Putative Insertion Sequence Research Articles

Genomic analysis of Burkholderia cenocepacia isolated from a liver abscess in a patient with cystic fibrosis.

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B.cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B.cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

DigIS: towards detecting distant and putative novel insertion sequence elements in prokaryotic genomes

BackgroundThe insertion sequence elements (IS elements) represent the smallest and the most abundant mobile elements in prokaryotic genomes. It has been shown that they play a significant role in genome organization and evolution. To better understand their function in the host genome, it is desirable to have an effective detection and annotation tool. This need becomes even more crucial when considering rapid-growing genomic and metagenomic data. The existing tools for IS elements detection and annotation are usually based on comparing sequence similarity with a database of known IS families. Thus, they have limited ability to discover distant and putative novel IS elements.ResultsIn this paper, we present digIS, a software tool based on profile hidden Markov models assembled from catalytic domains of transposases. It shows a very good performance in detecting known IS elements when tested on datasets with manually curated annotation. The main contribution of digIS is in its ability to detect distant and putative novel IS elements while maintaining a moderate level of false positives. In this category it outperforms existing tools, especially when tested on large datasets of archaeal and bacterial genomes.ConclusionWe provide digIS, a software tool using a novel approach based on manually curated profile hidden Markov models, which is able to detect distant and putative novel IS elements. Although digIS can find known IS elements as well, we expect it to be used primarily by scientists interested in finding novel IS elements. The tool is available at https://github.com/janka2012/digIS.

A Novel Arsenate-Resistant Determinant Associated with ICEpMERPH, a Member of the SXT/R391 Group of Mobile Genetic Elements.

ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its genetic structure, and its homology to related elements normally found in pathogenic Vibrio or Proteus species. Whole genome sequencing of Escherichia coli (E. coli) isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The “hotspot regions” of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes: orf69, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and orf70, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of Escherichia coli strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1–5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup.

Two identical nonylphenol monooxygenase genes linked to IS6100 and some putative insertion sequence elements in Sphingomonas sp. NP5

Sphingomonas sp. NP5 can degrade a wide range of nonylphenol (NP) isomers that have widely contaminated aquatic environments as major endocrine-disrupting chemicals. To understand the biochemical and genetic backgrounds of NP degradation, a gene library of strain NP5 was constructed using a broad-host-range vector pBBR1MCS-2 and introduced into Sphingobium japonicum UT26. Several transformants accumulated reddish brown metabolites on agar plates dispersed with a mixture of NP isomers. Two different DNA fragments (7.6 and 9.3 kb) involved in the phenotype were isolated from the transformants. Sequence analysis revealed that both fragments contained an identical 1593 bp monooxygenase gene (nmoA), the predicted protein sequence of which showed 83 % identity to the octylphenol-4-monooxygenase of Sphingomonas sp. PWE1. The nmoA gene in the 7.6 kb fragment was surrounded by an IS21-type insertion sequence (IS) and IS6100, while another in the 9.3 kb fragment was adjacent to an IS66-type IS, suggesting that they have been acquired through multiple transposition events. A fast-growing recombinant Pseudomonas putida strain harbouring nmoA was constructed and used for degradation of a chemically synthesized NP isomer, 4-(1-ethyl-1-methylhexyl)phenol. This strain converted the isomer into hydroquinone stoichiometrically. 3-Methyl-3-octanol, probably originating from the alkyl side chain, was also detected as the metabolite. These results indicate that these two nmoA genes are involved in the NP degradation ability of strain NP5.

Distribution of Putative Virulence Genes in Streptococcus mutans Strains Does Not Correlate with Caries Experience

Streptococcus mutans, a member of the human oral flora, is a widely recognized etiological agent of dental caries. The cariogenic potential of S. mutans is related to its ability to metabolize a wide variety of sugars, form a robust biofilm, produce copious amounts of lactic acid, and thrive in the acid environment that it generates. The remarkable genetic variability present within the species is reflected at the phenotypic level, notably in the differences in the cariogenic potential between strains. However, the genetic basis of these differences is yet to be elucidated. In this study, we surveyed by PCR and DNA hybridization the distribution of putative virulence genes, genomic islands, and insertion sequences across a collection of 33 strains isolated from either children with severe early childhood caries (S-ECC) or those who were caries free (CF). We found this genetically diverse group of isolates to be remarkably homogeneous with regard to the distribution of the putative virulence genes and genetic elements analyzed. Our findings point to the role of other factors in the pathogenesis of S-ECC, such as uncharacterized virulence genes, differences in gene expression and/or enzymatic activity, cooperation between S. mutans strains or with other members of the oral biota, and host factors.

