A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence.

Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons.

Origin and diversification of Xanthomonas citri subsp. citri pathotypes revealed by inclusive phylogenomic, dating, and biogeographic analyses.

BACKGROUND: Xanthomonas citri subsp. citri pathotypes cause bacterial citrus canker, being responsible for severe agricultural losses worldwide. The A pathotype has a broad host spectrum, while A* and Aw are more restricted both in hosts and in geography. Two previous phylogenomic studies led to contrasting well-supported clades for sequenced genomes of these pathotypes. No extensive biogeographical or divergence dating analytic approaches have been so far applied to available genomes. RESULTS: Based on a larger sampling of genomes than in previous studies (including six new genomes sequenced by our group, adding to a total of 95 genomes), phylogenomic analyses resulted in different resolutions, though overall indicating that A + AW is the most likely true clade. Our results suggest the high degree of recombination at some branches and the fast diversification of lineages are probable causes for this phylogenetic blurring effect. One of the genomes analyzed, X. campestris pv. durantae, was shown to be an A* strain; this strain has been reported to infect a plant of the family Verbenaceae, though there are no reports of any X. citri subsp. citri pathotypes infecting any plant outside the Citrus genus. Host reconstruction indicated the pathotype ancestor likely had plant hosts in the family Fabaceae, implying an ancient jump to the current Rutaceae hosts. Extensive dating analyses indicated that the origin of X. citri subsp. citri occurred more recently than the main phylogenetic splits of Citrus plants, suggesting dispersion rather than host-directed vicariance as the main driver of geographic expansion. An analysis of 120 pathogenic-related genes revealed pathotype-associated patterns of presence/absence. CONCLUSIONS: Our results provide novel insights into the evolutionary history of X. citri subsp. citri as well as a sound phylogenetic foundation for future evolutionary and genomic studies of its pathotypes.

Live neighbor-joining.

BACKGROUND: In phylogenetic reconstruction the result is a tree where all taxa are leaves and internal nodes are hypothetical ancestors. In a live phylogeny, both ancestral and living taxa may coexist, leading to a tree where internal nodes may be living taxa. The well-known Neighbor-Joining heuristic is largely used for phylogenetic reconstruction. RESULTS: We present Live Neighbor-Joining, a heuristic for building a live phylogeny. We have investigated Live Neighbor-Joining on datasets of viral genomes, a plausible scenario for its application, which allowed the construction of alternative hypothesis for the relationships among virus that embrace both ancestral and descending taxa. We also applied Live Neighbor-Joining on a set of bacterial genomes and to sets of images and texts. Non-biological data may be better explored visually when their relationship in terms of content similarity is represented by means of a phylogeny. CONCLUSION: Our experiments have shown interesting alternative phylogenetic hypothesis for RNA virus genomes, bacterial genomes and alternative relationships among images and texts, illustrating a wide range of scenarios where Live Neighbor-Joining may be used.

The complete genome sequence of 'Candidatus Liberibacter americanus', associated with Citrus huanglongbing.

Liberibacter spp. form a Rhizobiaceae clade of phloem-limited pathogens of limited host range. Two obligately parasitic species have been sequenced: 'Candidatus Liberibacter asiaticus', which causes citrus huanglongbing (HLB) worldwide, and 'Ca. L. solanacearum', which causes potato "zebra chip" disease. A third (proposed) species, Liberibacter crescens, was isolated from mountain papaya, grown in axenic culture, and sequenced. In an effort to identify common host determinants, the complete genomic DNA sequence of a second HLB species, 'Ca. L. americanus' strain 'São Paulo' was determined. The circular genome of 1,195,201 bp had an average 31.12% GC content and 983 predicted protein encoding genes, 800 (81.4%) of which had a predicted function. There were 658 genes common to all sequenced Liberibacter spp. and only 8 genes common to 'Ca. L. americanus' and 'Ca. L. asiaticus' but not found in 'Ca. L. solanacearum'. Surprisingly, most of the lipopolysaccharide biosynthetic genes were missing from the 'Ca. L. americanus' genome, as well as OmpA and a key regulator of flagellin, all indicating a 'Ca. L. americanus' strategy of avoiding production of major pathogen-associated molecular patterns present in 'Ca. L. asiaticus' and 'Ca. L. solanacearum'. As with 'Ca. L. asiaticus', one of two 'Ca. L. americanus' prophages replicated as an excision plasmid and carried potential lysogenic conversion genes that appeared fragmentary or degenerated in 'Ca. L. solanacearum'.

Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper.

BACKGROUND: Bacterial spot of tomato and pepper is caused by four Xanthomonas species and is a major plant disease in warm humid climates. The four species are distinct from each other based on physiological and molecular characteristics. The genome sequence of strain 85-10, a member of one of the species, Xanthomonas euvesicatoria (Xcv) has been previously reported. To determine the relationship of the four species at the genome level and to investigate the molecular basis of their virulence and differing host ranges, draft genomic sequences of members of the other three species were determined and compared to strain 85-10. RESULTS: We sequenced the genomes of X. vesicatoria (Xv) strain 1111 (ATCC 35937), X. perforans (Xp) strain 91-118 and X. gardneri (Xg) strain 101 (ATCC 19865). The genomes were compared with each other and with the previously sequenced Xcv strain 85-10. In addition, the molecular features were predicted that may be required for pathogenicity including the type III secretion apparatus, type III effectors, other secretion systems, quorum sensing systems, adhesins, extracellular polysaccharide, and lipopolysaccharide determinants. Several novel type III effectors from Xg strain 101 and Xv strain 1111 genomes were computationally identified and their translocation was validated using a reporter gene assay. A homolog to Ax21, the elicitor of XA21-mediated resistance in rice, and a functional Ax21 sulfation system were identified in Xcv. Genes encoding proteins with functions mediated by type II and type IV secretion systems have also been compared, including enzymes involved in cell wall deconstruction, as contributors to pathogenicity. CONCLUSIONS: Comparative genomic analyses revealed considerable diversity among bacterial spot pathogens, providing new insights into differences and similarities that may explain the diverse nature of these strains. Genes specific to pepper pathogens, such as the O-antigen of the lipopolysaccharide cluster, and genes unique to individual strains, such as novel type III effectors and bacteriocin genes, have been identified providing new clues for our understanding of pathogen virulence, aggressiveness, and host preference. These analyses will aid in efforts towards breeding for broad and durable resistance in economically important tomato and pepper cultivars.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii.

BACKGROUND: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. RESULTS: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. CONCLUSION: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

PAMDB, a multilocus sequence typing and analysis database and website for plant-associated microbes.

Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.

Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria.

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

A draft genome sequence of Pseudomonas syringae pv. tomato T1 reveals a type III effector repertoire significantly divergent from that of Pseudomonas syringae pv. tomato DC3000.

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

BACKGROUND: In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. RESULTS: Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml-1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. CONCLUSIONS: We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.

Comparative Genomics for Prokaryotes.

Bacteria and archaea, collectively known as prokaryotes, have in general genomes that are much smaller than those of eukaryotes. As a result, thousands of these genomes have been sequenced. In prokaryotes, gene architecture lacks the intron-exon structure of eukaryotic genes (with an occasional exception). These two facts mean that there is an abundance of data for prokaryotic genomes, and that they are easier to study than the more complex eukaryotic genomes. In this chapter, we provide an overview of genome comparison tools that have been developed primarily (sometimes exclusively) for prokaryotic genomes. We cover methods that use only the DNA sequences, methods that use only the gene content, and methods that use both data types.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

Draft Genome Sequences of Two Mycobacterium bovis Strains Isolated from Beef Cattle in Paraguay.

This work reports the draft genome sequences of the Mycobacterium bovis strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding M. bovis epidemiology in South America.

Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae.

The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.

Comparative genomics analyses of citrus-associated bacteria.

Xylella fastidiosa 9a5c (XF-9a5c) and Xanthomonas axonopodis pv. citri (XAC) are bacteria that infect citrus plants. Sequencing of the genomes of these strains is complete and comparative analyses are now under way with the genomes of other bacteria of the same genera. In this review, we present an overview of this comparative genomic work. We also present a detailed genomic comparison between XF-9a5a and XAC. Based on this analysis, genes and operons were identified that might be relevant for adaptation to citrus. XAC has two copies of a type II secretion system, a large number of cell wall-degrading enzymes and sugar transporters, a complete energy metabolism, a whole set of avirulence genes associated with a type III secretion system, and a complete flagellar and chemotatic system. By contrast, XF-9a5c possesses more genes involved with type IV pili biosynthesis than does XAC, contains genes encoding for production of colicins, and has 4 copies of Type I restriction/modification system while XAC has only one.