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. 2010 Nov 19;77(2):688–695. doi: 10.1128/AEM.01263-10

Polyphyletic Origin of Vibrio vulnificus Biotype 2 as Revealed by Sequence-Based Analysis ▿ †

Eva Sanjuán 1, Fernando González-Candelas 2,3, Carmen Amaro 1,*
PMCID: PMC3020543  PMID: 21097581

Abstract

A sequence-based analysis of seven housekeeping and virulence-related genes shows that the species Vibrio vulnificus is subdivided into three phylogenetic lineages that do not correspond with the biotypes and that biotype 2 is polyphyletic. These results support the reclassification of biotype 2 as a pathovar that would group the strains with pathogenic potential to develop vibriosis in fish.

Vibrio vulnificus is a pathogenic bacterial species that inhabits brackish waters in warm and tropical ecosystems (9, 11, 24). The species is highly heterogeneous and has been subdivided into three biotypes. Biotype 1 is the most abundant; it is distributed worldwide and causes sporadic cases of human vibriosis. Biotype 2 is also distributed worldwide, and it is the only one that harbors the genetic information to infect both fish and humans. Finally, biotype 3 is geographically restricted to Israel, and it causes outbreaks of human vibriosis after fish handling (1, 4, 13, 25). Biotype 2 is further subdivided into serovars A, E, and I (7; unpublished results), serovar E being the one associated with human vibriosis (1).

The genes essential for fish vibriosis are located in a recently described virulence plasmid that can be transmitted between strains cointegrated with a conjugative plasmid (12) that is present in almost 90% of biotype 2 isolates (17). In contrast, the genetic basis of human infection is poorly understood, since the putative virulence factors identified so far are found in both clinical and environmental isolates of the three biotypes (24, 27).

Several studies based on multilocus sequence typing (MLST) of housekeeping genes (2, 3, 6) and on ribotyping (21) suggest that the species is subdivided into two main evolutionary lineages with apparently different human pathogenic potential; one includes a majority of the human clinical isolates of biotype 1 (clinical branch), and the other a majority of the environmental isolates of the same biotype (environmental branch). The few isolates of biotype 2 studied are in the environmental branch, while biotype 3 strains are in a variable position depending on the study (2, 3, 6).

Given this scenario, the aim of this work has been to analyze the evolutionary origins of biotype 2, starting from the hypothesis that horizontal transfer of the virulence plasmid together with recombination events could have played a major role in the emergence of this biotype. To this end, a sequence-based analysis of three virulence-associated (vvhA, wzz, and pilF) and four housekeeping (glp, mdh, pyrC, and pntA) genes (the last ones selected from the MLST scheme for V. vulnificus [3]) was applied to a collection of 115 isolates that included strains of the three biotypes from clinical (humans and fish) and environmental sources (Table 1). The primer pairs for the genes are listed in Table S1 in the supplemental material. The genetic variability (θ) at the locus and biotype level was examined by using DnaSP4.09 (20). pilF and wzz (genes involved in surface antigen biogenesis) showed the highest levels of genetic variability (Table 2). Regarding the biotypes, biotype 1 showed the highest genetic variability, while biotype 2 was highly homogeneous, and no genetic diversity was observed among the biotype 3 isolates (see Table S2 in the supplemental material).

TABLE 1.

