Biomedical subjects

Fiona S L Brinkman

Publications and source records attributed to Fiona S L Brinkman.

At least 19 recordsLinked to original sources

PubMed2006

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.

Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.

Bacterial Proteins↗

PubMed2006

Methods for predicting bacterial protein subcellular localization.

The computational prediction of the subcellular localization of bacterial proteins is an important step in genome annotation and in the search for novel vaccine or drug targets. Since the 1991 release of PSORT I--the first comprehensive algorithm to predict bacterial protein localization--many other localization prediction tools have been developed. These methods offer significant improvements in predictive performance over PSORT I and the accuracy of some methods now rivals that of certain high-throughput laboratory methods for protein localization identification.

Amino Acid Sequence↗

PubMed2006

Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.

Active Transport, Cell Nucleus↗

PubMed2006

Improving the specificity of high-throughput ortholog prediction.

BACKGROUND: Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function. RESULTS: To address these issues, we present here a high-throughput computational method named Ortholuge that further evaluates previously predicted orthologs (including those predicted using an RBH-based approach) - identifying which orthologs most closely reflect species divergence and may more likely have similar function. Ortholuge analyzes phylogenetic distance ratios involving two comparison species and an outgroup species, noting cases where relative gene divergence is atypical. It also identifies some cases of gene duplication after species divergence. Through simulations of incomplete genome data/gene loss, we show that the vast majority of genes falsely predicted as orthologs by an RBH-based method can be identified. Ortholuge was then used to estimate the number of false-positives (predominantly paralogs) in selected RBH-predicted ortholog datasets, identifying approximately 10% paralogs in a eukaryotic data set (mouse-rat comparison) and 5% in a bacterial data set (Pseudomonas putida - Pseudomonas syringae species comparison). Higher quality (more precise) datasets of orthologs, which we term "ssd-orthologs" (supporting-species-divergence-orthologs), were also constructed. These datasets, as well as Ortholuge software that may be used to characterize other species' datasets, are available at http://www.pathogenomics.ca/ortholuge/ (software under GNU General Public License). CONCLUSION: The Ortholuge method reported here appears to significantly improve the specificity (precision) of high-throughput ortholog prediction for both bacterial and eukaryotic species. This method, and its associated software, will aid those performing various comparative genomics-based analyses, such as the prediction of conserved regulatory elements upstream of orthologous genes.

Algorithms↗

PubMed2006

Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37.

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (< or =1 microg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-alpha in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kappaB in the presence of LPS, including NFkappaB1 (p105/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappaB inhibitor, NFkappaBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kappaB subunits p50 and p65 was reduced > or =50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kappaB pathway. LL-37 almost completely prevented the release of TNF-alpha and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-alpha or IL-1beta. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.

Antimicrobial Cationic Peptides↗

PubMed2006

Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa.

When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg(2+) regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg(2+)-limited and Mg(2+)-replete conditions to isogenic phoP and pmrA mutants grown under Mg(2+)-limited conditions. Under Mg(2+)-limited conditions (0.02 mM Mg(2+)), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg(2+)-replete conditions (2 mM Mg(2+)). Only a modest subset of the Mg(2+)-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg(2+) in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.

Bacterial Proteins↗

PubMed2005

Evidence of a large novel gene pool associated with prokaryotic genomic islands.

