Bacteroidetes and gram-positive bacteria (Actino2008 Federation of European Microbiological Societies
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Presence of several tRNAs
tRNA-flanked
tRNA-flanked, presence of integrase
tRNA-flanked, presence of transposase
and integrase
Presence of transposase
Presence of transposase
Presence of transposase
tRNA-flanked, presence of transposase
bacteria and Firmicutes). However, in organisms other than
Xanthomonas these homologous genes are responsible for
the assembly of some other polysaccharide, because xanthan
gum is exclusive of this genus.
FEMS Microbiol Lett 281 (2008) 87–97
93
LGT islands in Xanthomonas
Fungi
Firmicutes
Xanthomonadales
10
Proteobacteria
Fig. 4. Distance tree of cysI gene computed by the neighbor-joining method. Numbers within the tree correspond to the bootstrap assessment (based
on 1000 replicates). Values below 50% are not shown.
Discussion
In this work, we present a concerted analysis of genomic
islands potentially originating through LGT events. The
initial and fast approach, the detection of clusters of genes
showing atypical ranking of similarity, is now complemented by analyses of unusual nucleotide composition and
atypical phylogenetic branching. Although the use of several
methods may fail to identify a common set of genes, as
previously observed in Escherichia coli (Ragan, 2001b), the
finding of two or more parameters indicating LGT in genes
within the islands supports the explanation of an alien
origin for those clustered genes.
As expected, the phylogenetic reconstruction of genes
within islands reinforced their potential foreign origin,
while the analysis of atypical nucleotide composition offered
some hints into the time of introgression for some of the
FEMS Microbiol Lett 281 (2008) 87–97
islands. Some authors consider codon bias and base composition as poor indicators for defining gene transfer events;
however, this method has been widely used in the search for
lateral transfer in prokaryotes, with the advantage of yielding information through the analysis of single genomes
(Lawrence & Ochman, 1998). In this work, atypical nucleotide composition and tetranucleotide usage variance were
used as additional criteria to validate cluster of genes
previously detected as a result of LGT. The high percentage
of genes featuring atypical nucleotide composition, with
consistent tetranucleotide distinct signatures, indicates that
a given island is a recently acquired island. In fact, many of
the islands with this feature were found as exclusive for one
of the two Xanthomonas genomes analyzed, and carry many
MGEs, confirming that they may be the result of recent
transfer events. In some cases, these islands contain phagerelated sequences, an indication of phage transduction.
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W.C. Lima et al.
(a)
Xanthomonas oryzae
gumG
Xanthomonas axonopodis
gumG
Xanthomonadales
Xanthomonas campestris
gumG
100
Xanthomonas campestris
gumF
Xylella fastidiosa
gumF
Xylella fastidiosa
gumG
Xanthomonas axonopodis
gumF
99
100
100
Staphylococcus saprophyticus
99
57
Xanthomonas oryzae
gumF
96
55
100
100
Staphylococcus aureus
Polaromonas sp.
Lactobacillus plantarum
Staphylococcus epidermidis
Clostridium perfringens
Oceanobacillus iheyensis
Bacteroides fragilis
76
100
Clostridium acetobutylicum
100
Firmicutes and
Bacteroidetes
Bacteroides thetaiotaomicron
79
Methanosarcina acetivorans
Bacteroides fragilis
_100
Methanosarcina mazei
Bacillus clausii
Bacillus licheniformis
(b)
Methanosarcina mazei
Methanosarcina acetivorans
Methanospirillum hungatei
Mycobacterium bovis
Archaea
Haloarcula marismortui
99
Mycobacterium tuberculosis
Pyrococcus abyssi
Frankia sp.
Archaeoglobus fulgidus
100
98
55
Mycobacterium paratuberculosis
100
Methanosarcina mazei
68
100
100
62
100
100
Streptomyces coelicolor
100
Methanosarcina barkeri
99
Streptomyces avermitilis
999
Brucella melitensis
Actinobacteria
Brucella suis
Xylella fastidiosa
Acetobacter xylinus
918
Xanthomonas campestris
Xanthomonadales
_100
Xanthomonas axonopodis
Xanthomonas oryzae
Fig. 5. Distance tree of gumF (a) and gumH (b) genes computed by the neighbor-joining method. Numbers within the tree correspond to the bootstrap
assessment (based on 1000 replicates). Values below 50% are not shown.
