# Acetic Acid Bacteria Genomes Reveal Functional Traits for Adaptation to Life in Insect Guts

Bessem Chouaia, Stefano Gaiarsa, Elena Crotti, Francesco Comandatore, Mauro Degli Esposti, Irene Ricci, Alberto Alma, Guido Favia, Claudio Bandi, Daniele Daffonchio

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Bessem Chouaia, Stefano Gaiarsa, Elena Crotti, Francesco Comandatore, Mauro Degli Esposti, et al.. Acetic Acid Bacteria Genomes Reveal Functional Traits for Adaptation to Life in Insect Guts. Genome Biology and Evolution, 2014, 6 (4), pp.912 - 920. 10.1093/gbe/evu062. hal-04472155

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# Acetic Acid Bacteria Genomes Reveal Functional Traits for Adaptation to Life in Insect Guts

Bessem Chouaia<fnref n="1" />, Stefano Gaiarsa<fnref n="1" />,<fnref n="2" />, Elena Crotti<fnref n="1" />, Francesco Comandatore<fnref n="2" />, Mauro Degli Esposti<fnref n="3" />, Irene Ricci<fnref n="4" />, Alberto Alma<fnref n="5" />, Guido Favia<fnref n="4" />, Claudio Bandi<fnref n="2" />, and Daniele Daffonchio<fnref n="1" />,*

Accepted: March 12, 2014

**Data deposition:** This project has been deposited at EMBL/GenBank under the accession numbers CBLX010000001–CBLX010000027 for *Asaia platycodi* genome and CBLY010000001–CBLY010000009 for *Saccharibacter* sp. genome.

## Abstract

Acetic acid bacteria (AAB) live in sugar rich environments, including food matrices, plant tissues, and the gut of sugar-feeding insects. By comparing the newly sequenced genomes of *Asaia platycodi* and *Saccharibacter* sp., symbionts of *Anopheles stephensi* and *Apis mellifera*, respectively, with those of 14 other AAB, we provide a genomic view of the evolutionary pattern of this bacterial group and clues on traits that explain the success of AAB as insect symbionts. A specific pre-adaptive trait, cytochrome *bo*₂ ubiquinol oxidase, appears ancestral in AAB and shows a phylogeny that is congruent with that of the genomes. The functional properties of this terminal oxidase might have allowed AAB to adapt to the diverse oxygen levels of arthropod guts.

**Key words:** symbiosis, acetic acid bacteria, cytochrome oxidase.

## Introduction

Besides plant tissues and food matrices, acetic acid bacteria (AAB) live in symbiosis with insects (reviewed in Crotti et al. 2010). Several research teams have investigated the relationship between AAB and their host (Crotti et al. 2010) focusing on the insect gut. In addition to the intestine, AAB could also be localized in other insect body compartments. For instance, the acetic acid bacterium *Asaia* colonizes not only the gut but also the salivary glands and the male and female reproductive systems, which are crucial sites for the bacterial transmission by horizontal and vertical routes (Damiani et al. 2008; Crotti et al. 2009; Gonella et al. 2012). Studies aiming to understand the nature of AAB symbiosis focused on the role of or potential advantages given by AAB to their respective hosts (Ryu et al. 2008; Chouaia et al. 2012; Lee et al. 2013). Key traits for intimately interacting with the insect host include, among others, the capacity to colonize host tissues and the interaction with the innate immunity and the developmental pathways of

the host (Ryu et al. 2008; Gross et al. 2009; Douglas et al. 2011; Shin et al. 2011; Lee et al. 2013; Login and Heddi 2013). For instance, *Asaia* exerts a beneficial role during the development of mosquito larvae (Chouaia et al. 2012; Mitraka et al. 2013) affecting the expression of genes related to the cuticle formation (Mitraka et al. 2013). In *Drosophila*, AAB are involved in the modulation of innate immunity, which keeps pathogenic strains under control (Ryu et al. 2008). Moreover, in the same host, *Acetobacter pomorum* modulates the insulin signaling, a pathway involved in the regulation of development, body size, energy metabolism, and intestinal stem cell activity of the host (Shin et al. 2011).

