Download Genetic transfer and genome evolution in MRSA

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Genetic transfer and
genome evolution in
MRSA
Whole genome sequences for a methicillinresistant Staphylococcus aureus (MRSA)
strain (N315) and a vancomycin-resistant S.
aureus strain (Mu50) have been reported
recently (16), and a number of partial genome
sequences for other MRSA strains are also
available (1, 19, 20), allowing comparison of
specific known sequences studied, and the
frequency of mutations within them. Three
regions of interest are the 16S–23S rDNA
intergenic spacer region (ISR), the seven
housekeeping genes used in multilocus sequence typing (MLST) (3) and the mecA gene.
The purpose of this article is to suggest that
the MRSA genome is subject to different
mechanisms of evolution ; this suggestion is
based on strain differences in these three
regions.
Variation in the type of ISR
found in MRSA strains
Interest in the ISR is due to its usefulness in
bacterial typing (11) and the identification of
highly conserved motifs within the ISR important for folding and for maturation of the
rRNA transcripts (12). Early reports (5, 6)
characterizing ISR sequences found a total of
10 different ISR types from three MRSA
strains (H11, ATCC 33952 and D46). Whole
genome sequence analysis from five other
strains (N315, 252, Mu50, 8325 and COL)
shown in Table 1a shows that (i) each of the
ISR types found in the five whole genomes
unambiguously matched one of the ISR types
found earlier in strains H11, ATCC 33952 and
D46 (5, 6) (no additional ISR types were
found), (ii) some ISR types were present in
some and absent in other strains (e.g. rrnA1
was only present in strain COL), (iii) some
ISR types were present in different copy
numbers in different strains (e.g. rrnC was
absent in strain COL and two copies were
present in strain N315) and (iv) there was a
variation in the total number of ISR types
between strains. Thus ISR type differences in
the whole genomes from the five strains listed
in Table 1a can be accounted for by the
presence or absence of the 10 ISR types
characterized earlier (5, 6).
Sequence variation within ISR
types between MRSA strains
A further contribution to differences in ISRs
between the whole genomes of the five strains
is from single nucleotide polymorphisms
(SNPs) between identical ISR types when in
an individual strain (see Table 1a), e.g. 10\546
(rrnA1), 3\473 (rrnC), 6\469 (rrnE), 7\460
(rrnF), 3\362 (rrnH) and 2\335 (rrnJ). Additionally, differences between alleles of the
same ISR type may occur within different
strains [e.g. rrnA1 (COL), rrnC (N315), rrnJ
(33952 and H11)]. In a previous study a ‘ T ’ at
nt 162 of rrnE was found in 92 % of MRSA
strains (7) ; this ‘ T ’ was found in rrnA1 of
COL and rrnE of H11, 252 and A48074 but
not in strains Mu50 and N315. Two copies of
the same ISR type that differ by a single
nucleotide difference isolated from one strain
could be explained by (i) the presence of
different alleles in the one genome or (ii)
possibly by the occurrence of different bacterial cells with different ISR types within the
one culture of one strain. Thus, further strain
differences are reflected in SNPs within strain-
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specific ISR types. It can be seen that the ISR,
a constant and possibly obligatory part of the
genome, is genetically dynamic and subject to
homogenization, consistent with concerted
evolution (5, 9, 10), which is only possible
because multiple copies of it occur in the one
genome.
Sequence variation within
seven housekeeping genes
MLST (sequencing and subsequent mutation
detection of parts of seven housekeeping, i.e.
essential, genes) represents a portable, precise
and sensitive method for typing MRSA strains
(3). The sequence data for parts of the seven
housekeeping genes from the whole genome
sequences of the five strains listed in Table 1b
is included as a standard because of the easy
availability of MLST data for a large number
of MRSA isolates (3). When the five genomes
are compared, the total number of SNPs
(31\2645) in all ISR types (Table 1a) is similar
to the total number of SNPs (43\3818) found
in the seven housekeeping genes (Table 1b). In
contrast to the ISR, only one copy of each
housekeeping gene is found on each whole
genome. Thus, the mechanisms for genetic
change are likely to be different to those with
ISRs.
