Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3349-3360 ◽  
Author(s):  
Alicia M. Gestal ◽  
Elissa F. Liew ◽  
Nicholas V. Coleman
Keyword(s):  
Natural Transformation ◽  
Multiple Displacement Amplification ◽  
Pseudomonas Stutzeri ◽  
Gene Cassette ◽  
Synthetic Gene ◽  
Catabolic Gene ◽  
Green Fluorescence ◽  
Gene Cassettes ◽  
New Methods ◽  
Gentamicin Resistance

Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (GmR or Lac+) were obtained at frequencies ranging from 101 to 106 c.f.u. (µg DNA)−1. Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter PC were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

scholarly journals Recombination Activity of a Distinctive Integron-Gene Cassette System Associated with Pseudomonas stutzeri Populations in Soil

2003 ◽  
Vol 185 (3) ◽  
pp. 918-928 ◽  
Author(s):  
Andrew J. Holmes ◽  
Marita P. Holley ◽  
Andrew Mahon ◽  
Blair Nield ◽  
Michael Gillings ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Hot Spot ◽  
Antibiotic Resistance Gene ◽  
Pseudomonas Stutzeri ◽  
Chromosome Mapping ◽  
Gene Cassette ◽  
Genomic Diversity ◽  
Natural Environments ◽  
Recombination Activity ◽  
Gene Cassettes

ABSTRACT Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. The intI and attI components of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily). The recombination activity of P. stutzeri integron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present in P. stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species.

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1853-1864 ◽  
Author(s):  
Nicholas V. Coleman ◽  
Andrew J. Holmes
Keyword(s):  
Insertion Sequence ◽  
Pseudomonas Stutzeri ◽  
Gene Cassette ◽  
Evolutionary Significance ◽  
Reporter Plasmid ◽  
Green Fluorescence ◽  
Rt Pcr ◽  
Site Specific ◽  
Site Specific Integration ◽  
Green Fluorescent

The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded GmR green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attI was detected by PCR in 8 % of GmR colonies and the frequency of attI integration was estimated as 2·0×10−8 per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (Pc) was localized to the 5′ end of the intI gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attI site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attI and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.

scholarly journals Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

F1000Research ◽  
2015 ◽  
Vol 2 ◽  
pp. 99
Author(s):  
Érica L. Fonseca ◽  
Ana Carolina Paulo Vicente
Keyword(s):  
Real Time ◽  
Northern Blot ◽  
Gene Cassette ◽  
Rt Pcr ◽  
Recombination Site ◽  
Gene Cassettes ◽  
Polycistronic Transcription ◽  
Polymerase Chain ◽  
Fused Gene ◽  
Gene Cassette Array

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

scholarly journals Characterization of the Metallo-β-Lactamase Determinant of Acinetobacter baumannii AC-54/97 Reveals the Existence of blaIMP Allelic Variants Carried by Gene Cassettes of Different Phylogeny

2000 ◽  
Vol 44 (5) ◽  
pp. 1229-1235 ◽  
Author(s):  
Maria Letizia Riccio ◽  
Nicola Franceschini ◽  
Letizia Boschi ◽  
Berardo Caravelli ◽  
Giuseppe Cornaglia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Kinetic Parameters ◽  
Gene Cassette ◽  
Global Risk ◽  
Allelic Variants ◽  
Gene Cassettes ◽  
Class 1 ◽  
Broad Array ◽  
Environmental Reservoir

ABSTRACT The metallo-β-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (namedbla IMP-2) of bla IMPfound in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla IMP,bla IMP-2 was also carried by an integron-borne gene cassette. However, the 59-base element of thebla IMP-2 cassette was unrelated to those of thebla IMP cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-bornebla IMP-2 gene in Escherichia coliresulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several β-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some β-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla IMP alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.

scholarly journals Molecular Characterization of a New Class 3 Integron in Klebsiella pneumoniae

2003 ◽  
Vol 47 (9) ◽  
pp. 2838-2843 ◽  
Author(s):  
Mário Correia ◽  
Filipa Boavida ◽  
Filipa Grosso ◽  
M. J. Salgado ◽  
L. M. Lito ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gene Cassette ◽  
Promoter Regions ◽  
Fusion Event ◽  
Integrase Gene ◽  
Gene Cassettes ◽  
New Class ◽  
Extended Spectrum ◽  
Class 1 ◽  
Cephalosporin Resistance

ABSTRACT Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum β-lactamase was encoded by bla GES-1, previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla GES-1 gene cassette was located on a new class 3 integron. The integron was 2,863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla GES-1 gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla OXA-10-type and aac(6′)-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla GES-1 gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

scholarly journals The aadB Gene Cassette Is Associated with blaSHV Genes in Klebsiella Species Producing Extended-Spectrum β-Lactamases

2005 ◽  
Vol 49 (2) ◽  
pp. 794-797 ◽  
Author(s):  
Louisa A. Jones ◽  
Christopher J. McIver ◽  
Mi-Jurng Kim ◽  
William D. Rawlinson ◽  
Peter A. White
Keyword(s):  
Gene Cassette ◽  
Amplicon Sequencing ◽  
Klebsiella Species ◽  
Class 1 Integron ◽  
Beta Lactamases ◽  
Gene Cassettes ◽  
Extended Spectrum ◽  
Class 1 ◽  
Extended Spectrum Beta Lactamases

ABSTRACT Integrons were detected in 37 (72.5%) of 51 Klebsiella spp. producing extended-spectrum beta-lactamases by PCR with primers that targeted integrase genes and cassette regions. PCR and amplicon sequencing of the cassette regions revealed aadB and aadA2 gene cassettes that confer resistance to a range of aminoglycosides. aadB was associated with a class 1 integron on a 28-kb plasmid, pES1, that also contained bla SHV-12 and IS26.

