Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism.

Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.     

Genetic organization of the lexA,recA and recX genes in Xanthomonas campestris

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.     

Comparative analyses of Xanthomonas and Xylella complete genomes

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.     

The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.     

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

A systematic method to identify genomic islands and its applications in analyzing the genomes of Corynebacterium glutamicum and Vibrio vulnificus CMCP6 chromosome I

MOTIVATION: Some genomic islands contain horizontally transferred genes, which play critical roles in altering the genotypes and phenotypes of organisms, and horizontal gene transfer has been recognized as a universal event throughout bacterial evolution. A windowless method to display the distribution of genomic GC content, the cumulative GC profile, is proposed to identify genomic islands in genomes whose complete genome sequences are available. Two new indices are proposed to assess the codon usage bias and amino acid usage bias in genomic islands. RESULTS: A 211 kb genomic island (CGGI-1) has been identified in the genome of Corynebacterium glutamicum, and three genomic islands VVGI-1, VVGI-2 and VVGI-3, with lengths 167, 40 and 33 kb, respectively, have been identified in the genome of Vibrio vulnificus CMCP6 chromosome I. The CGGI-1 is flanked by two approximately 500 bp direct repeats, and utilizes a Val-tRNA as the integration site. For the VVGI-1 and VVGI-2, each has an integrase gene at 5' junction. All the identified genomic islands show unusual GC content, codon usage and amino acid usage, compared with the rest of the genomes. In addition, it is found that genomic islands are fairly homogenous in terms of GC content variation. An index, h, to quantify the homogeneity of GC content for genomic islands is proposed, and it is shown that h is less than 0.1 for all the genomic islands analyzed. The cumulative GC profile, as well as various indices to assess the codon usage bias, amino acid usage bias and homogeneity of the genomic islands, will be useful in the analysis of other genomes. AVAILABILITY: Programs used in this work and numerical results are available upon request.     

Identification and categorization of horizontally transferred genes in prokaryotic genomes

Horizontal gene transfer （HGT）, a process through which genomes acquire genetic materials from distantly related organisms, is believed to be one of the major forces in prokaryotic genome evolution.However, systematic investigation is still scarce to clarify two basic issues about HGT： （1） what types of genes are transferred; and （2） what influence HGT events over the organization and evolution of biological pathways. Genome-scale investigations of these two issues will advance the systematical understanding of HGT in the context of prokaryotic genome evolution. Having investigated 82 genomes, we constructed an HGT database across broad evolutionary timescales. We identified four function categories containing a high proportion of horizontally transferred genes： cell envelope, energy metabolism, regulatory functions, and transport/binding proteins. Such biased function distribution indicates that HGT is not completely random;instead, it is under high selective pressure, required by function restraints in organisms. Furthermore, we mapped the transferred genes onto the connectivity structure map of organism-specific pathways listed in Kyoto Encyclopedia of Genes and Genomes （KEGG）. Our results suggest that recruitment of transferred genes into pathways is also selectively constrained because of the tuned interaction between original pathway members. Pathway organization structures still conserve well through evolution even with the recruitment of horizontally transferred genes. Interestingly, in pathways whose organization were significantly affected by HGT events, the operon-like arrangement of transferred genes was found to be prevalent. Such results suggest that operon plays an essential and directional role in the integration of alien genes into pathways.     

Speciation in <Emphasis Type="Italic">Chlamydia</Emphasis>: Genomewide Phylogenetic Analyses Identified a Reliable Set of Acquired Genes

Horizontal gene transfer (HGT), a process through which genomes acquire sequences from distantly related organisms, is believed to be a major source of genetic diversity in bacteria. A central question concerning the impact of HGT on bacterial genome evolution is the proportion of horizontally transferred sequences within genomes. This issue, however, remains unresolved because the various methods developed to detect potential HGT events identify different sets of genes. The present-day consensus is that phylogenetic analysis of individual genes is still the most objective and accurate approach for determining the occurrence and directionality of HGT. Here we present a genome-scale phylogenetic analysis of protein-encoding genes from five closely related Chlamydia, identifying a reliable set of sequences that have arisen via HGT since the divergence of the Chlamydia lineage. According to our knowledge, this is the first systematic phylogenetic inference-based attempt to establish a reliable set of acquired genes in a bacterial genome. Although Chlamydia are obligate intracellular parasites of higher eukaryotes, and thus suspected to be isolated from HGT more than the free-living species, our results show that their diversification has involved the introduction of foreign sequences into their genome. Furthermore, we also identified a complete set of genes that have undergone deletion, duplication, or rearrangement during this evolutionary period leading to the radiation of Chlamydia species. Our analysis may provide a deeper insight into how these medically important pathogens emerged and evolved from a common ancestor.     