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3195
Microbiology Comment
Table 1. Comparison of SNPs found in 18 alleles from the whole genome sequences of five MRSA strains
................................................................................................................................................................................................................................................................................................................................
Only those nucleotide positions (written directly below the ISR type or gene designation) that vary between strains are listed – all other
nucleotides are identical between strains.The early ISR sequence nomenclature (6) has been used and is recommended in further work on
rrns. Strains H11 and ATCC 33952 isolated from the Austin and Repatriation Medical Centre show further differences (strains ATCC
33592 and H11, rrnF at nt 443 and 453, rrnH at nt 8 and 24, and rrnJ at nt 15, 30, 34 and 151 ; strain H11 only has differences at nt 24 and
344 of rrnE). The nucleotide numbering for the ISR is according to the alignment in Gu$ rtler & Barrie (6). The ISR sequence block
designations (6) are shown directly below the sequences. Black shading indicates the absence of the entire ISR type within the whole
genome sequence of that strain. A dot indicates identity to the top nucleotide in each column ; k indicates absence of the nucleotide, allele
or ISR region and j indicates the presence of the whole sequence region or ISR type (e.g. VS5 and rrnA, G, K and L respectively). The
MLST housekeeping genes are shown with numbering according to Enright et al. (3). The nucleotide numbering for the coding sequence of
the mecA gene is according to GenBank X52593. The mecA sequence from strain COL is identical to mecA from S. sciuri (GenBank
SSK8MECA) and the mecA sequence from strain Mu50 is identical to the mecA sequences from S. epidermidis (GenBank SEMECAPB) and
MRSA strain 8325 (GenBank SAMECAPB).
(a) 16S–23S rDNA ISR
Strain
rrnA1
Length (nt) …
546
Position …
114444555
2565678011
4727624079
N315‡¶
252§
Mu50‡¶
8325§
COL‡§
CGTATAAAGC
TACGGGCTAA
cvvvvvvvvv
SSSSSSSSSS
1226666666
rrnC
473
rrnE
469
rrnF
460
rrnH
362
rrnJ
335
4
223
563
1 1 1 2V
95676S
072515
44455
7813922
9801568
44
422
367
3
48
36
GAC
..T
TC.
..T
TACAAj
.....j
AGTGG.....j
.....j
TC.
TC.
ccv
SSS
115
T--
iicvvvv
llSSSSS
ee25666
(b) Single copy genes – seven housekeeping genes and mecA
Strain
arcC
aroE
glp
Length (nt) …
570
536
576
Position …
N315¶
252§
Mu50¶
8325§
COL§
rrnG*
rrnK*
rrnL*
CTA
CTA
CTA
cvv
SSS
155
Copy
no.†
CA
5
.-
3
5
G-TTTTA
AACGCGG
vvvvvv
SSSSSS
122235
rrnA*
j
j
T.
5
7
cc
SS
12
gmk
488
pta
575
tpi
475
yqiL
598
mecA
2002
122223
245315562
314484568
1112345
3580581
35002739
1134
3484
4177
1334444
7360035
7465680
1234
33243
33925
223
057
428
1123345
2323673
3178820
7
73
59
CCAGTCACA
.TGAATGTG
.........
.T...TGT.
GT...TGT.
CGTATCGC
.A.GCA.T
........
A.A...A.
R
TCGC
ATAT
....
....
....
TTTCAGT
...TGAC
.......
CCATG..
CCATG..
AGGAA
GAACG
.....
...C.
...C.
TCA
CT.
...
.TT.
.TT
GTAGCTT
AC.AT.A
.......
..GAT.A
..GATCA
CG
A.
A.
A.
AA
* There are no SNPs in ISR types rrnA, G, K and L. ISR types rrnA, K and L are present in strain ATCC 33592 and ISR type rrnG is present in strain H11.
† The total number of ISR types found in the genome of each strain : there are seven and five for strains ATCC 33592 and H11, respectively.
‡ Strain N315 has two copies of ISR types rrnC and rrnE ; Mu50 has two copies of ISR type rrnE and COL has two copies of ISR types rrnA1 [A48073 in Gu$ rtler
(5)] and rrnH.