There are actually 14 genomes of AAB deposited in the databases but a genomic analysis of the evolutionary factors driving the association with insects is lacking. By including novel genome sequences of two AAB, *Asaia platycodi* and *Saccharibacter* sp., respectively, isolated from the malaria vector *Anopheles stephensi* and the honeybee *Apis mellifera*,

<footnote n="1">¹Department of Food, Environmental, and Nutritional Sciences (DeFENS), University of Milan, Italy</footnote>

<footnote n="2">²Dipartimento di Scienze Veterinarie e Sanità Pubblica (DIVET), University of Milan, Italy</footnote>

<footnote n="3">³Italian Institute of Technology (IIT), Genoa, Italy</footnote>

<footnote n="4">⁴Scuola di Bioscienze e Biotecnologie, Università degli Studi di Camerino, Camerino, Italy</footnote>

<footnote n="5">⁵Dipartimento di Scienze Agrarie (DISAFA), Forestali e Alimentari, University of Turin, Grugliasco, Italy</footnote>

<footnote>*Corresponding author: E-mail: daniele.daffonchio@unimi.it.</footnote>
we present a genomic evolutionary analysis of AAB for assess-
ing traits associated with the success of some of their mem-
bers as insect symbionts. We discuss the potential role of
alternative terminal oxidases as symbiotic factors favoring
the adaptation of AAB to the insect hosts.

**Results and Discussion**

Several Potential Symbiotic Traits Are Present in AAB

Annotation of the *A. platycodi* and *Saccharibacter* sp. ge-
nomes revealed a series of traits compatible with a symbiotic
life style in the insect gut. *Asaia platycodi* and *Saccharibacter*
present several secretion system (Sec-SRP and Tat for both
genomes and type IV in the case of *A. platycodi*) and ABC
transporters (in the case of *A. platycodi*) that may have roles in
the cross talk between the bacterium and the host. A series of
bacterial components for motility and cell surface structures
can be implicated in the colonization of the gut epithelium by
*A. platycodi* and *Saccharibacter*. These include the genes for
the flagellar machinery (e.g., *MotA*, *MotB*, *FlaA*, *FlaB*, *FlgC*,
*FlgD*, *FlgE2*, *FlgH*, *FtsI*) as well as genes encoding for fimbriae
(*sF-Chap* and *sF-UshP*) and glycan biosynthesis. Although
these features may help in the establishment of a symbiotic
relationship, they are not essential for it. The presence of these
traits was not associated with the ability to establish symbiosis.
In fact, genes for the flagellar machinery were also present in
*Ac. aceti*, *Gluconacetobacter diazotrophicus*, *Gluconobacter*
*frateurii*, *G. morbifer*, *G. oxydans*, and *G. thailandicus* but
not *Ac. pomorum* and *Commensalibacter intestini*. This
trend was also observed for the other traits.

Both genomes contain the operon for the production of
acetoin and 2,3-butandiol: These molecules have been shown
to play a role in insects' pheromone signaling (Tolasch et al.
2003). 2,3-Butandiol has been implicated in the modulation of
the innate immunity response of vertebrate hosts, facilitating
tissue colonization by pathogenic bacteria (Bari et al. 2011).
We can thus speculate that the production of metabolites
potentially interfering with insect physiology and innate im-
munity might have provided AAB with a pre-adaptive feature
toward symbiosis with insect hosts. This trait was observed in
other AAB including most of those described as insect symbi-
onts (i.e., *Ac. tropicalis*, *C. intestini*, *Glucona. diazotrophicus*,
*Glucona. europaeus*, *Glucona. oboediens*, *G. frateurii*, *G. mor-
bifer*, *G. Oxydans*, and *G. thailandicus*).

Adaptation to Diverse Oxic Conditions

AAB are aerobic organisms, consistent with their lifestyle in
oxygen rich environments (Kersters et al. 2006). On the other
hand, the oxygen levels in the guts of many arthropods may
vary from aerobic to completely anoxic (Sudakaran et al.
2012). We have thus focused our attention on the presence
and distribution of the oxygen-reacting systems of the elec-
tron transport chain (terminal oxidases). The genomes of both

*A. platycodi* and *Saccharibacter* sp. present all the genes for the operons of cytochrome *bo*₃ (*CyoA-D*) and *bd* (*CydAB*) ubiquinol oxidase, which have high affinity for oxygen. The operons of both cytochrome *bo*₃ and *bd* oxidase are present in all AAB genomes, often with two different versions for the *bd* oxidase. This implies that both *A. platycodi* and *Saccharibacter* sp., as well as all the other AAB, have the capacity to respire through an aerobic respiratory chain independent from the terminal cytochrome *aa*₃ oxidase, an enzyme with low affinity for oxygen, that is absent in AAB. Therefore, AAB have the potential to thrive at low oxygen concentrations like the enterobacteria colonizing animal guts, which also do not possess the cytochrome *aa*₃ oxidase.