Characteristics of the V. vulnificus isolates used in this study

Strain Country and yr of isolation Source Biotypea and serovar STb No. of allelic sequencesb of:
glp pntA pyrC pilF wzz vvhA mdh
YJ106 Taiwan Human blood BT1/3 1 1 1 30 18 1 21 5
CECT 4608 Spain, 1990 Eel farm water BT1/3 2 1 2 12 11 11 2 13
BT3-1033 Israel, 1996 Diseased human BT1/3 3 2 5 2 10 13 5 10
BT3-11028 Israel, 1996 Diseased human BT1/3 3 2 5 2 10 13 5 10
BT3-12 Israel, 1996 Diseased human BT1/3 3 2 5 2 10 13 5 10
BT3-162 Israel, 1997 Diseased human BT1/3 3 2 5 2 10 13 5 10
BT3-32 Israel Diseased human BT1/3 3 2 5 2 10 13 5 10
BT3-97 Israel, 1997 Diseased human BT1/3 3 2 5 2 10 13 5 10
A13 Spain, 2002 Diseased eel BT2 non-serovar E (serovar A) 4 3 7 3 1 2 7 6
CECT 5198 Spain, 1999 Diseased eel BT2 non-serovar E (serovar A) 5 3 7 3 1 2 7 8
CECT 7029 Denmark, 2004 Diseased eel BT2 non-serovar E (serovar A) 5 3 7 3 1 2 7 8
CECT 7030 Denmark, 2004 Diseased eel BT2 non-serovar E (serovar A) 5 3 7 3 1 2 7 8
A10 Spain, 2002 Diseased eel BT2 non-serovar E (serovar A) 5 3 7 3 1 2 7 8
CECT 5343 Spain, 2000 Diseased eel BT2 non-serovar E (serovar A) 6 3 7 3 1 2 7 28
A11 Spain, 2002 Diseased eel BT2 non-serovar E (serovar A) 7 3 7 3 1 2 7 32
CECT 5689 Spain, 2002 Diseased eel BT2 non-serovar E (serovar A) 8 3 7 17 1 2 7 29
CECT 5768 Spain, 2001 Diseased eel BT2 non-serovar E (serovar A) 9 3 7 17 1 2 7 30
CECT 5769 Spain, 2002 Diseased eel BT2 non-serovar E (serovar A) 10 3 7 17 1 2 7 31
L49 Japan Brackish water BT1/3 11 3 7 26 1 2 7 6
PD-2-66 Spain, 2003 Eel tank water BT1/3 12 3 8 26 25 28 15 8
JE USA Oyster BT1/3 13 3 15 13 2 24 1 25
CECT 5166 USA Human blood BT1/3 14 3 18 15 14 16 2 14
CECT 4867 Unknown Diseased eel BT1/3 15 4 22 31 26 3 15 33
534 Sweden Diseased eel BT1/3 16 5 8 4 2 4 12 8
CECT 4869 Belgium, 1990 Diseased eel BT1/3 17 5 8 13 2 14 19 1
A2 Spain, 2000 Eel tank water BT1/3 18 6 11 4 2 11 2 7
An4 Spain, 2000 Eel tank water BT1/3 18 6 11 4 2 11 2 7
An5 Spain, 2000 Eel tank water BT1/3 18 6 11 4 2 11 2 7
An6 Spain, 2000 Eel tank water BT1/3 18 6 11 4 2 11 2 7
An7 Spain, 2000 Eel tank water BT1/3 18 6 11 4 2 11 2 7
PD-1 Spain, 2001 Eel tank water BT1/3 18 6 11 4 2 11 2 7
PD-3 Spain, 2001 Eel tank water BT1/3 18 6 11 4 2 11 2 7
PD-5 Spain, 2001 Eel tank water BT1/3 18 6 11 4 2 11 2 7
V1 Spain, 2001 Eel tank water BT1/3 18 6 11 4 2 11 2 7
PD-12 Spain, 2001 Eel tank water BT1/3 19 6 11 4 23 11 2 7
CECT 4606 Spain, 1990 Healthy eel BT1/3 20 6 11 4 4 3 2 8
MLT 362 USA Environmental BT1/3 21 7 4 21 8 25 16 33
VV 1003 USA Environmental BT1/3 22 8 23 28 27 29 14 24
MLT 364 USA Environmental BT1/3 23 9 24 24 8 9 16 8
MLT 406 USA Environmental BT1/3 24 10 19 26 2 3 2 1
MLT404 USA Environmental BT1/3 25 11 20 25 21 26 7 33
ATCC 33816 USA Human blood BT1/3 26 12 9 12 16 12 7 4
CG100 Taiwan, 1993 Oyster BT1/3 27 12 9 12 15 20 9 2
V2 Unkown Environmental BT1/3 28 12 9 12 16 12 13 4
CG110 Taiwan, 1993 Seawater BT1/3 29 12 9 19 15 12 4 D3
VV 352 USA Environmental BT1/3 30 12 9 29 16 12 17 19
CG111 Taiwan, 1993 Seawater BT1/3 31 12 14 20 15 12 4 2
CG118 Taiwan, 1993 Seawater BT1/3 32 12 14 20 15 12 4 4
CG106 Taiwan, 1993 Oyster BT1/3 33 12 14 21 17 21 18 3
CECT 5167 Japan Human blood BT1/3 34 13 14 16 15 17 4 12
VV 425 USA Environmental BT1/3 35 13 21 4 18 31 5 27
CECT 5164 USA Human blood BT1/3 36 14 3 12 13 15 20 20
CECT 5169 USA Human blood BT1/3 37 15 7 4 17 19 11 22
CS9133 South Korea Human blood BT1/3 38 15 14 22 18 22 10 22
KH03 Japan, 2003 Human blood BT1/3 39 16 14 12 19 18 4 23
E4 USA Seafood BT1/3 40 16 14 21 19 18 4 26
V4 Australia Human blood BT1/3 41 17 14 27 18 30 22 23
94-9-118 Denmark, 1994 Expectoration from lungs BT1/3 42 18 22 5 2 5 1 8
G83 South Korea Fish BT1/3 43 19 16 23 20 23 8 8
CECT 5168 USA Human blood BT1/3 44 20 7 4 16 18 4 21