§ Taken from the partially completed whole genome sequences of strains COL (20), 8325 (1) and 252 (EMRSA-16) (19).
R The aroE sequence for strain COL was unavailable.
¶ Taken from the full genome sequences of strains N315 (methicillin-resistant) and Mu50 (vancomycin-resistant) reported in Kuroda et al. (16).
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Microbiology Comment
The mecA gene has less nucleotide
differences than other MRSA
sequences
The mecA gene is located on the staphylococcal cassette chromosome (SCC) which is a
new class of genetic element that is transferred
horizontally (2, 13–16, 21). Only two nucleotide positions vary in the mecA gene sequences
obtained from MRSA, Staphylococus epidermidis and Staphylococcus sciuri strains
(Table 1b). The number of differences in the
ISR types (31\2645) and the seven housekeeping genes (43\3818) is 15 times greater
than in the mecA gene sequence (2\2002).
Recombination or gene conversion between
rrn operons on a genome may play a role in
homogenizing the sequences of ISR types (5,
17). Similarly, it has been shown that recombination is responsible for SNPs in the
seven housekeeping genes of MRSA analysed
by MLST (4). If this is the case, the introduction of mutations by recombination
may be many times more frequent than during
or after the horizontal transfer of SCCmec.
While it is probable that the introduction of
mecA into S. aureus is a relatively recent
event, it is possible that the event is more
ancient and its introduction is more likely due
to increased transmission in hospitals and by
a higher exposure to antibiotics than occurs
in nature. However, the low rate of mutation
in mecA (compared to ISRs and housekeeping genes) must raise the question as to
whether there are different mechanisms of
mutation in different parts of the bacterial
genome.
Measurement of genome
evolution in MRSA
It has been proposed that the value and
stability of typing and taxonomy of bacteria
are highly dependent on mechanisms of
genome evolution, with different regions of
the bacterial chromosome undergoing different rates of genome evolution (8). The data
presented here suggest that at least three
mechanisms of genome evolution in MRSA
exist including (i) recombination and\or gene
conversion of the rrn operon resulting in
sequence homogeneity in some regions and
rearrangements, insertions or deletions in
others (5, 17), (ii) recombination of essential
housekeeping genes (4) and (iii) horizontal
transfer (2, 13, 15, 16). Based on sequence
data currently available, the mecA gene is a
poor candidate for typing because the sequence is highly stable between S. aureus, S.
epidermidis and S. sciuri. A more variable
region such as the ISR, which may undergo
frequent rearrangements, may be more suitable. Some of these questions may be resolved
by performing experiments (in S. aureus)
similar to those performed in Escherichia coli
to measure the mutation rates within different
genes (18) as a function of bacterial generation
times. Analysis of SNPs in different regions of
the S. aureus genome suggest that different
evolutionary mechanisms apply to different
regions of the genome and further studies to
elucidate these mechanisms may shed light on
the origin of MRSA.
Volker Gu$ rtler and Barrie C. Mayall
Department of Microbiology, Austin and
Repatriation Medical Centre, Studley Road,
Heidelberg 3084, Victoria, Australia.
Author for correspondence : V. Gu$ rtler.
Tel : j61 3 94963136. Fax : j61 3 94572590.
e-mail : volker!austin.unimelb.edu.au
1. Advanced Center for Genome Technology (2001).
http :\\www.genome.ou.edu\
2. Archer, G. L. & Niemeyer, D. M. (1994). Origin and
evolution of DNA associated with resistance to
methicillin in staphylococci. Trends Microbiol 2,
343–347.
3. Enright, M. C., Day, N. P., Davies, C. E., Peacock,
S. J. & Spratt, B. G. (2000). Multilocus sequence typing
for characterization of methicillin-resistant and
methicillin susceptible clones of Staphylococcus aureus.
J Clin Microbiol 38, 1008–1015.
4. Feil, E. J., Holmes, E. C., Bessen, D. E. & 9 other
authors (2001). Recombination within natural
populations of pathogenic bacteria : short-term empirical
estimates and long-term phylogenetic consequences. Proc
Natl Acad Sci U S A 98, 182–187.
