Identification of a putative infC-rpml-rplT operon flanked by long inverted repeats in Mycoplasma fermentons (incognitus strain)

Abstract

A specific 1542-bp DNA fragment was amplified from Mycoplasma fermentons (incognitus strain) using a unique 23-nucleotide nt) synthetic deoxyribonucleotide (oligo) (5'-TCCAAAAAGTCCGGAATTTGGGG) as the primer pair in the polymerase chain reaction (PCR). The 23-nt sequence is part of the 29-bp terminal inverted repeat (IR) which forms the left potential stem-and-loop (s&1) structure of the previously identified M. fermentans insertion-sequence(IS)-like genetic element [Hu et al., Gene 93 (1990) 67–72]. The amplified DNA was cloned and sequenced. A pair of 27-bp IR containing the 23-nt synthetic oligo was identified at both termini. Between the IR, there are four potential open reading frames (ORFs) which are arranged adjacent to each other in the order, ORF-1, ORF-2, ORF-3 and ORF-4, with parts of ORF-1 and ORF-2 overlapping. The deduced amino acid (aa) sequences of ORF-2, ORF-3 and ORF-4 are 34 to 60% identical to the translation initiation factor IF3 (encoded by the infC gene), ribosomal proteins L35 ( rpmI gene) and L20 ( rplT gene) of Escherichia coli and Bacillus stearothermophilus, respectively. In bacteria, the infC-rpml-rplT genes are organized to function as an operon. There are multiple sites with promoter-like sequences identified upstream from the putative infC gene in the mycoplasma closely resembling the gene arrangement in the bacterial operon. All three genes of ORF-2, ORF-3 and ORF-4 are preceded individually by a strong appropriately spaced (7 and 10 bp) putative Shine-Dalgarno sequence (5'-AAGGA). In addition, ORF-2 uses the unusual triplet, ATT, as the start codon, the same as that for infC in the bacterial operon. Thus, the cluster of genes (ORF-2, ORF-3 and ORF-4) is identified as a putative mycoplasma infC-rpml-rplT operon. Most interestingly, our study reveals that this operon potentially constitutes a part of a mobile genetic element in the incognitus strain of M. fermentans.