Microbial genes that are "novel" (no detectable homologs in other species) have become of increasing interest as environmental sampling suggests that there are many more such novel genes in yet-to-be-cultured microorganisms. By analyzing known microbial genomic islands and prophages, we developed criteria for systematic identification of putative genomic islands (clusters of genes of probable horizontal origin in a prokaryotic genome) in 63 prokaryotic genomes, and then characterized the distribution of novel genes and other features. All but a few of the genomes examined contained significantly higher proportions of novel genes in their predicted genomic islands compared with the rest of their genome (Paired t test = 4.43E-14 to 1.27E-18, depending on method). Moreover, the reverse observation (i.e., higher proportions of novel genes outside of islands) never reached statistical significance in any organism examined. We show that this higher proportion of novel genes in predicted genomic islands is not due to less accurate gene prediction in genomic island regions, but likely reflects a genuine increase in novel genes in these regions for both bacteria and archaea. This represents the first comprehensive analysis of novel genes in prokaryotic genomic islands and provides clues regarding the origin of novel genes. Our collective results imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. Moreover, there are more novel genes within the gene pool associated with genomic islands. Since genomic islands are frequently associated with a particular microbial adaptation, such as antibiotic resistance, pathogen virulence, or metal resistance, this suggests that microbes may have access to a larger "arsenal" of novel genes for adaptation than previously thought.

Bacteriophages↗

PubMed2005

Assessing the precision of high-throughput computational and laboratory approaches for the genome-wide identification of protein subcellular localization in bacteria.

BACKGROUND: Identification of a bacterial protein's subcellular localization (SCL) is important for genome annotation, function prediction and drug or vaccine target identification. Subcellular fractionation techniques combined with recent proteomics technology permits the identification of large numbers of proteins from distinct bacterial compartments. However, the fractionation of a complex structure like the cell into several subcellular compartments is not a trivial task. Contamination from other compartments may occur, and some proteins may reside in multiple localizations. New computational methods have been reported over the past few years that now permit much more accurate, genome-wide analysis of the SCL of protein sequences deduced from genomes. There is a need to compare such computational methods with laboratory proteomics approaches to identify the most effective current approach for genome-wide localization characterization and annotation. RESULTS: In this study, ten subcellular proteome analyses of bacterial compartments were reviewed. PSORTb version 2.0 was used to computationally predict the localization of proteins reported in these publications, and these computational predictions were then compared to the localizations determined by the proteomics study. By using a combined approach, we were able to identify a number of contaminants and proteins with dual localizations, and were able to more accurately identify membrane subproteomes. Our results allowed us to estimate the precision level of laboratory subproteome studies and we show here that, on average, recent high-precision computational methods such as PSORTb now have a lower error rate than laboratory methods. CONCLUSION: We have performed the first focused comparison of genome-wide proteomic and computational methods for subcellular localization identification, and show that computational methods have now attained a level of precision that is exceeding that of high-throughput laboratory approaches. We note that analysis of all cellular fractions collectively is required to effectively provide localization information from laboratory studies, and we propose an overall approach to genome-wide subcellular localization characterization that capitalizes on the complementary nature of current laboratory and computational methods.

Bacterial Proteins↗

PubMed2005

Molecular analyses of disease pathogenesis: application of bovine microarrays.

The molecular analysis of disease pathogenesis in cattle has been limited by the lack of availability of tools to analyze both host and pathogen responses. These limitations are disappearing with the advent of methodologies such as microarrays that facilitate rapid characterization of global gene expression at the level of individual cells and tissues. The present review focuses on the use of microarray technologies to investigate the functional pathogenomics of infectious disease in cattle. We discuss a number of unique issues that must be addressed when designing both in vitro and in vivo model systems to analyze host responses to a specific pathogen. Furthermore, comparative functional genomic strategies are discussed that can be used to address questions regarding host responses that are either common to a variety of pathogens or unique to individual pathogens. These strategies can also be applied to investigations of cell signaling pathways and the analyses of innate immune responses. Microarray analyses of both host and pathogen responses hold substantial promise for the generation of databases that can be used in the future to address a wide variety of questions. A critical component limiting these comparative analyses will be the quality of the databases and the complete functional annotation of the bovine genome. These limitations are discussed with an indication of future developments that will accelerate the validation of data generated when completing a molecular characterization of disease pathogenesis in cattle.

Animals↗

PubMed2005

Genetic and molecular analysis of GogB, a phage-encoded type III-secreted substrate in Salmonella enterica serovar typhimurium with autonomous expression from its associated phage.