5. Gu$ rtler, V. (1999). The role of recombination and
mutation in 16S–23S rDNA spacer rearrangements. Gene
238, 241–252.
6. Gu$ rtler, V. & Barrie, H. D. (1995). Typing of
Staphylococcus aureus strains by PCR-amplification of
variable-length 16S–23S rDNA spacer regions :
characterization of spacer sequences. Microbiology 141,
1255–1265.
7. Gu$ rtler, V., Barrie, H. D. & Mayall, B. C. (2001). Use
of denaturing gradient gel electrophoresis to detect
mutations in VS2 of the 16S–23S rDNA spacer amplified
from Staphylococcus aureus isolates. Electrophoresis 22,
1920–1924.
8. Gu$ rtler, V. & Mayall, B. C. (2001). Genomic
approaches to typing, taxonomy and evolution of
bacterial isolates. Int J Syst Evol Microbiol 51, 3–16.
9. Gu$ rtler, V. & Mayall, B. C. (1999). rDNA spacer
rearrangements and concerted evolution. Microbiology
145, 2–3.
10. Gu$ rtler, V., Rao, Y., Pearson, S. R., Bates, S. M. &
Mayall, B. C. (1999). DNA sequence heterogeneity in the
three copies of the long 16S–23S rDNA spacer of
Enterococcus faecalis isolates. Microbiology 145,
1785–1796.
11. Gu$ rtler, V. & Stanisich, V. A. (1996). New
approaches to typing and identification of bacteria using
the 16S–23S rDNA spacer region. Microbiology 142,
3–16.
12. Iteman, I., Rippka, R., Tandeau de Marsac, N. &
Herdman, M. (2000). Comparison of conserved
structural and regulatory domains within divergent 16S
rRNA–23S rRNA spacer sequences of cyanobacteria.
Microbiology 146, 1275–1286.
13. Ito, T., Katayama, Y., Asada, K., Mori, N.,
Tsutsumimoto, K., Tiensasitorn, C. & Hiramatsu, K.
(2001). Structural comparison of three types of
staphylococcal cassette chromosome mec integrated in
the chromosome in methicillin-resistant Staphylococcus
aureus. Antimicrob Agents Chemother 45, 1323–1336.
14. Ito, T., Katayama, Y. & Hiramatsu, K. (1999).
Cloning and nucleotide sequence determination of the
entire mec DNA of pre-methicillin-resistant
Staphylococcus aureus N315. Antimicrob Agents
Chemother 43, 1449–1458.
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15. Katayama, Y., Ito T. & Hiramatsu, K. (2000). A new
class of genetic element, staphylococcus cassette
chromosome mec, encodes methicillin resistance in
Staphylococcus aureus. Antimicrob Agents Chemother
44, 1459–1455.
16. Kuroda, M., Ohta, T., Uchiyama, I. and 34 other
authors (2001). Whole genome sequencing of
methicillin-resistant Staphylococcus aureus. Lancet 357,
1225–1240.
17. Liao, D. (2000). Gene conversion drives within genic
sequences : concerted evolution of ribosomal RNA genes
in bacteria and archaea. J Mol Evol 51, 305–317.
18. Papadopoulos, D., Schneider, D., Meier-Eiss, J.,
Arber, W., Lenski, R. E. & Blot, M. (1999). Genomic
evolution during a 10,000-generation experiment with
bacteria. Proc Natl Acad Sci U S A 96, 3807–3812.
19. Sanger Centre (2001). http :\\www.sanger.ac.uk\
20. TIGR (The Institute for Genomic Research) (2001).
http :\\www.tigr.org\tdb\mdb\mdbinprogress.html
21. Wu, S. W., de Lencastre, H. & Tomasz, A. (2001).
Recruitment of the mecA gene homologue of
Staphylococcus sciuri into a resistance determinant and
expression of the resistant phenotype in Staphylococcus
aureus. J Bacteriol 183, 2417–2424.
A second type III secretion
system in Burkholderia
pseudomallei : who is the
real culprit ?