Characterization of Small ColE-Like Plasmids Mediating Widespread Dissemination of the qnrB19 Gene in Commensal Enterobacteria

In this work, we have characterized two small ColE-like plasmids (pECY6-7, 2.7 kb in size, and pECC14-9, of 3.0 kb), encoding the QnrB19 quinolone resistance determinant, that were carried by several clonally unrelated quinolone-resistant commensal Escherichia coli strains isolated from healthy children living in different urban areas of Peru and Bolivia. The two plasmids are closely related to each other and carry the qnrB19 gene as the sole resistance determinant, located in a conserved genetic context between the plasmid RNAII sequence (which controls plasmid replication) and the plasmid Xer site (involved in plasmid dimer resolution). ISEcp1-like or other putative insertion sequences are not present in the qnrB19-flanking regions or elsewhere on the plasmids. Since we previously observed a high prevalence (54%) of qnrB genes in the metagenomes of commensal enterobacteria from the same population of healthy children, the presence of pECY6-7- and pECC14-9-like plasmids in those qnrB-positive metagenomes was investigated by PCR mapping. Both plasmids were found to be highly prevalent (67% and 16%, respectively) in the qnrB-positive metagenomes, suggesting that dissemination of these small plasmids played a major role in the widespread dissemination of qnrB genes observed in commensal enterobacteria from healthy children living in those areas.

A comparison of antibiotic resistance integrons in cattle from separate beef meat production systems at slaughter

To compare antibiotic resistance integrons in cattle from three separate grass-fed, grain-fed and certified organic cattle production systems at slaughter. In this study 198 samples from three separate cattle production systems were tested by PCR for the presence of class 1 and class 2 integrons. Integron-containing bacteria were readily isolated from pen faeces and hide samples regardless of production system. Lower numbers of integron-containing bacteria were isolated from the remaining sample types. Ninety-one class 1 and 34 class 2 integron-containing bacteria were isolated. Characterization of the integrons demonstrated a high degree of similarity across the three production systems with aadA1 and aadA2 routinely present. Integrons harbouring the cassette array cmlA5-bla(OXA-10)-aadA1 and the putative insertion sequence IS1066 were isolated from organic and grass-fed cattle and have not been described previously. Integrons carrying antibiotic resistance genes were common in cattle from differing production systems at slaughter and the likelihood of presence appears unrelated to the production system. Similar integron arrays are present in different cattle production systems suggesting that their presence may be independent of production practices. This is the first report of two novel integron structures present in Aeromonas.

Isolation and characterization of functional insertion sequences of rhizobia

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.

Pyrrolysine and Selenocysteine Use Dissimilar Decoding Strategies

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies.

Recombination in the genome of Chlamydia trachomatis involving the polymorphic membrane protein C gene relative to ompA and evidence for horizontal gene transfer.

Genome sequencing of Chlamydia trachomatis serovar D has identified polymorphic membrane proteins (Pmp) that are a newly recognized protein family unique to the Chlamydiaceae family. Cumulative data suggest that these diverse proteins are expressed on the cell surface and might be immunologically important. We performed phylogenetic analyses and statistical modeling with 18 reference serovars and 1 genovariant of C. trachomatis to examine the evolutionary characteristics and comparative genetics of PmpC and pmpC, the gene that encodes this protein. We also examined 12 recently isolated ocular and urogenital clinical samples, since reference serovars are laboratory adapted and may not represent strains that are presently responsible for human disease. Phylogenetic reconstructions revealed a clear distinction for disease groups, corresponding to levels of tissue specificity and virulence of the organism. Further, the most prevalent serovars, E, F, and Da, formed a distinct clade. According to the results of comparative genetic analyses, these three genital serovars contained two putative insertion sequence (IS)-like elements with 10- and 15-bp direct repeats, respectively, while all other genital serovars contained one IS-like element. Ocular trachoma serovars also contained both insertions. Previously, no IS-like elements have been identified for Chlamydiaceae. Surprisingly, 7 (58%) of 12 clinical isolates revealed pmpC sequences that were identical to the sequences of other serovars, providing clear evidence for a high rate of whole-gene recombination. Recombination and the differential presence of IS-like elements among distinct disease and prevalence groups may contribute to genome plasticity, which may lead to adaptive changes in tissue tropism and pathogenesis over the course of the organism's evolution.