Identification of plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens

2009 ◽  
Vol 58 (2) ◽  
pp. 217-221 ◽  
Author(s):  
Zhuting Hu ◽  
Wei-Hua Zhao
Keyword(s):  
Serratia Marcescens ◽  
Single Point ◽  
Gene Cassette ◽  
Clinical Isolates ◽  
Single Point Mutation ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
E Coli ◽  
Class 1 ◽  
P2 Promoter

The emergence of carbapenem-hydrolysing metallo-β-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid- and integron-borne bla IMP-1 and bla IMP-10 in clinical isolates of Serratia marcescens. The bla IMP-1 and bla IMP-10 gene cassettes were carried by a class 1 integron and followed by the aac(6′)-IIc gene cassette. The bla IMP-1 and bla IMP-10 gene cassettes were preceded by a weak Pant promoter, TGGACA(N)17TAAGCT, and an inactive P2 promoter, TTGTTA(N)14TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla IMP-1, the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64- and 128-fold, respectively. In comparison, after gaining bla IMP-10, the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256- and 64-fold, respectively. Strains harbouring bla IMP-10 showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla IMP-1, although the nucleotide sequences of the class 1 integrons carrying bla IMP-10 and bla IMP-1 were identical except for a single point mutation.

scholarly journals Characterization of In53, a Class 1 Plasmid- and Composite Transposon-Located Integron of Escherichia coli Which Carries an Unusual Array of Gene Cassettes

2001 ◽  
Vol 183 (1) ◽  
pp. 235-249 ◽  
Author(s):  
Thierry Naas ◽  
Yuzuru Mikami ◽  
Tamae Imai ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Gene Cassette ◽  
Promoter Sequence ◽  
Class 1 Integron ◽  
Integrase Gene ◽  
Chloramphenicol Resistance ◽  
Gene Cassettes ◽  
Class 1

ABSTRACT Further characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum β-lactamase, bla VEB-1, revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition tobla VEB-1. While the aadB and the arr-2 gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of the cmlAfamily, (ii) a qac allele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette,aacA1b/orfG, which encodes a novel 6′-N-acetyltransferase, and (iv) a fused gene cassette,oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition, oxa10 andaadA1 genes were expressed from their own promoter sequence present upstream of the oxa10 cassette.arr-2 coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by thearr-1 gene from Mycobacterium smegmatisDSM43756. While in M. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered from E. coli culture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26 insertion sequence, which was also present in the 3′ conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26 elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.

scholarly journals High Genetic Stability of Integrons in Clinical Isolates of Shigella spp. of Worldwide Origin

2007 ◽  
Vol 51 (4) ◽  
pp. 1333-1340 ◽  
Author(s):  
Véronique Dubois ◽  
Marie-Pierre Parizano ◽  
Corinne Arpin ◽  
Laure Coulange ◽  
Marie-Christine Bezian ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Gene Cassette ◽  
University Hospital ◽  
Ampicillin Resistance ◽  
Gene Cassettes ◽  
Class 1 Integrons ◽  
The World ◽  
Class 1 ◽  
Shigella Spp ◽  
History Of

ABSTRACT Over a 12-year period, 68 Shigella strains (31 S. sonnei, 30 S. flexneri, 4 S. dysenteriae, and 3 S. boydii strains) were collected in a French University Hospital from the stools of patients who generally had a recent history of travel to various parts of the world (91%), particularly Africa (67%). These strains were often resistant (streptomycin, spectinomycin, trimethoprim, tetracycline, and sulfonamides, 66 to 84%; ampicillin and chloramphenicol, 34 to 38%; nalidixic acid, 4%) and even multiresistant (87%), and they generally carried integrons (81%) of class 1 (21%), class 2 (47%), or both (13%). Class 1 integrons were associated with ampicillin resistance due to the production of an OXA-30 β-lactamase in S. flexneri and S. dysenteriae. Class 2 integrons were associated with trimethoprim resistance in S. sonnei. Class 1 and class 2 integrons were inserted within transposons Tn21 and Tn7, respectively, themselves located on the bacterial chromosome, except in one strain. Class 1 integrons showed an atypical organization consisting of the insertion sequence IS1 at the 3′ end instead of the typical 3′ conserved segment and two bla OXA-30 and aadA1 gene cassettes, despite the absence of epidemiological relationships between the strains, and an apparently functional integrase. Class 2 integrons showed the same albeit classical organization with the three dfrA1, sat, and aadA1 gene cassettes. Occasionally, the 3′ end was deleted and the aadA1 gene cassette was unexpressed. Thus, integrons contributed only in part to the multidrug resistance of the Shigella strains. The highly conserved organization of integrons might be related to their location within mobile genetic superstructures.

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