YN03 Japan, 2003 Human blood BT1/3 45 21 13 9 12 32 7 7
94385 Spain, 2001 Leg wound BT1/3 46 22 14 8 6 8 8 7
536 Sweden Diseased eel BT2 non-serovar E 47 23 10 3 3 2 7 8
535 Sweden Diseased eel BT2 non-serovar E 48 23 10 3 3 2 7 15
CECT 5165 USA Seawater BT1/3 49 23 21 14 14 3 2 8
Riu-1 Spain, 2003 Seawater BT1/3 50 24 16 8 20 7 8 7
94-9-130 Denmark, 1994 Water BT1/3 51 25 8 7 5 7 1 8
Riu-3 Spain, 2003 Seawater BT1/3 52 25 12 3 24 12 1 8
PD-2-58 Spain, 2003 Eel tank water BT2 non-serovar E 53 25 12 3 24 3 1 8
PD-2-52 Spain, 2003 Eel tank water BT2 non-serovar E 54 25 12 3 24 27 1 8
94-9-119 Denmark, 1994 Wound infection BT1/3 55 26 17 6 2 6 1 8
A14 Spain, 2002 Diseased eel BT2 non-serovar E 56 27 7 3 1 2 7 8
95-8-161 Denmark, 1995 Diseased eel BT2 non-serovar E (serovar I) 57 28 7 9 7 2 7 16
95-8-162 Denmark, 1995 Diseased eel BT2 non-serovar E (serovar I) 58 28 7 9 7 2 7 17
95-8-6 Denmark, 1995 Diseased eel BT2 non-serovar E (serovar I) 59 28 7 10 7 2 7 8
95-8-7 Denmark, 1995 Diseased eel BT2 non-serovar E (serovar I) 60 28 7 11 8 2 7 8
CIP 81.90 France Human blood BT2 serovar E 61 29 7 4 4 3 3 8
CECT 4862 Japan, 1979 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
90-2-11 Denmark, 1990 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
94-8-112 Denmark, 1994 Wound infection BT2 serovar E 62 29 8 4 4 3 3 8
94-9-123 Denmark, 1994 Seawater BT2 serovar E 62 29 8 4 4 3 3 8
CCUG 38521 Sweden, 1997 Wound infection BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4174 Japan, 1979 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4601 Spain, 1989 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4602 Spain, 1990 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4603 Spain, 1990 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4605 Spain, 1990 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4607 Spain, 1992 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4863 USA Leg wound infection BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4864 Spain, 1994 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4865 Taiwan Diseased shrimp BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4866 Australia Human blood BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4870 Sweden, 1991 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4917 Spain, 1997 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 897 Japan, 1979 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 898 Japan, 1979 Diseased eel BT2 serovar E 62 29 8 4 4 3 3 8
PD-2-47 Spain, 2003 Eel tank water BT2 serovar E 62 29 8 4 4 3 3 8
PD-2-55 Spain, 2003 Eel tank water BT2 serovar E 62 29 8 4 4 3 3 8
PD-2-56 Spain, 2003 Eel tank water BT2 serovar E 62 29 8 4 4 3 3 8
Riu-2 Spain, 2003 Seawater BT2 serovar E 62 29 8 4 4 3 3 8
UE516 Taiwan Diseased Japanese eel BT2 serovar E 62 29 8 4 4 3 3 8
CECT 4868 Norway, 1990 Diseased eel BT2 serovar E 63 29 8 4 4 3 3 10
CECT 4999 Spain, 1999 Diseased eel BT2 serovar E 64 29 8 4 4 3 3 16
C1 Spain, 2003 Healthy eel BT2 serovar E 65 29 8 4 4 3 3 17
CECT 4604 Spain, 1990 Diseased eel BT2 serovar E 66 29 8 4 4 3 3 18
CECT 4998 Spain, 1997 Diseased eel BT2 serovar E 66 29 8 4 4 3 3 18
CECT 5139 Spain, 1998 Diseased eel BT2 serovar E 66 29 8 4 4 3 3 18
PD-2-50 Spain, 2003 Eel tank water BT2 serovar E 67 29 8 4 4 3 3 32
PD-2-51 Spain, 2003 Eel tank water BT2 serovar E 67 29 8 4 4 3 3 32
CECT 5762 Spain, 2002 Healthy eel BT2 serovar E 68 29 8 18 4 3 3 17
CECT 5763 Spain, 2002 Eel tank water BT2 serovar E 69 29 8 18 4 3 3 8
CECT 529T USA Human blood BT1/3 70 29 18 15 14 16 2 14
960717-1/2F Denmark, 1996 Diseased eel BT2 non-serovar E 71 30 6 4 9 6 1 11
960426-1/4C Denmark, 1996 Diseased eel BT2 non-serovar E 72 30 6 4 9 10 6 11
N87 Japan, 1987 Human blood BT1/3 73 31 4 1 22 33 4 9
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a