The phylogenetic tree of the protein subunits of cyto-
chrome *bo*₃ oxidase of AAB matches that inferred using 70
proteins from the core genome (supplementary table S1,
Supplementary Material online, and fig. 1), indicating that
*bo*₃ oxidase evolution followed the differentiation of AAB spe-
cies from a common ancestor. On the other hand, the phylo-
genetic tree of the cytochrome *bd* oxidases has a topology
that is different from the phylogenomics inferred from core
genes (fig. 2). In the *bd* oxidase tree, *Saccharibacter* branches
with *Acetobacter* species rather than with *Gluconobacter*
(fig. 2) suggesting that lateral gene transfer of *bd* oxidase
genes might have occurred along the evolution of AAB.

In sum, although AAB are usually described as strictly aer-
obic organisms thriving in normoxic environments, our results
show that most of these organisms also possess ubiquinol
oxidases that should allow their survival under micro-oxic con-
ditions, such as those existing in the insect gut (Sudakaran
et al. 2012). Moreover, phylogenetic comparisons show that
these terminal oxidases were present in the common ancestor
of AAB, thereby constituting an ancestral character. We thus
propose that the capacity to thrive at low oxygen concentra-
tion conferred by ubiquinol oxidases has provided AAB organ-
isms with a constitutive propensity for thriving in micro-oxic
environments including the insect gut, an environment with
ample variation in its oxygen levels (Sudakaran et al. 2012).
The deep branching of the AAB family contains pathogens
such as *Granulibacter bethesdensis* (figs. 1 and 2) further sup-
ports that capacity to establish intimate associations with
animal hosts is an ancestral trait in these bacteria. On the
other hand, the association of AAB with phylogenetically di-
verse insect species (Crotti et al. 2010) can be considered
rather recent, in view of the phylogenetic proximity of symbi-
ont and free-living bacteria (Chouaia et al. 2010).

The analysis of the genomes presented here thus provides
new clues indicating ancient pre-adaptation traits to symbiosis
in AAB organisms that might have helped, and are still help-
ing, establishing association with insects.

A cluster analysis carried on the ortholog gene groups that
were not part of the core genes of the 16 AAB species showed
that, in terms of gene acquisitions and losses, there was a
coherence at the genus level: All of the members of a given
<figure><img src="image_2.png" /><figcaption>**Fig. 1.**—Comparison of species (AAB) (A) and operon (ubiquinol oxidase *bo*₃) (B) in phylogenetic trees. The scientific names reported at the terminal nodes are those of the bacterial species. The tree of AAB (top) is based on the results of 70 concatenated protein (supplementary table S1, Supplementary Material online) phylogenetic analyses. Operon tree (bottom) was derived from the phylogeny inferred from the *bo*₃ operon. The topology shown was obtained by the program RAxML using a partitioned ML model after reconstruction with 1,000 rapid bootstrap.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.**—Comparison of species (AAB) (A) and operon (cytochrome oxidase *bd*) (B) in phylogenetic trees. The scientific names at the terminal nodes are those of the bacteria species. The tree of AAB (top) is based on the results of 70 concatenated protein (supplementary table S1, Supplementary Material online) phylogenetic analyses. Operon tree (bottom) was derived from the phylogeny inferred from the *bd* operon. The tree topology and other details were as given in figure 1.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.**—Cluster analysis carried on the total number of ortholog groups after removal of those present in all genomes (i.e., core genome). The analysis shows that groups cluster at the genus level. Numbers on the branches indicate the number of ortholog groups specific to the cluster.</figcaption></figure>

genus clustered together, except for *G. diazotrophicus* and *A. aceti* (fig. 3). However, when only orthologs present in at least 50% of the genomes are considered, the clustering resulting from the analysis of shared genes is congruent to the phylogeny based on the 70 coding sequences (CDS), including the positioning of *G. diazotrophicus* and *A. aceti* (figs. 1A and 4). In other words, phenetic analysis based on gene presence/absence produced a tree comparable to that generated by phylogenetics. This result supports the robustness of the phylogenomics here presented for AAB. It is noteworthy that the phylogenomic tree based on 70 CDS was not congruent with 16S rRNA-based phylogeny (supplementary fig. S1, Supplementary Material online)