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FEMS Microbiol Lett 281 (2008) 87–97
95
LGT islands in Xanthomonas
These results confirm previous observations where MGES
were associated to genomic islands that distinguish XAC and
XCC genomes, defined by a different approach (MonteiroVitorello et al., 2005).
On the other hand, islands carrying most of the
genes with nucleotide content similar to the rest of the
genome may still result from LGT events. These genes may
result from an ancient event, but their nucleotide composition may have evolved to a composition similar to that of
the host genome, or, in other words, may have ameliorated
(Lawrence & Ochman, 1997). However, these islands may
also have been recently acquired, albeit from genomes with a
nucleotide composition similar to Xanthomonas. For these
islands, the concomitant atypical phylogenetic reconstruction supports their origin via LGT, which would not be
detected simply by genome composition analyses, certifying
the methods of best-match screenings for the search of
LGT events.
Functional analyses of genes found in islands corroborate
previous observations in whole-sequenced genomes (Merkl,
2004; Nakamura et al., 2004; Lima et al., 2005). One
interesting feature is certainly the high number of genes
related to virulence and pathogenicity that accounts for a
metabolic role in the life style of the phytopathogens.
Another relevant consideration is the under-representation
of informational genes (those related to macromolecule
metabolism) in these islands. However, the most striking
finding is the strong bias of genes playing roles in the
core metabolism of bacteria (intermediate and cellular
processes categories), especially in islands that carry genes
with low nucleotide composition discrepancy compared
with the whole genome. The presence of gene clusters, such
as those related to amino acid biosynthesis (arginine and
cysteine), NAD and energetic metabolism, points to the
potential relevance of LGT contributing to primary functions in bacterial genomes, and supports the selfish-operon
model (Lawrence & Roth, 1996). According to this model,
transfer of clustered genes confers a selectable function,
facilitating its maintenance in the recipient genome. Transfer of single genes that would provide only part of a
metabolic function would most likely be lost during evolution. This seems to be the case for the clusters reported in
this work, as the genes analyzed present a clear phylogenetic
proximity to organisms unrelated to Xanthomonadales.
Transfer of a single gene within the integron/gene cassette
system has been reported, including for the Xanthomonas
species (Gillings et al., 2005), but this was not detected by
our strategy.
Although the characteristics of a genome (such as nucleotide composition features) usually place a threshold in the
search for atypical genes, the evolutionary traits of a gene
(similarity and phylogeny) are more direct to identify
potential LGT events, making it possible to identify gene
FEMS Microbiol Lett 281 (2008) 87–97
clusters normally not detected by the traditional compositional approaches. Islands from potentially recent LGT
events carry genes that enlarge the adaptive responses of
bacteria in the environment, and are maintained, as they
probably facilitate the pathogenic life of these bacteria in
plants. The findings of this work are in agreement with
observations from literature that recently transferred genes
are under fast and relaxed evolution, and many of them may
be lost quickly from the genome, when compared with
ancient genes (Hao & Golding, 2006). On the other hand,
some of the other islands seem to provide genes related to
core metabolic pathways, replacing and maintaining functions that are normally acquired by vertical inheritance.
These data support the mosaic structure of Xanthomonas
genomes and the dynamic exchange of genetic information
among bacteria.
Acknowledgements
This work was supported by FAPESP (São Paulo, SP, Brazil)
and CNPq (Brası́lia, DF, Brazil). W.C.L. has a fellowship
from FAPESP, and A.C.M.P. and A.M.V have fellowships
from CAPES (Brası́lia, DF, Brazil). C.F.M.M. is a Fellow of
the John Simon Guggenheim Memorial Foundation (New
York, USA).
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Supplementary material
The following supplementary material is available for this
article online:
Table S1. Genomic coordinates and size of Xanthomonas
islands.
Fig. S1. Tetranucleotide usage variance of three more
deviant islands in XAC (A) and XCC (B).
Fig. S2. Prokaryotic phylogenetic tree based on rRNA 16S
sequences, computed by neighbor-joining (data obtained
FEMS Microbiol Lett 281 (2008) 87–97
97
LGT islands in Xanthomonas
through the Ribosomal Database Project website, http://
rdp.cme.msu.edu).
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/10.1111/j.15746968.2008.01083.x (This link will take you to the article
abstract).
FEMS Microbiol Lett 281 (2008) 87–97
Please note: Blackwell Publishing are not responsible
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Published by Blackwell Publishing Ltd. All rights reserved
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