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process.

Amino Acid Sequence↗

PubMed2005

Phylogeny of Na+/Ca2+ exchanger (NCX) genes from genomic data identifies new gene duplications and a new family member in fish species.

The Na+/Ca2+ exchanger (NCX) is a member of the cation/Ca2+ antiporter (CaCA) family and plays a key role in maintaining cellular Ca2+ homeostasis in a variety of cell types. NCX is present in a diverse group of organisms and exhibits high overall identity across species. To date, three separate genes, i.e., NCX1, NCX2, and NCX3, have been identified in mammals. However, phylogenetic analysis of the exchanger has been hindered by the lack of nonmammalian NCX sequences. In this study, we expand and diversify the list of NCX sequences by identifying NCX homologs from whole-genome sequences accessible through the Ensembl Genome Browser. We identified and annotated 13 new NCX sequences, including 4 from zebrafish, 4 from Japanese pufferfish, 2 from chicken, and 1 each from honeybee, mosquito, and chimpanzee. Examination of NCX gene structure, together with construction of phylogenetic trees, provided novel insights into the molecular evolution of NCX and allowed us to more accurately annotate NCX gene names. For the first time, we report the existence of NCX2 and NCX3 in organisms other than mammals, yielding the hypothesis that two serial NCX gene duplications occurred around the time vertebrates and invertebrates diverged. In addition, we have found a putative new NCX protein, named NCX4, that is related to NCX1 but has been observed only in fish species genomes. These findings present a stronger foundation for our understanding of the molecular evolution of the NCX gene family and provide a framework for further NCX phylogenetic and molecular studies.

Amino Acid Sequence↗

PubMed2005

PSORTdb: a protein subcellular localization database for bacteria.

Information about bacterial subcellular localization (SCL) is important for protein function prediction and identification of suitable drug/vaccine/diagnostic targets. PSORTdb (http://db.psort.org/) is a web-accessible database of SCL for bacteria that contains both information determined through laboratory experimentation and computational predictions. The dataset of experimentally verified information (approximately 2000 proteins) was manually curated by us and represents the largest dataset of its kind. Earlier versions have been used for training SCL predictors, and its incorporation now into this new PSORTdb resource, with its associated additional annotation information and dataset version control, should aid researchers in future development of improved SCL predictors. The second component of this database contains computational analyses of proteins deduced from the most recent NCBI dataset of completely sequenced genomes. Analyses are currently calculated using PSORTb, the most precise automated SCL predictor for bacterial proteins. Both datasets can be accessed through the web using a very flexible text search engine, a data browser, or using BLAST, and the entire database or search results may be downloaded in various formats. Features such as GO ontologies and multiple accession numbers are incorporated to facilitate integration with other bioinformatics resources. PSORTdb is freely available under GNU General Public License.

Bacterial Proteins↗

PubMed2005

Pseudomonas aeruginosa Genome Database and PseudoCAP: facilitating community-based, continually updated, genome annotation.

Using the Pseudomonas aeruginosa Genome Project as a test case, we have developed a database and submission system to facilitate a community-based approach to continually updated genome annotation (http://www.pseudomonas.com). Researchers submit proposed annotation updates through one of three web-based form options which are then subjected to review, and if accepted, entered into both the database and log file of updates with author acknowledgement. In addition, a coordinator continually reviews literature for suitable updates, as we have found such reviews to be the most efficient. Both the annotations database and updates-log database have Boolean search capability with the ability to sort results and download all data or search results as tab-delimited files. To complement this peer-reviewed genome annotation, we also provide a linked GBrowse view which displays alternate annotations. Additional tools and analyses are also integrated, including PseudoCyc, and knockout mutant information. We propose that this database system, with its focus on facilitating flexible queries of the data and providing access to both peer-reviewed annotations as well as alternate annotation information, may be a suitable model for other genome projects wishing to use a continually updated, community-based annotation approach. The source code is freely available under GNU General Public Licence.