## Electron Chain Transport and Symbiotic Traits

The analysis of the phylogeny of the cytochrome oxidase operons *bo*₃ and *bd* showed that both of them were ancestral. The analysis showed also that cytochrome oxidase *bo*₃ had an evolutionary history similar to that of the AAB genomes. These results suggest that these two operons may have played a role in the symbiotic potential of the AAB.

In order to investigate the possible implication of other proteins of the electron transport chain in the pre-adaptation of AAB to a symbiotic life, a further cluster analysis was carried on the different orthologs involved in the electron transport chain that were present in the different genomes. This analysis showed that the pattern of gain, loss, and duplication of these

genes was coherent and allowed to identify two groups (fig. 5), although there was no correlation between the presence of certain groups of orthologs and the ability to establish symbiosis. The two groups that were identified were the same that emerged from the phylogenomic study. The first group was formed by members of the *Acetobacter* and *Gluconacetobacter* genera, whereas the second group was formed by members of the *Gluconobacter* genus in addition to *Asaia* and *Saccharibacter*.

## Materials and Methods

### Strains

*Asaia platycodi* strain SF2.1 was isolated from *An. stephensi* (Favia et al. 2007). *Saccharibacter* sp. strain AM169 was isolated from an adult gut of *Ap. mellifera* using the pre-enrichment medium ABEM (2.0% D-sorbitol, 0.5% peptone, 0.3% yeast extract pH 3.5; Favia et al. 2007) supplemented with 100 µg ml⁻¹ of cycloheximide, followed by a plating on CaCO₃-containing plates (1.0% D-glucose, 1.0% glycerol, 1.0% ethanol, 1.0% peptone, 0.5% yeast extract, 0.7% CaCO₃, and 1.5% agar, pH 6.8). *Saccharibacter* sp. strain AM169 colony was selected based on the capability to clear CaCO₃. Both strains were characterized as aerobic, Gram-negative, and rod-shaped bacteria belonging to the family Acetobacteraceae.
<figure><img src="image_6.png" /><figcaption>**Fig. 4.**—Cluster analysis carried on the subset ortholog groups that were present in at least 50% of the genome. The analysis shows that the clustering of the different groups is congruent with the phylogenomic analysis carried on 70 CDS (fig. 1A).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5.**—Gene presence-absence analysis of the oxidative phosphorylation chain orthologs in the genomes of AAB. A hierarchical clustering tree (left) was inferred based on the Kulczynski dissimilarity matrix calculated on the presence-absence matrix of genes in the examined genomes. The heatmap to the right of the tree represents the values of the Kulczynski dissimilarity matrix.</figcaption></figure>
## Genome Sequencing, Assembly, and Annotation

The whole genome DNAs of *A. platycodi* SF2.1 and *Saccharibacter* sp. AM169 were purified using the DNeasy® Blood and Tissue kit (QIAGEN) and sequenced by Macrogen Korea institute. The genome sequence of *A. platycodi* SF2.1 was determined using a 3-kb paired-end library (~200 × 10³ reads, ~80 Mb) with the Genome Sequencer FLX system (Roche, Diagnostics, Branford, CT) and a 100-bp library (~28 × 10⁶ reads, ~3 Gb) with Genome AnalyzerIIx (Illumina, San Diego, CA). Raw data were assembled into 27 contigs— generated using Mira (version 3.4) (Chevreux et al. 1999); total coverage over the whole genome reached ~500-fold. The draft genome was 3,420,092 bp in length and contained 3,137 open reading frames (ORFs). The G+C content of the genome was 59.9%. The genome of *Saccharibacter* sp. AM169 was obtained from a tenth if a lane of Illumina Hiseq2000 platform generating 101-pb-long pair-end reads (~24 × 10⁶ reads, ~2.4 Gb). The nine contigs were generated using Velvet (version 1.2) (Zerbino and Birney 2008); total coverage over the whole genome reached ~1000-fold. The draft genome was 1,978,091 bp in length and contained 1,877 ORFs. The G+C content of the genome was 59.3% (table 1).

