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PubMed2000

Functional genomics of Helicobacter pylori: identification of a beta-1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide.

A previously annotated open reading frame (ORF) (HP0826) from Helicobacter pylori was cloned and expressed in Escherichia coli cells and determined to be a beta-1,4-galactosyltransferase that used GlcNAc as an acceptor. Mutational analysis in H. pylori strains demonstrated that this enzyme plays a key role in the biosynthesis of the type 2 N-acetyl-lactosamine (LacNAc) polysaccharide O-chain backbone, by catalysing the addition of Gal to GlcNAc. To examine the potential role of this O-chain structure in bacterial colonization of the host stomach, the mutation was introduced into H. pylori strain SS1 which is known to be capable of colonizing the gastric mucosa of mice. Compared with the parental strain, mutated SS1 was less efficient at colonizing the murine stomach.

Alleles↗

PubMed2005

NPRD: Nucleosome Positioning Region Database.

Nucleosome Positioning Region Database (NPRD), which is compiling the available experimental data on locations and characteristics of nucleosome formation sites (NFSs), is the first curated NFS-oriented database. The object of the database is a single NFS described in an individual entry. When annotating results of NFS experimental mapping, we pay special attention to several important functional characteristics, such as the relationship between type of gene activity and nucleosome positioning, the influence of non-histone proteins on nucleosome formation, type of the variant of nucleosome positioning (translational or rotational), indication of tissue types and states of cell activity, description of experimental methods used and accuracy of nucleosome position determination, and the results of applying theoretical and computer methods to the analysis of contextual and conformational DNA properties. At present, the NPRD database contains 438 entries and integrates the data described in 124 original papers. The database URL: http://srs6.bionet.nsc.ru/srs6/. Then click the button 'Databank' and open the link NUCLEOSOME.

Chromosome Mapping↗

PubMed2006

Cellular response of Shewanella oneidensis to strontium stress.

The physiology and transcriptome dynamics of the metal ion-reducing bacterium Shewanella oneidensis strain MR-1 in response to nonradioactive strontium (Sr) exposure were investigated. Studies indicated that MR-1 was able to grow aerobically in complex medium in the presence of 180 mM SrCl2 but showed severe growth inhibition at levels above that concentration. Temporal gene expression profiles were generated from aerobically grown, mid-exponential-phase MR-1 cells shocked with 180 mM SrCl2 and analyzed for significant differences in mRNA abundance with reference to data for nonstressed MR-1 cells. Genes with annotated functions in siderophore biosynthesis and iron transport were among the most highly induced (>100-fold [P < 0.05]) open reading frames in response to acute Sr stress, and a mutant (SO3032::pKNOCK) defective in siderophore production was found to be hypersensitive to SrCl2 exposure, compared to parental and wild-type strains. Transcripts encoding multidrug and heavy metal efflux pumps, proteins involved in osmotic adaptation, sulfate ABC transporters, and assimilative sulfur metabolism enzymes also were differentially expressed following Sr exposure but at levels that were several orders of magnitude lower than those for iron transport genes. Precipitate formation was observed during aerobic growth of MR-1 in broth cultures amended with 50, 100, or 150 mM SrCl2 but not in cultures of the SO3032::pKNOCK mutant or in the abiotic control. Chemical analysis of this precipitate using laser-induced breakdown spectroscopy and static secondary ion mass spectrometry indicated extracellular solid-phase sequestration of Sr, with at least a portion of the heavy metal associated with carbonate phases.

Bacterial Proteins↗

PubMed2005

Peripheral genotype-phenotype correlations in Asian Indians with type 2 diabetes mellitus.