BT, biotype.

b

Sequences from each locus were aligned using Vector NTI 9.0.0 software (Infomax) and were edited manually by visual inspection. Different allelic sequences within a locus were assigned arbitrary numbers. Each isolate was consequently given a 7-number sequence designated a sequence type (ST).

TABLE 2.

Genetic diversity parameters for the 4 housekeeping and 3 virulence-associated loci studied in 115 V. vulnificus isolates

Locus Chromosome Sequence length No. of haplotypes Haplotype diversity Nucleotide diversity (π) Polymorphic sites (S) θ (from S) Pairwise nucleotide differences (k)
mdh I 489 32 0.82 8.5·10−3 23 0.01 4.2
pilF I 480 27 0.87 3.3·10−2 57 0.025 15.72
wzz I 460 33 0.84 2.7·10−2 61 0.027 12.65
glp I 479 31 0.87 1.8·10−2 50 0.02 8.55
pyrC II 423 31 0.79 1.6·10−2 39 0.02 6.99
pntA II 396 24 0.85 1.5·10−2 33 0.018 5.95
vvhA II 432 22 0.85 1.8·10−2 32 0.015 7.921
Value for:
    All genes 3,159 73 0.95 2·10−2 295 0.017 20.87
    Chromosome I 1,908 66 0.94 2.1·10−2 191 0.02 41.11
    Chromosome II 1,251 53 0.91 1.6·10−2 104 0.017 20.88
    Housekeeping genes 1,787 68 0.95 1.4·10−2 145 0.016 25.69
    Virulence genes 1,372 49 0.89 2.6·10−2 150 0.023 36.3
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To analyze the phylogeny of the V. vulnificus collection, we constructed a maximum likelihood (ML) tree from the 3,159-bp concatenated sequence of the seven loci (Fig. 1) by using PHYML 2.4.4 (8). The most appropriate model for nucleotide substitution was assessed with Modeltest version 3.7 (16). The concatenated tree shows the isolates clustered into three main lineages (Fig. 1). Lineage I (LI) contained isolates of biotypes 1 and 2 from fish farms and isolates from diseased fish and humans infected through fish handling or water contact. This lineage is enriched in European isolates, probably because the fish-farming industry is especially developed in Europe, whose countries apply specific-pathogen-control programs. LII was formed by biotype 3 strains from Israel, and LIII included biotype 1 isolates mostly recovered from environmental samples or from human septicemic cases registered in the United States and Asia. The nucleotide diversity within each lineage was then examined, and it was found that LI and LIII have similar values (π in Table S2 in the supplemental material). The human isolates are genetically more diverse than those from environmental origins, and both are much more diverse than isolates from diseased animals. This result would suggest that multiple environmental clones have the ability to infect humans, which correlates with human cases being presented as sporadic infections worldwide, and that only a few clones are able to infect fish, although they are overrepresented by clone amplification after epizootics in fish farms. The exception would be the clone formed by biotype 3 isolates, the only ones capable of causing outbreaks of human vibriosis.