Burkholderia pseudomallei is a Gram-negative motile bacillus that is the causative agent
of melioidosis, a severe emerging infection
that is endemic in South-East Asia and Northern Australia. Antibiotic therapy of melioidosis is long and difficult, because of the
resistance of the bacterium to many antibiotics and a tendency to relapse after recovery from clinical disease (3, 4). Although
some potential virulence factors have been
suggested, the pathogenesis of the disease is
poorly understood (1). Here we report on the
presence of a second, Salmonella SPI-1-like,
type III secretion gene cluster in B. pseudomallei.
In more than a dozen major Gram-negative bacterial pathogens of animals and plants,
virulence is largely dependent on type III
secretion (TTS) systems. The TTS, triggered
by a close contact of the bacterium with
eukaryotic host cells, involves the assembly of
a dedicated secretion\translocation apparatus
enabling the injection of pathogenicity (effector) proteins directly into the host cells.
The system components are encoded by a set
of approximately 20 genes which are usually
clustered in the bacterial genome, forming socalled pathogenicity islands (PIs). A major
difference between animal and plant pathogens is that the latter interact with the cell
cytoplasm by piercing from outside the
200 nm thick plant cell wall, while animal
pathogens have to deal with only about 5 nm
thick cell membranes. Accordingly, the protein composition and structure of what is
often called the ‘ translocator’ seem to be
quite different in these two pathogen groups.
3197
Microbiology Comment
..................................................................................................................................................................................................................
Fig. 1. Gene organization of TTS-associated clusters of B. pseudomallei and comparison with SPI-1 of
S. typhimurium and the HRP cluster of R. solanacearum. ORFs are represented as arrows according to
the pattern code of predicted proteins as indicated. Information on the HRP-like locus of B.
pseudomallei and the HRP cluster of R. solanacearum are available from GenBank (accession nos
AF074878 and RSO245811, respectively). ORFs in the new SPI-1-like locus of B. pseudomallei are
annotated as follows : conserved secretion apparatus components are named Sct (7) or Sct2 when the
gene already exists within the HRP-like locus ; putative homologues of Salmonella Sip, Sic and Sop
proteins are designated Bip, Bic and Bop, respectively ; other ORFs are annotated by the name of their
putative homologue, with a Bpm suffix (e.g. prgHBpm). All putative homologues within the new SPI-1like locus are from Salmonella, with the exception of BpH3 (Bordetella pertussis), GacA and PA4094
(Pseudomonas aeruginosa), and IcsB (Shigella flexneri).
animal pathogen Salmonella typhimurium,
in terms of both gene organization (Fig. 1) and
sequence similarity (Table 1). Although the
GjC content of this ‘ SPI-1-like’ locus is
close to that of the core genome of B. pseudomallei (about 68 mol %), the presence of
sequences similar to transposases at one
boundary suggests a possibility of horizontal
transfer.
The predicted proteins encoded at the B.
pseudomallei SPI-1-like locus include the conserved secretion apparatus components [annotated here as Scts, as proposed by Hueck (7)]
but also putative homologues of the translocators SipC and SipD and of the effectors SipB
and SopE (6). By analogy with their function
in Salmonella infection, the two latter
proteins might be involved in the B.
pseudomallei-induced apoptosis, cell fusion
and actin-associated membrane protrusion,
the phenotypes recently reported (8). No
putative homologues of other SPI-1 TTS
effectors SipA, SopA, SopB, SptP and AvrA
(6) were found in the B. pseudomallei genome.
It is possible that genes encoding additional
TTS-delivered effectors are scattered throughout the B. pseudomallei genome and await
molecular characterization.
B. pseudomallei is the first bacterial pathogen apparently armed with one TTS system
dedicated to infecting animal cells and another one to interact with plants. This represents a novel level of diversity and complexity
in the bacterial world, and challenges us to
elucidate the respective origins and roles of
these two TTS systems.
In several animal pathogens, mutants deficient in TTS have been shown to be avirulent
or attenuated in their ability to provoke
disease in animal models, thus making TTSsecreted proteins attractive targets for antimicrobial therapy and vaccine development
(9).
Acknowledgements
In addition, the effector proteins are speciesspecific, thus contributing to differences in
pathogenicity phenotypes (2, 7).