OBJECTIVE: A genome-wide scan of gene expression in leucocytes in Asian Indians with type 2 diabetes was performed and correlated with their known phenotype. METHODS: Microarray gene profiling of 13,474 sequence-verified, non-redundant human cDNAs was done to study leukocyte gene expression in Asian Indians with type 2 diabetes (DM: n=3) and matched controls (n=3). RESULTS: Significant differential expression (fold change <0.3 or >3) was noted for 897 genes in DM vs. controls. The 147 known genes in this category belonged to following broad functional groups (%): enzyme (32), nucleic acid binding (22), ligand binding or carrier (10), signal transducer (9), transporter (7), structural protein (6), cell adhesion (3), tumor suppressor (3), transcription factor binding (2), enzyme inhibitor (2), chaperone (2), cell cycle regulator (1), and defense/immunity protein (1). The 20 genes with at least a 3-fold change, annotated with known phenotypic associations in the current gene databank (phenotype association, fold change) were aspartoacylase (Canavan disease, 9.96), growth hormone receptor (Laron dwarfism, idiopathic short stature, 8.25), lipoprotein lipase (familial chylomicronemia syndrome, lipoprotein lipase deficiency, 8.00), vitamin D (1,25- dihydroxyvitamin D3) receptor (involutional osteoporosis, vitamin D resistant rickets, 7.94), intercellular adhesion molecule 1 human rhinovirus receptor (cerebral malaria susceptibility, 7.16), peroxisomal membrane protein 3 35-kDa (Refsum disease, infantile form, Zellweger syndrome-3, 6.00), Bardet-Biedl syndrome 2 (Bardet-Biedl syndrome, 5.87), ribosomal protein S19 (Diamond Blackfan anemia, 5.85), apolipoprotein C-III (hypertriglyceridemia, 5.44), argininosuccinate lyase (argininosuccinicaciduria, 5.22), myosin VA (Griscelli syndrome-type pigmentary dilution with mental retardation, 4.92), lysozyme (renal amyloidosis, 4.17), SAM domain, SH3 domain and nuclear localisation signals 1 (Cherubism, 4.12 ), von Hippel-Lindau syndrome (hemangioblastoma, cerebellar, somatic, von Hippel-Lindau syndrome, 3.94), early-onset breast cancer 1 (BRCA1, papillary serous carcinoma of the peritoneum, 3.73), UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (inclusion body myopathy, autosomal recessive, sialuria, 3.53), apolipoprotein A-I (amyloidosis, 3 or more types, hypoalphalipoproteinemia, 3.29), midline 1 Opitz/BBB syndrome (Opitz G syndrome, type I, 3.28), ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide (familial hemiplegic migraine, 3.05). Canavan disease, Zellweger syndrome, infantile Refsum disease, Griscelli syndrome, cherubism, breast cancer, peritoneal papillary serous carcinoma, Opitz G/BBB syndrome, and familial hemiplegic migraine (FHM) are phenotypes not previously reported in association with type 2 DM, but whose underlying genes were up-regulated in this peripheral genome scan of Asian Indians. CONCLUSION: Rare and/or previously unknown phenotypes linked to known genes with significant differential expression in type 2 DM are reported. Further testing of heterogeneity in diabetes phenotype syndromes may reveal common pathogenic mechanisms and potential candidate genes responsible for type 2 DM.

Asian People↗

PubMed2006

Expression changes in tolerant murine cardiac allografts after gene therapy with a lentiviral vector expressing alpha1,3 galactosyltransferase.

Comparison of intragraft gene expression changes in tolerant cardiac allograft models may provide the basis for identifying pathways involved in graft survival. Our laboratory has previously demonstrated that tolerance to the gal alpha1,3 gal epitope, the major target of rejection of wild-type pig hearts in human cardiac transplantation, can be achieved after transplantation with bone marrow transduced with a lentiviral vector expressing alpha1,3 galactosyltransferase. We now present intracardiac gene expression changes associated with long-term tolerance in this model. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip 430 2.0 Mouse Genome Arrays. Data were subjected to functional annotation analysis to identify genes of known function in which expression was increased or decreased by at least 2-fold (t-test, P < .05) in tolerant gal+/+ wild-type hearts as compared to transplanted syngeneic controls. Tolerant hearts demonstrated increased expression of genes associated with the stress response, modulation of immune function and cell survival (HSPa9a, CD56, and Akt1s1), and decreased expression of several immunoregulatory genes (CD209, CD26, and PDE4b). These data suggest that tolerance may be associated with activation of immunomodulatory and survival pathways.

Animals↗

PubMed2006

Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens.

