attI1 -Located Small Open Reading Frames ORF-17 and ORF-11 in a Class 1 Integron Affect Expression of a Gene Cassette Possessing a Canonical Shine-Dalgarno Sequence

Abstract

By searching the Integrall integron and GenBank databases, a novel open reading frame (ORF) of 51 nucleotides (nts) (ORF-17) overlapping the previously described ORF-11 was identified within the attI1 site in virtually all class 1 integrons. Using a set of isogenic plasmid constructs carrying a single gene cassette (bla GES-1 ) and possessing a canonical translation initiation region, we found that ORF-17 contributes to GES-1 expression.KEYWORDS class 1 integrons, attI1 sequence, gene cassettes, expression (2); however, the underlying mechanism remains unclear. We observed that ORF-11 was also present upstream in about 40% of gene cassettes possessing a canonical TIR. ORF-11 overlaps the inner boundary of the 5= conserved segment (CS) of class 1 integrons, with the stop codon of the ORF located in the 7-bp core site (G2TTRRRY) (3). ORF-11 can be created during recombination, provided that the inserted cassette sequence introduces an in-frame stop codon. Interestingly, a second ORF (ORF-17), comprising 51 nts and overlapping with ORF-11, was observed within the attI1 site in virtually all class 1 integrons ( Fig. 1; see also representative integrons in Table S1 in the supplemental material). Unlike ORF-11, formation of ORF-17 does not depend on the sequence of the gene cassette inserted downstream of attI1. These two ORFs possess canonical TIRs (Fig. 1).In this study, we found indications that ORF-17 and ORF-11 contribute to the expression of TIR-containing gene cassettes.A set of six plasmid variants of the previously described GES-1-encoding pHP329 was used (Table 1). pHP329 was a pACYC177 derivative containing a bla GES-1 gene cassette preceded by a hybrid 1 Pc promoter; the cloned sequence had been obtained from In58-G1, a Pseudomonas aeruginosa class 1 integron with a variable region possessing bla GES-1 as a single gene cassette (4). Shine-Dalgarno (SD) sequences and/or initiation codons of ORF-17, ORF-11, and bla GES-1 were subjected to site-specific mutagenesis using 12 mutagenic primers (see Table S2 in the supplemental material) and a QuikChange site-directed mutagenesis kit (Stratagene). Also, pCTRL, a promoterless variant of pHP329 constructed by reverting the 29-bp Pc sequence and deleting five of the 17 bp between the Ϫ35 and Ϫ10 hexamers, was used for control purposes (4).

Mobile Genetic Elements Associated with Antimicrobial Resistance

“…This means that cassettes can be carried at less cost at the "back" of an array but still have the potential to be shuffled to the "front" of the array. Some cassette genes lack a ribosome binding site (RBS), and ORF-11 (118) and the recently identified ORF-17 (119) in attI1 may contribute to expression if the cassette is the first in the array.…”

Integron activity accelerates the evolution of antibiotic resistance

“…Instead of a sharp drop, we observed a steady decrease depending on the cassette position ( Figure 1—figure supplement 1a ). Previous work has shown that two short open-reading frames contained within the attI site can substantially enhance the translation of a cassette lacking a Shine–Dalgarno (SD) sequence when the cassette is located in first position ( Hanau-Berçot et al, 2002 ; Papagiannitsis et al, 2017 ), showing that cassette position can also modulate translation levels ( Hanau-Berçot et al, 2002 ; Jacquier et al, 2009 ). Interestingly, the aadB cassette contains a reduced SD box ( Figure 1—figure supplement 1b ), suggesting that the steep gradient in gentamicin resistance between first and second positions was mostly due to decreased translation.…”

“…( B ) Representation of the genomic environment of the aadB cassette when aadB is in last (WTA3) or first position (WTA5) in our arrays. The two ORFs overlapping the attI , which have been shown to improve cassette translation ( Hanau-Berçot et al, 2002 ; Papagiannitsis et al, 2017 ), are represented in color. …”

Integron activity accelerates the evolution of antibiotic resistance

“…Instead of a sharp drop, we observed a steady decrease depending on the cassette position ( Figure S1a). Previous work has shown that two short open reading frames contained within the attI site can substantially enhance the translation of a cassette lacking a Shine-Dalgarno (SD) sequence when the cassette is located in first position (Hanau-Berçot et al, 2002;Papagiannitsis et al, 2017), showing that cassette position can also modulate translation levels (Hanau-Berçot et al, 2002;Jacquier et al, 2009). Interestingly, the aadB cassette contains a reduced SD box ( Figure S1b), suggesting that the steep gradient in gentamicin resistance between first and second position was mostly due to decreased translation.…”

“…Representation of the genomic environment of the aadB cassette when aadB is in last (WTA3) or first position (WTA5) in our arrays. The two ORFs overlapping the attI which have been shown to improve cassette translation (Hanau-Berçot et al, 2002;Papagiannitsis et al, 2017) are represented in colour.…”