Genetic environment and transcription of ampC in an Acinetobacter baumannii clinical isolate.

An ampC gene was cloned from a clinical isolate of Acinetobacter baumannii (strain RAN). DNA sequencing and primer extension studies showed that ampC is transcribed from a promoter contained within a putative insertion sequence element which has been found to abut several different genes in Acinetobacter spp.

Osmoregulatory systems of Escherichia coli: identification of betaine-carnitine-choline transporter family member BetU and distributions of betU and trkG among pathogenic and nonpathogenic isolates.

Multiple transporters mediate osmoregulatory solute accumulation in Escherichia coli K-12. The larger genomes of naturally occurring strains such as pyelonephritis isolates CFT073 and HU734 may encode additional osmoregulatory systems. CFT073 is more osmotolerant than HU734 in the absence of organic osmoprotectants, yet both strains grew in high osmolality medium at low K(+) (micromolar concentrations) and retained locus trkH, which encodes an osmoregulatory K(+) transporter. Both lacked the trkH homologue trkG. Transporters ProP and ProU account for all glycine-betaine uptake activity in E. coli K-12 and CFT073, but not in HU734, yet elimination of ProP and ProU impairs the growth of HU734, but not CFT073, in high osmolality human urine. No known osmoprotectant stimulated the growth of CFT073 in high osmolality minimal medium, but putative transporters YhjE, YiaMNO, and YehWXYZ may mediate uptake of additional osmoprotectants. Gene betU was isolated from HU734 by functional complementation and shown to encode a betaine uptake system that belongs to the betaine-choline-carnitine transporter family. The incidence of trkG and betU within the ECOR collection, representatives of the E. coli pathotypes (PATH), and additional strains associated with urinary tract infection (UTI) were determined. Gene trkG was present in 66% of the ECOR collection but only in 16% of the PATH and UTI collections. Gene betU was more frequently detected in ECOR groups B2 and D (50% of isolates) than in groups A, B1, and E (20%), but it was similar in overall incidence in the ECOR collection and in the combined UTI and PATH collections (32 and 34%, respectively). Genes trkG and betU may have been acquired by lateral gene transfer, since trkG is part of the rac prophage and betU is flanked by putative insertion sequences. Thus, BetU and TrkG contribute, with other systems, to the osmoregulatory capacity of the species E. coli, but they are not characteristic of a particular phylogenetic group or pathotype.

Tandem duplication of alkaline phosphatase genes and polymorphism in the intergenic sequence in Bombyx mori.

Alkaline phosphatases are ubiquitous in organisms from bacteria to human. Two alkaline phosphatase genes, Alp-m and Alp-s, were independently cloned from the silkworm Bombyx mori. They were mapped to a small DNA region and shown to be organized in tandem. Exon-intron structures of the two genes were highly conserved, with the exception of the second intron in Alp-m, which has no counterpart in Alp-s. The similarity between the nucleotide sequences of the exons of the two genes was strikingly high (60-79%), suggesting that Alp-m and Alp-s originated from a duplication of their common ancestor gene. The intergenic sequence between the two Alp genes shows length polymorphism in different B. mori strains, which can be explained by presence/absence of two putative insertion sequences. This structural variation suggests a possible scenario for the divergence of the two Alp genes after the duplication event.

Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri.

The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.

Tissue-specific gene expression identifies a gene in the lysogenic phage Gifsy-1 that affects Salmonella enterica serovar typhimurium survival in Peyer's patches.

In vivo expression technology was used to identify Salmonella enterica serovar Typhimurium genes that are transcriptionally induced when the bacteria colonize the small intestines of mice. These genes were subsequently screened for those that are transcriptionally inactive during the systemic stages of disease. This procedure identified gipA, a gene that is specifically induced in the small intestine of the animal. The gipA gene is carried on the lambdoid phage Gifsy-1. Consistent with the expression profile, the sole defect conferred by a gipA null mutation is in growth or survival in a Peyer's patch. The gipA strain is wild type in its ability to initially colonize the small intestine and invade the intestinal epithelium. The mutant also survives and propagates at wild-type levels during the systemic stages of disease. The gipA open reading frame is homologous to a family of putative insertion sequence elements, although our evidence shows that transposition is not required for gipA function in the Peyer's patch. These results suggest that the bacteria sense and respond to the particular environment of the Peyer's patch, a critical site for the replication of Salmonella serovar Typhimurium.