It has been reported that B. pseudomallei
contains a cluster of five genes with high
similarity to genes of a HRP (hypersensitive
response and pathogenicity) locus in the plant
pathogen Ralstonia solanacearum (10). The
same authors recently expanded this locus,
identifying several ORFs showing significant
similarity with TTS-associated genes of R.
solanacearum, Xanthomonas campestris or
Pseudomonas syringae (Fig. 1), thus completing the picture of a plant pathogen-like
TTS gene cluster in B. pseudomallei. Although the role of this locus is unknown, an
involvement in either symbiotic or pathogenic
bacterium–plant interactions can be speculated. Indeed, the predominant environment
for B. pseudomallei is rice fields and its
relationship with plants and the rhizosphere
has already been suggested (5).
B. pseudomallei is clearly a human pathogen, and it is unexpected that a plant
pathogen-like TTS system would be used for
human-cell intoxication. We searched the B.
pseudomallei genome using the  program with PcrD, a highly conserved component
of the secretion apparatus of the human
pathogen Pseudomonas aeruginosa, as the
query sequence. This search, performed
on the assembly contigs generated through
the B. pseudomallei genome sequencing project (www.sanger.ac.uk\Projects\BIpseudomallei), led to the identification of a second
cluster of about 25 genes with similarities to
TTS systems. The new locus resembles most
the TTS-associated SPI-1 of the human\
3198
We are grateful to Dr Sylvie Elsen for critical
reading of the manuscript.
Olivier Attree and Ina Attree1
1
Laboratoire de Biochimie et Biophisique des
Syste' mes Inte! gre! s, (UMR5092
CNRS/CEA/UJF), DBMS, CEA, 17 rue des
Martyrs, 38054 Grenoble, France.
Author for correspondence : Ina Attree.
Tel : j33 438783483. Fax : j33 438785185.
e-mail : iattreedelic!cea.fr
1. Brett, P. J. & Woods, D. E. (2000). Pathogenesis of
and immunity to melioidosis. Acta Trop 74, 201–210.
2. Cornelis, G. R. & Van Gijsegem, F. (2000). Assembly
and function of type III secretory systems. Annu Rev
Microbiol 54, 735–774.
3. Dance, D. A. (2000). Burkholderia pseudomallei
infections. Clin Infect Dis 30, 235–236.
4. Dance, D. A. (2000). Melioidosis as an emerging
global problem. Acta Trop 74, 115–119.
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Microbiology Comment
Table 1. Relatedness of B. pseudomallei predicted proteins to proteins encoded
within the SPI-1 locus of S. typhimurium and to B. pseudomallei putative proteins
from the HRP-like cluster, and the relatedness of B. pseudomallei HRP-like
putative proteins to proteins encoded by the HRP cluster of R. solanacearum
..................................................................................................................................................................................................................
Relatedness is measured as percentage of identical amino acid matches (percentage of
similar amino acid matches).
S. typhimurium
SPI-1 cluster
InvF
InvG
InvE
InvA
InvB
InvC
SpaO
SpaP
SpaQ
SpaR
SpaS
SicA
SipB
SipC
SipD
PrgH
PrgI
PrgJ
PrgK
B. pseudomallei
SPI-1-like cluster
33 (48)
38 (52)
30 (44)
55 (72)
26 (44)
54 (66)
24 (37)
58 (75)
54 (77)
38 (55)
43 (61)
58 (72)
31 (46)
16 (30)
22 (36)
20 (33)
52 (62)
28 (44)
39 (55)
InvFBpm
SctC2
SctW
SctV2
InvBBpm
SctN2
SctQ2
SctR2
SctS2
SctT2
SctU2
BicA
BipB
BipC
BipD
PrgHBpm
SctF
SctI
SctJ2
B. pseudomallei
HRP-like cluster
R.
solanacearum
HRP cluster
26 (43)
SctC
44 (60)
HrcC
36 (56)
SctV
57 (70)
HrcV
45 (62)
21 (33)
35 (60)
33 (57)
24 (36)
25 (44)
SctN
SctQ
SctR
SctS
SctT
SctU
63 (77)
23 (39)
64 (78)
56 (71)
41 (59)
49 (67)
HrcN
HrcQ
HrcR
HrcS
HrcT
HrcU
24 (41)
SctJ
48 (62)
HrcJ
5. Dharakul, T. & Songsivilai, S. (1999). The many
facets of melioidosis. Trends Microbiol 7, 138–140.