Whole-genome analysis of gene expression in Geobacter sulfurreducens revealed 474 genes with transcript levels that were significantly different during growth with an electrode as the sole electron acceptor versus growth on Fe(III) citrate. The greatest response was a more than 19-fold increase in transcript levels for omcS, which encodes an outer-membrane cytochrome previously shown to be required for Fe(III) oxide reduction. Quantitative reverse transcription polymerase chain reaction and Northern analyses confirmed the higher levels of omcS transcripts, which increased as power production increased. Deletion of omcS inhibited current production that was restored when omcS was expressed in trans. Transcript expression and genetic analysis suggested that OmcE, another outer-membrane cytochrome, is also involved in electron transfer to electrodes. Surprisingly, genes for other proteins known to be important in Fe(III) reduction such as the outer-membrane c-type cytochrome, OmcB, and the electrically conductive pilin "nanowires" did not have higher transcript levels on electrodes, and deletion of the relevant genes did not inhibit power production. Changes in the transcriptome suggested that cells growing on electrodes were subjected to less oxidative stress than cells growing on Fe(III) citrate and that a number of genes annotated as encoding metal efflux proteins or proteins of unknown function may be important for growth on electrodes. These results demonstrate for the first time that it is possible to evaluate gene expression, and hence the metabolic state, of microorganisms growing on electrodes on a genome-wide basis and suggest that OmcS, and to a lesser extent OmcE, are important in electron transfer to electrodes. This has important implications for the design of electrode materials and the genetic engineering of microorganisms to improve the function of microbial fuel cells.

Bacterial Outer Membrane Proteins↗

PubMed2001

Cell migration and cerebral cortical development.

This annotation describes the clinical and pathological features of several conditions believed to result from a primary defect in cell migration which include the lissencephalies, pachygria, polymicrogyrias, and focal cortical dysplasia. A variety of factors must be considered in pathogeneses, including cellular proliferation, cell death, post-migrational intracortical growth and development, axonogenesis and dendritogenesis. At least two distinct types of lissencephaly exist. Classic (also known as Type I) lissencephaly is the prototypic pattern being seen in autosomal dominant Miller-Dieker syndrome, in addition to autosomal recessive and X-linked forms. The Miller-Dieker syndrome locus (LIS-1) encodes the platelet activating factor acetylhydrolase-1, beta1 subunit. The gene for an X-linked form of lissencephaly (XLIS) encodes a protein called doublecortin. Cobblestone (type II) lissencephaly is most commonly seen in patients with the Walker-Warburg syndrome, and also occurs in a group of disorders associated with congenital muscular dystrophy, including Finnish 'muscle-eye-brain' disease and Fukuyama muscular dystrophy. Controversy exits as to whether polymicrogyria is a malformation or a disruption of development. The answer is likely both. Polymicrogyria is believed to arise from defects occurring between 17 and 25 or 26 weeks gestation. Heterotopia can be sporadic, inherited as a simple Mendelian trait, or may be part of a more complex syndrome being characterized by collections of disorganized grey matter in inappropriate places. X-linked periventricular heterotopia syndrome is caused by mutations in filamin-1. In addition to those described above, many other syndromes show lissencephaly, pachygyria and polymicrogyria and many cases are not easily classified into any particular syndrome.

Brain Diseases↗

PubMed2006

pSTIING: a 'systems' approach towards integrating signalling pathways, interaction and transcriptional regulatory networks in inflammation and cancer.

pSTIING (http://pstiing.licr.org) is a new publicly accessible web-based application and knowledgebase featuring 65 228 distinct molecular associations (comprising protein-protein, protein-lipid, protein-small molecule interactions and transcriptional regulatory associations), ligand-receptor-cell type information and signal transduction modules. It has a particular major focus on regulatory networks relevant to chronic inflammation, cell migration and cancer. The web application and interface provide graphical representations of networks allowing users to combine and extend transcriptional regulatory and signalling modules, infer molecular interactions across species and explore networks via protein domains/motifs, gene ontology annotations and human diseases. pSTIING also supports the direct cross-correlation of experimental results with interaction information in the knowledgebase via the CLADIST tool associated with pSTIING, which currently analyses and clusters gene expression, proteomic and phenotypic datasets. This allows the contextual projection of co-expression patterns onto prior network information, facilitating the identification of functional modules in physiologically relevant systems.