The functional annotation of the predicted genes was performed using the RAST server (Aziz et al. 2008) combined with KEGG (Kanehisa et al. 2004) and COG (Tatusov et al. 2003) databases.

Genomes of *A. platycodi* and *Saccharibacter* sp. were also checked against the genomes of *G. oxydans* and *Glucona. diazotrophicus* using the KEGG database. The search for specific genes was carried out by local BLAST against the different genomes downloaded from the NCBI website using well-characterized and annotated genes. Confirmation or rectification for genes' annotation was obtained by additional DeltaBLAST

analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed April 8, 2014).

The result of this whole-genome project has been deposited at EMBL/GenBank database under the accession numbers CBLX010000001–CBLX010000027 for *A. platycodi* and CBLY010000001–CBLY010000009 for *Saccharibacter* sp. The material described in this article corresponds to the first version of the submitted genomes.

## Phylogenomics and Phylogenetic Reconstruction of Specific Operons

For the phylogenetic reconstruction of the AAB, 14 available (table 2), complete or draft, genomes were downloaded from the NCBI database along with our two genomes. A standardized ORF calling using Prodigal (Hyatt et al. 2010) was performed on all the nucleotidic sequences and nontruncated proteins longer than 50 residues were kept for the following analysis. Orthologs present in a single copy in any given genome were then selected using OrthoMCL (Li et al. 2003)

<caption>**Table 1**</caption>

General Genome Features of *Asaia platicody* and *Saccharibacter* sp

<table><caption>**Table 2**</caption><thead><tr><th>Organism</th><th>A. platicody SF2.1</th><th>Saccharibacter sp. AM169</th></tr></thead><tbody><tr><td>Genome size (pb)</td><td>3,420,092</td><td>1,978,091</td></tr><tr><td>Number of contigs</td><td>27</td><td>9</td></tr><tr><td>GC%</td><td>59.9</td><td>59.3</td></tr><tr><td>CDS</td><td>3,134</td><td>1,877</td></tr><tr><td>tRNAs</td><td>56</td><td>58</td></tr><tr><td>rRNAs</td><td>3</td><td>3</td></tr><tr><td>Accession number</td><td>CBLX010000001:27</td><td>CBLY010000001:9</td></tr><tr><td>Isolation year</td><td>2005</td><td>2010</td></tr></tbody></table>

