What are superintegrons?
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Chromosomes, Bacterial↗
Biomedical subjects
Paul H Roy
Publications and source records attributed to Paul H Roy.
Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn.
In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA(Asn) but by specific tRNA-dependent transamidation of aspartyl-tRNA(Asn). This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA(Asp) and tRNA(Asn) with aspartic acid. Recently, it has been shown for the first time in an organism (Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA(Asn) but does not participate in Gln-tRNA(Gln) formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA(Asn). Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA(Asp) charging over that of tRNA(Asn) by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA(Asp) and tRNA(Asn). Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.
Amino Acid Sequence↗
Genetic characterization of Vibrio cholerae isolates from Argentina by V. cholerae repeated sequences-polymerase chain reaction.
We have developed a novel typing method based on Vibrio cholerae repeat sequences (VCR) using primers directed out of the VCR sequences. To evaluate the VCR-polymerase chain reaction (PCR) as a typing system, 2 categories, efficacy and efficiency, were analyzed in 69 strains of human and environmental V. cholerae O1 toxigenic and nontoxigenic, and non-O1 strains isolated since 1992-2000 from Argentina. The discriminatory power (0.91), stability (0.95), reproducibility (1), typeability (1), rapidity, accessibility, as well ease of use, indicated that the VCR-PCR method provides an alternative useful tool for molecular epidemiology of V. cholerae. The VCR-PCR of V. cholerae isolates showed 29 patterns, of which pattern 1 represented 68% of the V. cholerae O1 isolates, supporting the hypothesis that a clone with epidemic behavior was responsible for the epidemic in Latin America. These results showed a good correlation and a better epidemiologic analysis when the results were compared in parallel with repetitive extragenic palindromic sequences-PCR. In conclusion, VCR-PCR showed excellent performance as a typing method for cholera surveillance programs.
Argentina↗
Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase beta-subunit.
The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase beta-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.
Amino Acid Sequence↗
Crystal structure of chloramphenicol acetyltransferase B2 encoded by the multiresistance transposon Tn2424.
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Binding Sites↗
Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1.
The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT. On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.
Amino Acid Sequence↗
Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species.
A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.
DNA Primers↗
Direct sequencing and PCR mapping of integrons reveals multiple class 1 integrons in the multiresistant strain Enterobacter cloacae SCH88040794.
We have characterized the variable region of three integrons found in the multiresistant strain Enterobacter cloacae SCH88040794, using polymerase chain reaction and direct sequencing of amplicons. The first integron has the gene cassette arrangement dfrAI-aadA1 in its variable region and the second aadB-oxa9. The third integron is In40 (aacA4-qacF-cmlB-oxa9), which has recently been described. The multiresistance expressed by this strain to amikacin, gentamicin, trimethoprim, sulfonamides, oxacillin, chloramphenicol and quaternary ammonium compounds is due, at least in part, to these three integrons.
Anti-Bacterial Agents↗
An integron cassette encoding erythromycin esterase, ere(A), from Providencia stuartii.
We have mapped the variable region of the two class 1 integrons found in the multiresistant strain Providencia stuartii 1723. Integron 1 contains a new arrangement of gene cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides used for clinical treatment. Integron 2 contains a variant of the gene cassette ere(A), coding for an erythromycin esterase, whose nucleotide sequence shares 93.7% DNA identity with ere(A) from Escherichia coli BM2195 plasmid pIP1100.
Carboxylic Ester Hydrolases↗
Molecular epidemiology of orf513-bearing class 1 integrons in multiresistant clinical isolates from Argentinean hospitals.
The spread of orf513-bearing class 1 integrons is associated with bla(CTX-M-2) in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without bla(CTX-M-2), only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.
Argentina↗
Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea.
We found in the environmental strain Nitrosomonas europaea a chromosomal integron-like structure with an integrase gene, intI(Neu). We have tested the capacity of the IntINeu integrase to excise and integrate several resistance gene cassettes. The results allow us to consider IntINeu a new functional integron integrase.
Attachment Sites, Microbiological↗
Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus.
Resistance to fluoroquinolones among clinical isolates of Staphylococcus aureus has become a clinical problem. Therefore, a rapid method to identify S. aureus and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of S. aureus and mutations at codons Ser-80 and Glu-84 of the grlA gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-S. aureus bacterial species tested. A total of 85 S. aureus isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the S. aureus isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of S. aureus and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of S. aureus and its susceptibility to fluoroquinolones.
Anti-Infective Agents↗
Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion.
We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3")-Ia cassettes. The first cassette in integron 2 contains the ant(2")-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3")-Ii-aac(6')-IId, which has been characterized for its bifunctional ANT(3")-I and AAC(6')-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla(OXA-10) cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla(OXA-10) cassette is incomplete and that the 3' conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3")-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.
Amino Acid Sequence↗
blaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513.
Examination of the bla(CTX-M-2) gene in plasmid pMAR-12 by sequencing and PCR analysis revealed that the bla gene and the surrounding DNA, which is closely related (99% homology) to the Kluyvera ascorbata chromosomal DNA that contains the bla(KLUA-1) gene, are located in a complex sul1-type integron, termed In35, that includes Orf513. It is possible that bla(CTX-M-2) was acquired by plasmid pMAR-12 through an uncharacterized recombinational event in which Orf513 could be involved.
Base Sequence↗
IntI2 integron integrase in Tn7.
Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.
3' Untranslated Regions↗
The IntI-like tyrosine recombinase of Shewanella oneidensis is active as an integron integrase.
We have found an integron-like integrase gene and an attI site in Shewanella oneidensis as part of a small chromosomal integron. We have cloned this gene and tested the ability of the integrase to excise cassettes from various integrons. Most cassettes flanked by two attC sites are readily excised, while cassettes in the "first" position, with an attI1 or attI3 site on one end, are not excised. An exception is a cassette with attI2 on one end. The attI2 site, from Tn7, has greater similarity to the attI site adjacent to the integrase of S. oneidensis than do attI1 or attI3. We cloned the attI site of S. oneidensis and observed the integration of two different cassettes. We have, therefore, demonstrated the function of this integron-like integrase.
Amino Acid Sequence↗