PT3.2I, the Smallest Plasmid of Thiobacillus T3.2

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3.2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.

Repeated extragenic sequences in prokaryotic genomes: a proposal for the origin and dynamics of the RUP element in Streptococcus pneumoniae.

A survey of all Streptococcus pneumoniae GenBank/EMBL DNA sequence entries and of the public domain sequence (representing more than 90% of the genome) of an S. pneumoniae type 4 strain allowed identification of 108 copies of a 107-bp-long highly repeated intergenic element called RUP (for repeat unit of pneumococcus). Several features of the element, revealed in this study, led to the proposal that RUP is an insertion sequence (IS)-derivative that could still be mobile. Among these features are: (1) a highly significant homology between the terminal inverted repeats (IRs) of RUPs and of IS630-Spn1, a new putative IS of S. pneumoniae; and (2) insertion at a TA dinucleotide, a characteristic target of several members of the IS630 family. Trans-mobilization of RUP is therefore proposed to be mediated by the transposase of IS630-Spn1. To account for the observation that RUPs are distributed among four subtypes which exhibit different degrees of sequence homogeneity, a scenario is invoked based on successive stages of RUP mobility and non-mobility, depending on whether an active transposase is present or absent. In the latter situation, an active transposase could be reintroduced into the species through natural transformation. Examination of sequences flanking RUP revealed a preferential association with ISs. It also provided evidence that RUPs promote sequence rearrangements, thereby contributing to genome flexibility. The possibility that RUP preferentially targets transforming DNA of foreign origin and subsequently favours disruption/rearrangement of exogenous sequences is discussed.

Identification of a virulence-associated protein homolog gene and IS Ra1 in a plasmid of Riemerella anatipestifer

Riemerella anatipestifer is the causative agent of polyserositis of ducks and geese. We have previously reported that a 3.9-kb plasmid, pCFC1, carries protein genes ( vapD1 and vapD2) that are similar to virulence-associated genes of other bacteria. In the present study, we report the complete sequence of a second plasmid of 5.6 kb, pCFC2. pCFC2 has a 28% G-C content and three large open reading frames (ORFs). One of the ORFs (designated asVapD1) encodes a polypeptide that shares 53.9, 53.9, 48.3, 48.3 and 46.1% identity with virulence-associated proteins of Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Neisseria gonorrhoeae, Helicobacter pylori and Haemophilus influenzae, respectively. The second ORF encodes a putative DNA replication protein (RepA3) with 309 amino acids and a molecular mass of approximately 36 kDa. A novel insertion sequence (IS) element, designated IS Ra1, was found on the plasmid pCFC2. IS Ra1 was flanked by 15-bp imperfect inverted repeats (only one mismatched nucleotide). IS Ra1 contained an ORF encoding a putative transposase of 292 amino acids. Southern blot analysis indicated that in R. anatipestifer strains examined, IS Ra1 was present with 2–20 copies (at least). IS Ra1 displayed a sequence approximately 35% homologous to the putative IS 982 and RSBst-α from Lactococcus lactis ssp. cremoris SK11 and Bacillus stearothermophilus CU21. Three hybridization patterns of genomic DNA of eight R. anatipestifer strains with an IS Ra1 probe indicated that IS Ra1 might be a useful tool for epidemiological studies.

Cloning and characterization of type III iodothyronine deiodinase from the fish Oreochromis niloticus.

Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131. The deduced amino acid sequence shows 57-67% identity with frog, chicken, and mammalian D3, 33-39% identity with frog, fish (Fundulus heteroclitus) and mammalian D2, and 30-35% identity with fish (tilapia), chicken, and mammalian D1. The 3' UTR contains a putative Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) expressed in COS-1 cells and native tD3 in tilapia brain microsomes show identical catalytic activities, with a strong preference for IRD of T3 (Km approximately 20 nM). IRD of [3,5-125I]T3 by native and recombinant tD3 are equally sensitive to inhibition by substrate analogs (T3 > T4 >> rT3) and inhibitors (gold thioglucose >> iodoacetate > propylthiouracil). Northern analysis using a tD3 riboprobe shows high expression of a 1.6-kb messenger RNA in gill and brain, although D3 activity is much higher in brain than in gill. The characterization of tD3 cDNA provides new information about the structure-activity relationship of iodothyronine deiodinases and an important tool to study the regulation of thyroid hormone bioactivity in fish.