List of Bacterial Genomes Used for the Phylogenetic Studies

<table><thead><tr><th>Organism</th><th>Accession Number</th><th>Reference</th><th>Origin</th></tr></thead><tbody><tr><td>Acetobacter aceti NBRC 14818</td><td>PRJNA70715/PRJDA52649</td><td>Sakurai et al. (2011)</td><td>Reference strain</td></tr><tr><td>Ac. pasteurianus IFO 3283-01</td><td>PRJNA59279/PRJDA31129</td><td>Azuma et al. (2009)</td><td>Cocoa bean heap fermentation</td></tr><tr><td>Ac. pomorum DM001</td><td>PRJNA65823/PRJNA60787</td><td>Shin et al. (2011)</td><td>Drosophila melanogaster</td></tr><tr><td>Ac. tropicalis NBRC 101654</td><td>PRJNA68643/PRJDA46891</td><td>Matsutani et al. (2011)</td><td>Fruits</td></tr><tr><td>Asaia platycodi SF2.1</td><td>CBLX010000001:27</td><td>This study</td><td>Anopheles stephensi</td></tr><tr><td>Commensalibacter intestini A911</td><td>PRJNA75109/PRJNA73359</td><td>Roh et al. (2008)</td><td>Drosophila melanogaster</td></tr><tr><td>Gluconacetobacter diazotrophicus PAI 5</td><td>PRJNA61587/PRJNA377</td><td>Bertalan et al. (2009)</td><td>Sugarcane plants</td></tr><tr><td>Glucona. europaeus LMG 18494</td><td>PRJNA73763/PRJEA61325</td><td>Andrés-Barrao et al. (2011)</td><td>Reference strain</td></tr><tr><td>Glucona. oboediens 174Bp2</td><td>PRJNA73765/PRJEA61333</td><td>Andrés-Barrao et al. (2011)</td><td>Spirit vinegar</td></tr><tr><td>Glucona. xylinus NBRC 3288</td><td>PRJNA46523/PRJDA64985</td><td>Ogino et al. (2011)</td><td>Vinegar</td></tr><tr><td>Gluconobacter frateurii NBRC 101659</td><td>PRJNA178735/PRJDB2</td><td>Hattori et al. (2012)</td><td>Reference strain</td></tr><tr><td>G. morbifer G707</td><td>PRJNA76941/PRJNA73361</td><td>Roh et al. (2008)</td><td>Drosophila melanogaster</td></tr><tr><td>G. oxydans H24</td><td>PRJNA179202/PRJNA173388</td><td>Ge et al. (2013)</td><td>Reference strain</td></tr><tr><td>G. thailandicus NBRC 3255</td><td>PRJDB753/PRJNA191942</td><td>Matsutani et al. (2013)</td><td>Strawberry</td></tr><tr><td>Granulibacter bethesdensis CGDNIH1</td><td>PRJNA58661/PRJNA17111</td><td>Greenberg et al. (2007)</td><td>Chronic granulomatous disease patient</td></tr><tr><td>Saccharibacter sp. AM169</td><td>CBLY010000001:9</td><td>This study</td><td>Apis mellifera</td></tr></tbody></table>
and a custom script designed to keep only those matching
with a single or no COG entry. The amino acid sequences of
CDS belonging to each ortholog family were aligned using
MUSCLE (Edgar 2004); the alignments were subsequently
retro-transcribed to their respective nucleic acid sequences,
which were checked for the probability of recombination
and lateral gene transfer using the phi-test under the Phi-
pack (Bruen et al. 2006). At the end of this screening, 70
proteins were kept for phylogenetic analysis (listed in supple-
mentary table S1, Supplementary Material online). The align-
ment for the remaining CDS was Gblocked, keeping only the
proteins that had <3 misaligned residues. For each of the
aligned CDS, an evolutionary model was predicted using
ProtTest (Darriba et al. 2011); then all the protein sequences
were concatenated and their phylogenetic tree was con-
structed with RAxML (Stamatakis et al. 2005) using a parti-
tioned Maximum Likelihood model that takes into account the
evolutionary model predicted for each of the CDS. The phy-
logenetic trees were tested with 1,000 rapid bootstraps. The
phylogenetic trees of the concatenated protein subunits of
the cytochrome bo₃ and bd oxidases were constructed with
the same method. A phylogenetic tree based on 16S rRNA
was also inferred (see supplementary material, Supplementary
Material online)

Cluster Analysis on the Orthologs

The data obtained from OrthoMCL (5,488 ortholog groups)
were transformed into a matrix reporting the presence of each
ortholog group. The orthologs present in all of the genomes
(i.e., core genome) were removed from the data set, leaving
4,575 ortholog groups, and a cluster analysis (Murtagh 1985)
was carried out using R (cran.r-project.org, last accessed April
8, 2014). A second cluster analysis was carried out on a subset
consisting only of the ortholog groups (1,167 ortholog
groups) present in at least 50% of the genomes. The pattern
of presence/absence was reconstructed also for the subset of
genes involved in the oxidative phosphorylation chain. The
oxidative phosphorylation chain genes (listed in supplemen-
tary table S2, Supplementary Material online) were identified
on the basis of KEGG annotation of the *Glucona. diazotrophi-
cus* PAI 5, *G. oxydans* H24, *Ac. pasteurianus* IFO 3283-01, and
*Granulibacter bethesdensis* strain genomes; the relative pres-
ence/absence informations were retrieved from the
OrthoMCL matrix generated above and organized in a new
matrix. This new matrix was subjected to hierarchical cluster-
ing analysis using the Kulczynski distance index and the heat-
map graphic representation was generated with R.

**Supplementary Material**

Supplementary methods, tables S1 and S2, and figure S1 are
available at *Genome Biology and Evolution* online (http://
www.gbe.oxfordjournals.org/).

Acknowledgments

This work was supported by the project BIODESERT GA-
245746 "Biotechnology from Desert Microbial
Extremophiles for Supporting Agriculture Research Potential
in Tunisia and Southern Europe" (European Union) and the
Prin 2009 (grant 009L27YC8_003), from the Italian Ministry of
Education, University and Research (MIUR). C.B. and B.C.
thank Massimo Pajoro for inspirations.

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Associate editor: Bill Martin