Expression of antibiotic resistance genes in the integrated cassettes of integrons

Abstract

Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter P ant , and alterations in the sequence of P ant affected the level of resistance expressed by cassette genes. When both P ant and the secondary promoter P2 were present, transcription from both promoters was detected. When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene. In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P ant , and was reduced to different extents by the presence of individual upstream cassettes. In Northern (RNA) blots, multiple discrete transcripts originating at P ant were detected, and only the longer transcripts contained the distal genes. Together, these data suggest that premature transcription termination occurs within the cassettes. The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3 to the coding region) not only function as recombination sites but may also function as transcription terminators.Integrons are genetic elements that contain the determinants of a site-specific recombination system by means of which they are able to capture genes (21). The captured genes, most commonly antibiotic resistance genes, are part of discrete mobile cassettes which contain the gene coding region and a recombination site, known as a 59-base element, located at the 3Ј end of the gene ( Fig. 1) (6-8, 10). The 59-base elements are recombination sites recognized by the integron integrase, and cassettes are inserted at attI (6), a unique integrase recognition site located in the integron adjacent to the integrase gene (19). Once integrated, cassettes are formally part of the integron, and in naturally occurring integrons there appear to be no restrictions on the number or order of inserted cassettes (see reference 10 for a compilation). Moreover, as the cassettes are discrete units that can be independently mobilized by the integron integrase (7), the arrangement of the cassettes in the insert region can be altered by excision of individual cassettes or reassortment (7, 8), or new cassettes can be precisely inserted (6).In general, the initiation codons of the genes found in cassettes are located very close to one boundary of the cassette and a promoter is not included in the cassette. The cassettes are all inserted in the same orientation with respect to their coding regions and are believed to be expressed from a common promoter, P ant , which is the only region with strong similarity to the Escherichia coli promoter consensus present in the conserved segment of the integron (5Ј-conserved segment) located 5Ј to the ...

Gene cassettes: a new class of mobile element

“…All originate at Pant but end prematurely at discrete points that appear to correspond to the ends of cassettes. These observations raise the possibility that the 59-base elements, which generally include inverted repeats, either act as transcription terminators or as processing signals for endonucleolytic cleavage of transcripts (Collis & Hall) 1995). This effect leads to differences in the levels of transcripts from promoter proximal and promoter distal genes.…”

“…Recent studies have demonstrated the existence of long transcripts covering several cassettes (Collis & Hall, 1995). However, the majority of transcripts are shorter.…”

“…This effect leads to differences in the levels of transcripts from promoter proximal and promoter distal genes. These differences reflect differences in the level of resistance conferred by the same cassette gene when its cassette is first or located downstream of other cassettes, and in general the first cassette is expressed at the highest level (Collis & Hall, 1995).…”

“…In class 1 integrons, four variants of this promoter are known (Stokes & Hall, 1989;Bunny etal., 1995). These variants differ in strength over at least a 20-fold range (Levesque e t al., 1994;Collis & Hall, 1995), and the strongest has an activity equivalent to or greater than that of the tac promoter (Lkvesque e t al., 1994; C. Tomaras, H. W. Hall, unpublished observations). In a few integron variants a second promoter, P,, has been created by the insertion of three bases between otherwise poorly spaced -35 and -10 signals.…”

“…In a few integron variants a second promoter, P,, has been created by the insertion of three bases between otherwise poorly spaced -35 and -10 signals. P, is a strong promoter (Lkvesque et al, 1994) and increases the level of resistance expressed when the weakest version of Pant is present (Collis & Hall, 1995).…”