19Integrons are genetic elements consisting of a functional platform for recombination and expression 20 of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding 21 proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly 22 on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less 23 GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA 24 mediated gene regulation. 25In this study, promoter sequences were identified, using in silico analysis, within GCs PCR amplified 26 from the oral metagenome. The promoter activity of ORF-less GCs was verified by cloning them 27 upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria 28 to adapt to multiple stresses within the complex physico-chemical environment of the human oral 29 cavity. A bi-directional promoter detection system was also developed allowing direct identification of 30 clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified 31 from the human oral metagenomic DNA using this construct, called pBiDiPD. 32 This is the first demonstration and detection of promoter activity of ORF-less GCs and the development 33 of an agar plate-based detection system will enable similar studies in other environments. 34
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Introduction 36Integrons are bacterial genetic elements able to integrate and express genes present on gene cassettes 37 (GCs) [1][2][3]. They consist of two main components; a functional platform and a variable array of GCs. 38The functional platform, located on the 5' end of an integron, consists of an integrase gene (intI), and 39 its promoter (PintI), an attI recombination site and a constitutive cassette promoter (PC) for the 40 expression of GCs [4]. IntI is a site-specific tyrosine integrase that catalyses the insertion and excision 41 of GCs via recombination mainly at attI and the attC, the latter located at the joint of excised, 42 circularised GCs. The integrase gene; intI, is normally transcribed in the opposite direction to GCs 43 within an integron ( Fig 1A). However, some integrons have integrase genes transcribed in the same 44 directions as their GCs. These are called unusual integrons or reverse integrons ( Fig 1B), and have been 45 identified in Treponema denticola, Chlorobium phaeobacteroides and Blastopirellula marina [5, 6]. 46The second part of an integron is an array of GCs. Each usually contains a single promoterless open 47 reading frame (ORF) and an attC recombination site [7]. The proteins encoded by GCs are diverse and 48 include those associated with antibiotic resistance, virulence, and metabolism [2, 8]. When a GC is 49 excised from integron, it forms a non-replicative mobile genetic element, which can be a substrate for 50 integrase mediated recombination between attI (on the integrons) and attC (on the circular GC). This 51 directionality of recombination is favoured over attC:attC...