Amino Acid Motifs↗

PubMed2026

Large-scale functional annotation establishes a reference framework for human LRRK2 variants.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2)1are among the most frequent monogenic causes of Parkinson's disease (PD)2 and act through a gain-of-function mechanism of increased kinase activity. LRRK2-targeted therapies are in clinical development, but interpretation of the rapidly expanding catalogue of rare LRRK2 variants remains a barrier to translation. Here, we present functionally annotated data on >350 LRRK2 coding variants using a standardized cellular assay with Rab10 phosphorylation as a readout of kinase activity and integrated these data with curated genetic and clinical annotations from the Movement Disorders Society Genetic Mutation Database (MDSGene). Variants differed in activation magnitude, ranging from modest increases (e.g., p.G2019S) to strongly activating substitutions such as p.Y1699C or p.L1795F. Activating variants occurred across the full length of LRRK2, although the largest effects clustered within the ROC-COR regulatory hub, where structural analysis identified subdomains forming an allosteric scaffold controlling kinase output. All known/established pathogenic variants showed increased activity, whereas benign and likely benign variants remained within the wild-type range. Functional effect sizes correlated with pathway activation in patient-derived immune cells, altogether providing a framework for ACMG-based variant interpretation in which kinase activation can support PS3 functional evidence for reclassification of variants.

Protein phosphorylation↗

PubMedDate not supplied

Domain graph of Arabidopsis proteome by comparative analysis.

The domain graph of domains and domain combinations of Arabidopsis thaliana is established based on pfam 14.0 database and analyzed via comparison with 10 eukaryotic, 30 bacterial, and 16 archaeal proteomes. The comparative analysis of the domain graphs provides a useful platform for revealing global insights on the evolution of plant kingdom. More importantly, it is a powerful tool for searching not only the possible new function of both plant-specific and nonspecific domains via specific domain combinations in Arabidopsis thaliana but also the functional role of unknown domains. As an example, we present the functional link between ubiquitin and Myb_DNA-binding domains via Bromodomain as the plant specific evidence for the association between transcription and ubiquitin. We further show that PentatricoPeptide Repeats (PPR) proteins have plant-specific links with a wide variety of domains responsible for RNA binding/metabolism, modulation of protein-protein interactions, ubiquitin-conjugation, cell growth/maintenance, catalysis, and others. This further supports the recently proposed association of PPR proteins with specific RNA transcripts and defined effector proteins. Moreover, the domain graph built from tissue-specific genes is frequently associated with DNA binding domains, suggesting that the differentiation of tissue cell types is contributed mostly by tissue-specific transcriptional process. DOGMA (DOmain Graph via coMparitive analysis for Arabidopsis thaliana) is available on-line with a variety of search tools at http://theory.med.buffalo.edu/DOGMA. The database, which allows user-specified search for plant specific domains and their combinations, will be useful as an additional tool for annotation of the proteins that play specific roles in plants and other organisms.

Arabidopsis↗

PubMed2004

Characterization of the type I interferon locus and identification of novel genes.

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.

Amino Acid Sequence↗

PubMed2005

Evolution of distinct EGF domains with specific functions.

EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional inter-domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues.

Amino Acid Sequence↗

PubMed2006

Modeling a whole organ using proteomics: the avian bursa of Fabricius.

While advances in proteomics have improved proteome coverage and enhanced biological modeling, modeling function in multicellular organisms requires understanding how cells interact. Here we used the chicken bursa of Fabricius, a common experimental system for B cell function, to model organ function from proteomics data. The bursa has two major functional cell types: B cells and the supporting stromal cells. We used differential detergent fractionation-multidimensional protein identification technology (DDF-MudPIT) to identify 5198 proteins from all cellular compartments. Of these, 1753 were B cell specific, 1972 were stroma specific and 1473 were shared between the two. By modeling programmed cell death (PCD), cell differentiation and proliferation, and transcriptional activation, we have improved functional annotation of chicken proteins and placed chicken-specific death receptors into the PCD process using phylogenetics. We have identified 114 transcription factors (TFs); 42 of the bursal B cell TFs have not been reported before in any B cells. We have also improved the structural annotation of a newly sequenced genome by confirming the in vivo expression of 4006 "predicted", and 6623 ab initio, ORFs. Finally, we have developed a novel method for facilitating structural annotation, "expressed peptide sequence tags" (ePSTs) and demonstrate its utility by identifying 521 potential novel proteins from the chicken "unassigned chromosome".