6. Hansen-Wester, I. & Hensel, M. (2001). Salmonella
pathogenicity islands encoding type III secretion systems.
Microb Infect 3, 549–559.
7. Hueck, C. J. (1998). Type III protein secretion systems
in bacterial pathogens of animals and plants. Microbiol
Mol Biol Rev 62, 379–433.
8. Kespichayawattana, W., Rattanachetkul, S., Wanun,
T., Utaisincharoen, P. & Sirisinha, S. (2000).
Burkholderia pseudomallei induces cell fusion and actinassociated membrane protrusion : a possible mechanism
for cell-to-cell spreading. Infect Immun 68, 5377–5384.
9. Turbyfill, K. R., Hartman, A. B. & Oaks, E. V. (2000).
Isolation and characterization of a Shigella flexneri
invasin complex subunit vaccine. Infect Immun 68,
6624–6632.
10. Winstanley, C., Hales, B. A. & Hart, C. A. (1999).
Evidence for the presence in Burkholderia pseudomallei
of a type III secretion system-associated gene cluster. J
Med Microbiol 48, 649–656.
Lipoxygenase in bacteria :
a horizontal transfer
event ?
Lipoxygenase (LOX ; EC 1;13;11;12) is a
non-haem iron-containing dioxygenase that
catalyses the addition of molecular oxygen to
polyunsaturated fatty acids with a (Z,Z)-1,4pentadiene system to give an unsaturated fatty
acid hydroperoxide. The oxygen can be added
to either end of the pentadiene system (regiospecificity).
In plants, linoleic and linolenic acids are
the most abundant fatty acids and the principal substrates of LOX. Its products have
several functions which include mediators of
the stress response and products with bactericidal activity (2). In animals the predominant substrate of LOX is arachidonic acid. Its
products include bioregulators, mainly prostaglandins, thromboxanes and leukotrienes,
with a role in the maintenance of the homeostasis of the animal cell (4).
Until now, LOX had been found in plants,
fungi and animals, but it was not known to be
present in yeast and bacteria. A careful search
of reported LOX sequences showed two
bacterial LOXs, one from Pseudomonas aeruginosa (accession no. AE004547) annotated as
a putative LOX, and the other from Sorangium cellulosum (accession no. AX024393).
The sequences of S. cellulosum and P. aeruginosa were obtained with the  search
program of the bacterial database (http :\\
www.ncbi.nlm.nih.gov\BLAST\), using as
template the Arabidopsis thaliana LOX2 (1).
The identification of a LOX gene in P.
aeruginosa may not be surprising, since LOX
activity has been reported in the periplasmic
Microbiology 147, December 2001
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space of Pseudomonas (3). Besides, for S.
cellulosum, an -dopa decarboxylase has been
reported that was thought to be present only
in eukaryotes (6) and LOX seems to be
another example of a eukaryote-related gene
present in this bacterium.
Although LOX activity has not been demonstrated for the specific sequences of S.
cellulosum and P. aeruginosa, results from
primary amino acid sequence analysis showed
that the residues involved in the iron binding
of soybean L-1 [His499, His504, His690,
Asn694 and Ile839 (5)], are conserved in an
equivalent position in S. cellulosum and P.
aeruginosa LOXs, suggesting that the active
site of bacterial and plant LOX is similar (Fig.
1a). The comparison of the S. cellulosum
sequence with other LOXs reveals 39n7 % and
42n3 % similarity with soybean L-3 and human
LOX5, respectively. P. aeruginosa LOX has
36n3 %, 36n3 % and 36n5 % similarity with
potato H3 LOX, human LOX5 and S. cellulosum LOX, respectively.
