Abstract
Integrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation. In this study, the promoter activity of ORF-less GCs, previously recovered from the oral metagenome, was verified by cloning them upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD. This is the first demonstration and detection of promoter activity of ORF-less GCs from Treponema bacteria and the development of an agar plate-based detection system will enable similar studies in other environments.
Highlights
Integrons are bacterial genetic elements able to integrate and express genes present on gene cassettes (GCs)[1,2,3]
63 GCs previously identified from human oral metagenomic DNA, 13 were predicted to be open reading frames (ORFs)-less GCs29
Even though ORF-less GCs have been found in the previous studies[25,28,29,42,43], their functions have not been fully understood
Summary
Integrons are bacterial genetic elements able to integrate and express genes present on gene cassettes (GCs)[1,2,3] They consist of two main components; a functional platform and a variable array of GCs. The functional platform, located on the 5′ end of an integron, consists of an integrase gene (intI), and its promoter (PintI), an attI recombination site and a constitutive cassette promoter (PC) for the expression of GCs4. When a GC is excised from integron, it forms a non-replicative mobile genetic element, which can be a substrate for integrase mediated recombination between attI (on the integrons) and attC (on the circular GC) This directionality of recombination is favoured over attC:attC recombination, resulting in the usual insertion of a newly integrated GC immediately next to the PC promoter in the first position of the GC array, ensuring maximal expression[9,10,11]. There are some GCs that contain their own promoters, ensuring constitutive expression of their genes regardless of the PC promoter and their position within the integron array; examples include cmlA1 (chloramphenicol resistance), qnrVC1 (quinolone resistance), ere(A) (erythromycin resistance) and many of the GCs encoding toxin-antitoxin (TA) systems[21,22,23,24]
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