Horizontal gene transfers as metagenomic gene duplications

While it is well accepted that horizontal gene transfer plays an important role in the evolution and the diversification of prokaryotic genomes, many questions remain open regarding its functional mechanisms of action and its interplay with the extant genome. This study addresses the relationship between proteome innovation by horizontal gene transfer and genome content in Proteobacteria. We characterize the transferred genes, focusing on the protein domain compositions and their relationships with the existing protein domain superfamilies in the genome. In agreement with previous observations, we find that the protein domain architectures of horizontally transferred genes are significantly shorter than the genomic average. Furthermore, protein domains that are more common in the total pool of genomes appear to have a proportionally higher chance to be transferred. This suggests that transfer events behave as if they were drawn randomly from a cross-genomic community gene pool, much like gene duplicates are drawn from a genomic gene pool. Finally, horizontally transferred genes carry domains of exogenous families less frequently for larger genomes, although they might do it more than expected by chance.     

Association between translation efficiency and horizontal gene transfer within microbial communities

Horizontal gene transfer (HGT) is a major force in microbial evolution. Previous studies have suggested that a variety of factors, including restricted recombination and toxicity of foreign gene products, may act as barriers to the successful integration of horizontally transferred genes. This study identifies an additional central barrier to HGT-the lack of co-adaptation between the codon usage of the transferred gene and the tRNA pool of the recipient organism. Analyzing the genomic sequences of more than 190 microorganisms and the HGT events that have occurred between them, we show that the number of genes that were horizontally transferred between organisms is positively correlated with the similarity between their tRNA pools. Those genes that are better adapted to the tRNA pools of the target genomes tend to undergo more frequent HGT. At the community (or environment) level, organisms that share a common ecological niche tend to have similar tRNA pools. These results remain significant after controlling for diverse ecological and evolutionary parameters. Our analysis demonstrates that there are bi-directional associations between the similarity in the tRNA pools of organisms and the number of HGT events occurring between them. Similar tRNA pools between a donor and a host tend to increase the probability that a horizontally acquired gene will become fixed in its new genome. Our results also suggest that frequent HGT may be a homogenizing force that increases the similarity in the tRNA pools of organisms within the same community.     

野油菜黄单胞菌野油菜致病型胞外多糖合成有关的DNA序列的克隆

以从野油菜黄单胞菌野油菜致病型（Xanthomonascampestrispv.campestris）胞外多糖突变体T117中克隆到的含有突变序列的DNA片段作探针,从野生型菌株中鉴定和克隆了含有相应序列的9．4kbHindⅢ片段。该片段可反式互补突变体T117,完全恢复其胞外多糖的合成,说明该片段至少含有一个完整的与该菌胞外多糖合成有关的基因。     

Mutagenesis of all eight avr genes in Xanthomonas campestris pv. campestris had no detected effect on pathogenicity, but one avr gene affected race specificity

Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.     

A computational tool for the genomic identification of regions of unusual compositional properties and its utilization in the detection of horizontally transferred sequences

Similarity Plot (S-plot) is a Windows-based application for large-scale comparisons and 2-dimensional visualization of compositional similarities between genomic sequences. This application combines 2 approaches widely used in genomics: window analysis of statistical characteristics along genomes and dot-plot visual representation. S-plot is effective in identifying highly similar regions between genomes as well as regions with unusual compositional properties (RUCPs) within a single genome, which may be indicative of horizontal gene transfer or of locus-specific selective forces. We use S-plot to identify regions that may have originated through horizontal gene transfer through a 2-step approach, by first comparing a genomic sequence to itself and, subsequently, comparing it to the genomic sequence of a closely related taxon. Moreover, by comparing these suspect sequences to one another, we can estimate a minimum number of sources for these putative xenologous sequences. We illustrate the uses of S-plot in a comparison involving Escherichia coli K12 and E. coli O157:H7. In O157:H7, we found 145 regions that have most probably originated through horizontal gene transfer. By using S-plot to compare each of these regions with 277 completely sequenced prokaryotic genomes, 1 sequence was found to have similar compositional properties to the Yersinia pseudotuberculosis genome, indicating a transfer from a Yersinia or Yersinia relative. Based upon our analysis of RUCPs in O157:H7, we infer that there were at least 53 sources of horizontally transferred sequences.     

A new computational method for the detection of horizontal gene transfer events

In recent years, the increase in the amounts of available genomic data has made it easier to appreciate the extent by which organisms increase their genetic diversity through horizontally transferred genetic material. Such transfers have the potential to give rise to extremely dynamic genomes where a significant proportion of their coding DNA has been contributed by external sources. Because of the impact of these horizontal transfers on the ecological and pathogenic character of the recipient organisms, methods are continuously sought that are able to computationally determine which of the genes of a given genome are products of transfer events. In this paper, we introduce and discuss a novel computational method for identifying horizontal transfers that relies on a gene's nucleotide composition and obviates the need for knowledge of codon boundaries. In addition to being applicable to individual genes, the method can be easily extended to the case of clusters of horizontally transferred genes. With the help of an extensive and carefully designed set of experiments on 123 archaeal and bacterial genomes, we demonstrate that the new method exhibits significant improvement in sensitivity when compared to previously published approaches. In fact, it achieves an average relative improvement across genomes of between 11 and 41% compared to the Codon Adaptation Index method in distinguishing native from foreign genes. Our method's horizontal gene transfer predictions for 123 microbial genomes are available online at http://cbcsrv.watson.ibm.com/HGT/.