Amino Acid Sequence↗

PubMed2026

Matching genomic evidence to claims about Mycobacterium avium subsp. paratuberculosis: Host association, host adaptation, mechanism, and virulence.

Whole-genome sequencing (WGS) permits high-resolution comparison of Mycobacterium avium subsp. paratuberculosis (MAP) isolates and pangenome analysis. Combined with animal-movement data, WGS can support transmission inference, but resolution alone does not establish the biological meaning of genomic variation. This focused narrative review applies a two-dimensional framework to purposively selected MAP studies, separating claim targets from support profiles. Claim targets include lineage identity, host-source or lineage characterization, host association, transmission, candidate genomic features, measured bacterial or host-cell phenotypes, natural-host infection fitness, disease or damage, shedding, and control outcomes. Depending on the claim, evidence operations may include characterization, context-aware comparative inference, direct endpoint ascertainment, and controlled feature perturbation; these are non-ordinal and may co-occur. On-target attribution, independent replication, and transportability are reported separately. The claim, not the study, is the unit of assessment. Typing markers support isolate or lineage discrimination, whereas phylogenomics supports evolutionary inference; neither alone establishes host adaptation. Pangenome comparisons and microbial genome-wide association studies nominate candidate features rather than establish adaptation. Cell-envelope and iron-associated studies support specified biochemical, transcriptional, or physiological phenotypes under defined conditions, while macrophage and calf models support only the endpoints measured. Annotated sequence variation alone nominates pathogenicity hypotheses. Across the illustrative studies selected here, MAP genomics most directly supported lineage classification, candidate discovery, measured bacterial phenotypes, bounded transmission inference, and natural-host infection-fitness claims. Claims about adaptation, mechanism, virulence, or control require endpoints and comparisons matched to the stated claim and model; feature-specific causal claims additionally require evidence linking the bacterial feature to the measured endpoint.

Claim boundaries↗

PubMed2004

Information assessment on predicting protein-protein interactions.

BACKGROUND: Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. RESULTS: Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. CONCLUSIONS: In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the Bayesian methods decreased classification performance.

Artificial Intelligence↗

PubMed2005

Identification of germ plasm-enriched mRNAs in Drosophila melanogaster by the cDNA microarray technique.

The development of embryonic germ cells in Drosophila depends on the germ plasm, the most posterior part of the ooplasm. The germ plasm is devoted to the formation of future germ cells and is known to contain all the factors that are necessary to induce germ cell fate. Besides having a characteristic organelle and protein distribution, the germ plasm also contains a large number of localized RNA species that have been shown to play crucial roles in germ cell determination. To identify germ plasm-enriched, localized transcripts, we used a two-step method composed of cDNA microarray (containing 3200 annotated Drosophila cDNAs) and in situ RNA hybridization techniques. We compared germ plasm deficient, normal and ectopic germ plasm conditions in the cDNA microarray experiments. RNA species whose concentration increased when ectopic germ plasm was present and decreased when the germ plasm was missing were selected. These candidates were then subjected to a second screen which compared the distribution of the given RNA in wild type embryos and in eggs with ectopic germ plasm. Finally, 17 RNA species were found to be enriched in the germ plasm. Based on these data, we estimate that around 1% of the Drosophila genes encode for germ plasm-enriched, localized transcripts. We conclude that this combination of microarray and in situ hybridization techniques is a simple but powerful experimental design for the genome-wide identification of genes coding for germ plasm localized transcripts.

Animals↗

PubMed2005

Recent innovations in tissue-specific gene modifications in the mouse.

Annotating the functions of individual genes in in vivo contexts has become the primary task of mouse genetics in the post-genome era. In addition to conventional approaches using transgenic technologies and gene targeting, the recent development of conditional gene modification techniques has opened novel opportunities for elucidating gene function at the level of the whole mouse to individual tissues or cell types. Tissue-specific gene modifications in the mouse have been made possible using site-specific DNA recombinases and conditional alleles. Recent innovations in this basic technology have facilitated new types of experiments, revealing novel insights into mammalian embryology. In this review, we focus on these recent innovations and new technical issues that impact the success of these conditional gene modification approaches.

Animals↗

PubMed2006

Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa.

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

Bacterial Proteins↗