DNA, Bacterial↗

PubMed2005

Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen.

The Gram-negative pathogen Pseudomonas aeruginosa encodes multiple protein export systems, the substrates of which contain export signals such as N-terminal signal peptides. Here we report the first genome-wide computational and laboratory screen for N-terminal signal peptides in this important opportunistic pathogen. The computational identification of signal peptides was based on a consensus between multiple predictive tools and showed that 38% of the P. aeruginosa PAO1 proteome was predicted to encode exported proteins, most of which utilize cleavable type I signal peptides or uncleavable transmembrane helices. In addition, known and novel lipoproteins (type II), twin arginine transporter (TAT), and prepilin peptidase substrates (type IV) were also identified. A laboratory-based screen using the alkaline phosphatase (PhoA) fusion method was then used to test our predictions. In total, 310 nonredundant PhoA fusions were successfully identified, 296 of which possess a predicted export signal. Analysis of the PhoA fusion proteins lacking an export signal revealed that three proteins have alternate translation start sites that encode signal peptides, two proteins may use an unknown export signal, and the remaining nine proteins are likely cytoplasmic proteins and represent false positives associated with the PhoA screen. Our approach to identify exported proteins illustrates how computational and laboratory-based methods are complementary, where computational analyses provide a large number of accurate predictions while laboratory methods both confirm predictions and reveal unique cases meriting further analysis.

Alkaline Phosphatase↗

PubMed2005

Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes.

Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived) infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, -55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology.

Antimicrobial Cationic Peptides↗

PubMed2004

ArrayPipe: a flexible processing pipeline for microarray data.

A number of microarray analysis software packages exist already; however, none combines the user-friendly features of a web-based interface with potential ability to analyse multiple arrays at once using flexible analysis steps. The ArrayPipe web server (freely available at www.pathogenomics.ca/arraypipe) allows the automated application of complex analyses to microarray data which can range from single slides to large data sets including replicates and dye-swaps. It handles output from most commonly used quantification software packages for dual-labelled arrays. Application features range from quality assessment of slides through various data visualizations to multi-step analyses including normalization, detection of differentially expressed genes, andcomparison and highlighting of gene lists. A highly customizable action set-up facilitates unrestricted arrangement of functions, which can be stored as action profiles. A unique combination of web-based and command-line functionality enables comfortable configuration of processes that can be repeatedly applied to large data sets in high throughput. The output consists of reports formatted as standard web pages and tab-delimited lists of calculated values that can be inserted into other analysis programs. Additional features, such as web-based spreadsheet functionality, auto-parallelization and password protection make this a powerful tool in microarray research for individuals and large groups alike.

Gene Expression Profiling↗

PubMed2004

ProbeLynx: a tool for updating the association of microarray probes to genes.

As genome sequence data and gene prediction improve, probes developed for a given microarray experiment should be continuously re-evaluated for their specificity for given genes. ProbeLynx(www.pathogenomics.ca/probelynx) is a new web service which uses current genomic sequence information to re-examine microarray probe specificity and provide annotation updates relevant to determining which gene(s) and transcript(s) are associated with a given probe. Probe sequences (either oligonucleotide- or cDNA-based) are uploaded in FASTA format and the results returned as a tab-delimited flat file for insertion into a spreadsheet application or database management system for further analysis. ProbeLynx has been initially developed to focus on arrays derived from human, mouse, chicken and bovine genomes, but may be expanded to handle other genomic datasets. ProbeLynx offers microarray users the important ability to continuously assess the potential of a probe to cross-hybridize to paralogous genes and the suitability of a given probe to investigate a transcript of interest. By also including the latest gene function annotation information in the output, ProbeLynx provides the critical first step in updating microarray data annotation.

Animals↗

PubMed2004

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1.

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.

Amino Acid Sequence↗