Pc promoter from class 2 integrons and the cassette transcription pattern it evokes

Fonseca, Érica L.; Freitas, Fernanda dos Santos; Vicente, Ana Carolina Paulo

doi:10.1093/jac/dkr011

Cited by 16 publications

(19 citation statements)

References 12 publications

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“…The use of the bla gene eliminates any interference of plasmid copy number on relative quantification of cloned genes. The criteria to interpret the results are described elsewhere (5). Similar relative quantification values were obtained for the aadA2 (2.68 Ϯ 0.04 for the wild-type strain and 2.77 Ϯ 0.09 for recombinant strains) and qnrVC1 (2.51 Ϯ 0.1 for the wild-type strain and 2.60 Ϯ 0.07 for recombinant strains) genes, indicating similar transcript amounts.…”

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confidence: 60%

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Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-Associated qnrVC1 Gene

Fonseca

Vicente

2012

Antimicrob Agents Chemother

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Integrons are natural expression vectors due to the presence of an intrinsic promoter (P c ). Although rare, gene cassettes can harbor their own promoter. This study determined the functionality of an internal promoter in the qnrVC1 cassette whose presence was suggested by a level of transcription similar to that of the preceding cassette (aadA2) and confirmed by in silico analysis. Its functionality was determined by 5= rapid amplification of cDNA ends (RACE) and cloning into promoter-probe vectors. P qnrVC was found in the qnrVC cassette family, stressing its role in contributing to resistance manifestation.T he integron is a genetic element which produces an integrase capable of inserting, excising, and rearranging gene cassettes by site-specific recombination. This element is also considered a natural expression system due to the presence of a promoter (P c ) that controls the transcription of gene cassettes inserted in the array (12). Although gene cassettes are generally promoterless functional units, internal cassette-specific promoters have been identified by in silico analyses and by the resistance phenotype changes of transformed Escherichia coli (1,2,9,11,13). In general, the expression level of cassette-associated genes is influenced by their relative position in the array (cassette position effect), where genes closer to P c are more expressed than distal ones (3), and by the presence of cassette-specific promoters.The qnrVC1 cassette was previously found in a class 1 integron, preceded by the aadA2 cassette, from a clinical Brazilian Vibrio cholerae strain (4). Different from other gene cassettes, which have a specific attC recombination site, qnrVC1 was associated with an attC typically found in Vibrio chromosomal integrons (CIs). More precisely, its attC corresponded to a Vibrio parahaemolyticus repeat (VPR), which is one of the signatures of the V. parahaemolyticus CI. Moreover, although qnrVC1 has been found in a class 1 integron, it is more similar to the chromosomal qnr genes from Vibrionaceae than to the plasmid-borne determinants, such as qnrA1.This work fully characterized and determined the functionality of an internal cassette promoter distributed among the qnrVC alleles that was present in several genetic elements, ensuring their expression and the emergence of the resistance phenotype.The transcription of the aadA2-qnrVC1 array was assessed by real-time reverse transcription (RT)-PCR using Power SYBR green PCR master mix (Applied Biosystems). The transcription of gene cassettes was measured in both the wild V. cholerae strain (VC627) and in an E. coli DH5␣ strain transformed with the pGEM-T Easy vector (Promega) harboring the aadA2-qnrVC1 insert. The single-copy housekeeping rpoA gene from V. cholerae and the ␤-lactamase (bla) gene, present in the pGEM vector as a selective marker, were used for normalizing the transcription of wild-type and cloned genes, respectively (Table 1). The use of the bla gene eliminates any interference of plasmid copy number on relative quantification of clo...

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confidence: 60%

“…In order to determine whether qnrVC1 transcription begins under the control of its putative internal promoter, its transcription start site (TSS) was assessed by the 5= rapid amplification of cDNA ends (RACE) strategy as previously described (5). Sequence analysis revealed a TSS with the ϩ1 position located 14 bp upstream from the initiation codon of qnrVC1.…”

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confidence: 99%

Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-Associated qnrVC1 Gene

Fonseca

Vicente

2012

Antimicrob Agents Chemother

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“…In the integrons studied to date, the promoter driving the expression of the cassette genes is typically located either in the intI gene or in the region between the attI recombination site and the intI translational start site [17], [19], [21], [22]. However, it remained to be characterized in V. cholerae .…”

Section: Resultsmentioning

confidence: 99%

“…However, most of the resistance cassettes found in mobile integrons involved in antibiotic resistance development and spread lack a promoter [16]. Instead, their expression relies on a strong promoter located upstream of the recombination point, either in the attI itself, as in class 2 integrons [17], [18] or in the intI gene, as in class 1 and 3 integrons [19]–[22]. In V. cholerae most uncharacterized cassettes carry an open reading frame with a potential start codon located very close to the recombination site, leaving no space for a promoter.…”

Section: Introductionmentioning

confidence: 99%

The Superintegron Integrase and the Cassette Promoters Are Co-Regulated in Vibrio cholerae

Krin

Cambray²,

Mazel³

2014

PLoS ONE

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Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI) and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter) partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

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“…Three different gene cassette arrays, dfrA1-sat2-aadA1 , dfrA1-catB2-sat2-aadA1 , and sat2-aadA1 , were first detected in M. morganii isolates. The cassette arrays dfrA1-sat2-aadA1 and sat2-aadA1 have been reported in many Gram-negative bacteria, including E. coli , Salmonella , Campylobacter spp., P. mirabilis , P. vulgaris , K. pneumoniae , and Vibrio cholerae [29,30]. The cassette array dfrA1-catB2-sat2-aadA1 was observed for the first time in 2014 [31].…”

Section: Discussionmentioning

confidence: 99%

Characterization of Integrons and qnr Genes in Proteeae from a Teaching Hospital in China

Cao

Li²,

Xu³

et al. 2016

Chemotherapy

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Background: Proteeae isolates displaying multidrug-resistance (MDR) are the second most common causes of hospital-associated infections. The aim of this study was to screen class 1-3 integrons and plasmid-mediated quinolone resistance (PMQR) genes in Proteeae isolates from the First Affiliated Hospital of the Wenzhou Medical University. Materials and Methods: 176 Proteeae isolates were collected from clinical specimens of inpatients between January 2011 and December 2013. Susceptibility testing was determined by the agar dilution method. Class 1-3 integrons and PMQR genes were amplified by polymerase chain reaction, and the variable regions of integrons were determined by restriction fragment length polymorphisms. Results: 68.2% Proteeae isolates exhibited MDR phenotypes: 46.6 and 10.8% Proteeae isolates were positive for intI1 and intI2, respectively. The resistance rate of integron-positive isolates to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole was significantly higher than integron-negative isolates. Sequence analysis revealed that dfrA1-sat2-aadA1, dfrA1-catB2-sat2-aadA1, and sat2-aadA1 were first detected in Morganella morganii strains isolated from China. PMQR was determined by qnrD in 40 strains (22.7%). Conclusion: Our results indicate that class 1 and 2 integrons are common among Proteeae isolates. Meanwhile, qnrD are highly prevalent in Proteeae isolated from our hospital.

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Pc promoter from class 2 integrons and the cassette transcription pattern it evokes

Abstract: The Pc from class 2 integrons was characterized for the first time and the cassette position effect on transcription was demonstrated.

Cited by 16 publications

References 12 publications

Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-Associated qnrVC1 Gene

Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-Associated qnrVC1 Gene

The Superintegron Integrase and the Cassette Promoters Are Co-Regulated in Vibrio cholerae

Characterization of Integrons and <b><i>qnr</i></b> Genes in Proteeae<b> </b>from a Teaching Hospital in China

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