FIG. 1.

FIG. 1.

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Maximum likelihood phylogenetic tree of 115 V. vulnificus isolates obtained from the alignment of 7 concatenated loci. Black, biotype 1 isolates; blue, biotype 2 serovar E isolates; red, biotype 2 non-serovar E isolates; green, biotype 3 isolates; #, human isolates; §, diseased fish isolates. Branches where recombination events involving the indicated loci might have occurred are indicated by arrows. The numbers at the nodes represent the percentage values given by bootstrap analysis of 1,000 replicates.

Furthermore, LI can be subdivided into four main groups, as follows: LI-A, grouping all biotype 2 serovar E isolates plus a Spanish biotype 1 isolated from a fish tank (CECT 4606); LI-B, formed by two atypical isolates of biotype 2 from Denmark; LI-C, clustering all non-serovar E biotype 2 isolates together with biotype 1 isolates from fish farms and humans; and lastly, LI-D, grouping biotype 1 isolates from the environment and humans (mainly from blood). Thus, biotype 2 is polyphyletic and appears to be divided into serovar-related subgroups, the isolates in each subgroup being more related to biotype 1 isolates from fish farms than to each other. This result is compatible with the hypothesis that biotype 2 emerged by acquisition of the virulence plasmid by V. vulnificus strains from fish farms.

The groupings of strains changed when only virulence or housekeeping genes were considered (Fig. 2). In both cases, the position of biotype 3 changed, and this also occurred in the individual gene trees (Fig. S1 to S5 in the supplemental material). Previous studies of the phylogeny of V. vulnificus (based on different techniques, multilocus enzyme electrophoresis, MLST, or 16S rRNA or gene sequencing) divided the species into two main lineages, the clinical and the environmental lineages (5, 6, 10, 15, 19, 23, 26). In our study, the division in the two lineages was observed in the phylogenetic trees from virulence-related or from housekeeping genes but not in the concatenated phylogenetic tree. Thus, it can be concluded that combining four housekeeping and three virulence gene sequences in the analysis gives enough resolution to show biotype 3 as an independent lineage. Bisharat et al. (3) proposed that biotype 3 contains a mosaic genome that would have evolved by hybridization of genomes of representative strains of the other two lineages. Our results support this hypothesis, although the change of position in the trees affected not only biotype 3 isolates but also biotype 2 non-serovar E isolates and biotype 1 strains, mostly from fish-farming environments (Fig. 2; also see Fig. S1 to S5 in the supplemental material).

FIG. 2.

FIG. 2.

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Maximum likelihood phylogenetic tree of 115 V. vulnificus isolates obtained from the alignment of 4 housekeeping (A) and 3 virulence-associated (B) concatenated loci. Symbols indicate isolates that cluster in LI-A (•), LI-B (▪), LI-C (▴), LI-D (⧫), LII (○), or LIII (□) in the concatenated tree. The numbers at the nodes represent the percentage values given by bootstrap analysis of 1,000 replicates.

The Recombination Detection Program, version 3 (RDP3) (14) detected recombination in chromosome I by at least four of the implemented methods, involving two of the four loci (pilF and wzz). The putative recombinant events were mapped onto the phylogenetic tree of the seven concatenated loci (Fig. 1). To further corroborate the RDP3 results, two different ML trees were constructed for each recombinant locus. The tree obtained from the multiple sequence alignment of each locus was compared with the one obtained from the concatenated sequences of all the other loci by using TreePuzzle version 5.2 (22). In both cases, Shimodaira-Hasegawa (SH) and expected likelihood weight (ELW) tests revealed significant differences between the two topologies (Table 3). Figures 3 and 4 each show the phylogenetic tree of one of the loci involved in recombination (pilF and wzz) and the tree derived from the remaining aligned loci. In both cases, biotype 2 serovar E strains are involved in the recombination events, either as predicted parental strains, in wzz, or as daughter strains, in pilF. The other strains involved in recombination events are biotype 2 nontypeable strains and clinical and environmental biotype 1 isolates. This result suggests that the conditions of aquaculture settings (e.g., high nutrient loads and high host density) might favor the exchange of genetic material among strains of V. vulnificus, originating new variants in the V. vulnificus species.