A phylogenetic tree was constructed from
the aligned protein sequences of LOX from
different origins : 19 plant LOXs with 13-,
13\9-, and 9-LOX proven activities, three
human LOXs with 5-, 12-, and 15-LOX
activity (accession numbers P09917, O75342,
O15296, respectively) ; a red alga Porphyra
purpurea LOX (accession number U08842) ;
and P. aeruginosa and S. cellulosum LOXs.
The alignment used for the phylogenetic
analysis was generated with the 
program (http :\\www.ebi.ac.uk\clustalw\).
The tree was constructed with the  search
program (Wisconsin Package Version 10.1,
Genetics Computer Group, Madison, WI),
and drawn with the  program. This
tree is the best tree by parsimony and it was
also the shortest distance tree. Results showed
that plant LOXs can be divided into five
groups representing four different plant families : Solanaceae, Fabaceae, Curcubitaceae,
Poaceae and, in a different group, chloroplastic LOXs from several plant families.
There is not phylogenetic relationship between the plant LOXs based on regiospecificity. Bacterial and red algal LOXs were
grouped with human LOXs. This result indicates that bacterial and red algal LOXs
appear to be more closely related to human
than to plant LOXs (Fig. 1b). Two methods
(parsimony and distance) agree on the tree
topology with moderate to high bootstrap
support from 1000 replicas. The presence
of LOX gene in S. cellulosum and P. aeruginosa may be interpreted as separate events
of horizontal transfer. It is not an artefact
of the phylogenetic method, because different methods agree, and there is statistical support. Furthermore, the  search
did not report another LOX sequence from
other reported bacterial genomes. A horizontal gene transfer has been also suggested
for the -dopa decarboxylase gene of
S. cellulosum (6).
3199
Microbiology Comment
(a)
(b)
.....................................................................................................
Fig. 1. (a) Alignment of Glycine max (G. max),
S. cellulosum, P. aeruginosa and human LOX5.
Numbers in the right margin refer to nucleotide
residues. The asterisks indicate residues involved
in iron-atom binding at the active site of LOX
(5). (b) Phylogenetic tree of LOX amino acid
sequences including 19 plant LOXs, human 5-,
12- and 15-LOXs, red alga, and bacterial LOXs.
This tree is the best tree by parsimony and it
was also the shortest distance tree. The numbers
indicate the bootstrap values (parsimony\
distance) from 1000 replicas.
Helena Porta and Mario Rocha-Sosa
Departamento de Biologia Molecular de
Plantas, Instituto de Biotecnologia,
Universidad Nacional Auto! noma de Me! xico,
Apdo. Postal 510-3, Cuernacava, Mor. 62250,
Me! xico.
Author for correspondence : Mario RochaSosa. Tel : j52 7 329 1652. Fax : j52 73
172388. e-mail : rocha!ibt.unam.mx
1. Bell, E. & Mullet, J. E. (1993). Characterization of
an Arabidopsis lipoxygenase gene responsive to
methyl jasmonate and wounding. Plant Physiol 103,
1133–1137.
2. Creelman, R. A. & Mullet, J. E. (1997). Biosynthesis
and action of jasmonates in plants. Ann Rev Plant
Physiol Plant Mol Biol 48, 355–381.
3. Guerrero, A., Casals, I., Busquets, M., Leo! n, Y. &
Manresa, A. (1997). Oxidation of oleic acid to (E)-10hydroperoxy-8-octadecenoic and (E)-10-hydroxy-8octadecenoic acids by Pseudomonas sp. 42A2. Biochim
Biophys Acta 12, 75–81.
3200
4. Kuhn, H. & Thiele, B. (1999). The diversity of the
lipoxygenase family : many sequence data but little
information on biological significance. FEBS Lett 449,
7–11.
5. Minor, W., Steczko, J., Stec, B., Otwinowski, Z.,
Bolin, T. J., Walter, R. & Axelrod, B. (1996). Crystal
structure of soybean lipoxygenase L-1 at 1n4 AH resolution.
Biochemistry 35, 10687–10701.
6. Muller, R., Gerth, K., Brandt, P., Blocker, H. &
Beyer, S. (2000). Identification of an -dopa
decarboxylase gene from Sorangium cellulosum So ce90.
Arch Microbiol 173, 303–306.
Microbiology 147, December 2001
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