TABLE 3.

Likelihood scores for the loci identified as involved in recombination eventsa

Alignment −lnL for ML tree of the:
Locus Concatenated loci
pilF −3,136.12 −4,148.14*/*
Concatenated loci without pilF −11,095.79*/* −9,753.16
wzz −3,125.20 −4,001.95*/*
Concatenated loci without wzz −11,302.35*/* −9,503.07
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a

For each recombinant locus, we considered two multiple alignments, the one from that locus and that obtained from the concatenated alignment of the other 6 loci. These two alignments were used to evaluate the likelihood (−lnL, negative natural log of the likelihood) of the ML trees obtained with each of them, and the tree obtained with each alignment is compared with that derived from the other by the Shimodaira-Hasegawa (SH) and expected likelihood weight (ELW) tests. Levels of significance for the SH/ELW tests: *, P < 0.001.

FIG. 3.

FIG. 3.

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Maximum likelihood phylogenetic trees from 6 of the 7 concatenated loci, excluding region pilF (A) and the pilF locus (B). Isolates identified as recombinants are marked with a filled circle, while the putative parental isolates are marked with an open circle. The numbers at the nodes represent the percentage values given by bootstrap analysis of 1,000 replicates.

FIG. 4.

FIG. 4.

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Maximum likelihood (ML) phylogenetic trees from 6 of the 7 concatenated loci, excluding region wzz (A) and the wzz locus (B). Isolates identified as recombinants are marked with a filled circle, while the putative parental isolates are marked with an open circle. The numbers at the nodes represent the percentage values given by bootstrap analysis of 1,000 replicates.

Interestingly, the pilF ML tree splits the strains into two groups, one that clusters most clinical biotype 1 isolates from humans together with biotype 3 and biotype 2 serovar E isolates from human origins and another that groups serovar A and I isolates together with environmental biotype 1 isolates (Fig. 3B), suggesting that pilF could be used as a genetic marker to distinguish isolates potentially dangerous to humans. A PCR-based protocol to distinguish V. vulnificus isolates with pathogenic potential against humans based on the polymorphism in pilF has been designed and validated in our laboratory (18).

Conclusion.

The V. vulnificus species is subdivided into three different phylogenetic lineages which do not correspond to the current intraspecific biotype classification. LI and LII seem to have evolved in fish-farming-related environments where recombination or/and horizontal transfer phenomena would have favored the emergence of pathogenic clones for fish or humans, which would have been amplified after outbreaks of fish (biotype 2) or human (biotype 3) vibriosis. The polyphyletic origin of the so-called biotype 2 supports its reclassification within the species as a pathovar that would group the strains with pathogenic potential to infect and develop vibriosis in fish.

Supplementary Material

[Supplemental material]
supp_77_2_688__index.html (2.2KB, html)

Acknowledgments

This work was financed by grants AGL2008-03977/ACU, BFU2008-03000, and Programa Consolider-Ingenio 2010 CSD2009-00006 from MICINN and by ACOMP/2009/240 from Conselleria d'Educació (Generalitat Valenciana).

We thank the SCSIE of the University of Valencia for technical support in determining the sequences.

Footnotes

▿

Published ahead of print on 19 November 2010.

†

Supplemental material for this article may be found at http://aem.asm.org/.

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Supplementary Materials

[Supplemental material]
supp_77_2_688__index.html (2.2KB, html)
supp_77_2_688__Supplementary_Table_1.doc (28.5KB, doc)
supp_77_2_688__supplement_table_2.doc (152KB, doc)
supp_77_2_688__supfig1.zip (448KB, zip)
supp_77_2_688__supfi2.zip (430.2KB, zip)
supp_77_2_688__supfig3.zip (480.2KB, zip)
supp_77_2_688__supfig4.zip (408.9KB, zip)
supp_77_2_688__supfig5.zip (453KB, zip)
supp_77_2_688__legend_to_the_supplementary_figures.doc (23.5KB, doc)

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