Construction Of An Escherichia Coli Strain With Single Copy Of RRNA Operon And The Plasmid Rearrangement During Homologous Recombination

Since the discovery of the ribosome as the machinery essential for protein synthesis in the mid-1950s, extensive studies have been carried out with respect to ribosomal structure, function, biosynthesis, and regulation. For obvious historical reasons, these studies, especially those on the structure-function relationship, have been done mostly by using Escherichia coli, leading to enormous amounts of information on E. coli ribosomes. Regarding the presence of multiple copies of rRNA genes and their chromosomal location in bacteria, the first question concerned the heterogeneity of rRNA sequences among multiple copies of the rRNA genes and its functional significance.Using Red recombination system, E. coli strains BW25113 and DH5αwere selected as the target strains to do the knockout of 16S rRNA gene, several recombinants were picked up and verified by PCR and sequencing. The E. coli strain SQ88, which was sequentially inactivated from one to five of rRNA operons using X Red recombination system was selected as the target strain. Using Red Recombination system, SQ88C-Kan, a strain just carrying one copy of rRNA operon was constructed by screening Kan resistance mutant. The Kan resistant gene between two FRT recognizing sites was then eliminated designedly. The manipulated strain SQ88C showed no obvious defects in growth rates, and the rRNA/protein ratio of it remained as SQ88. The results suggested that one copy of rRNA operon could support the growth of E. coli in some conditions. This strain is of potential in the research of in 16S rRNA gene mutations. An E. coli strain just with one 16S rRNA gene can grow reasonably well indicates that any differences among different rRNA operons, if they exist, must be small and inessential for growth under standard culture conditions. This was a reasonable support for following study and fine strain for the knockout of the last copy of 16S rRNA gene. .There was one copy of rrn operon on the chromosome of E. coli SQ110. A foreign 16S rRNA gene of B. subtilis was introduced into the target strain SQ110 using the same strategy. A Kan resistance recombinant SQ110BSP was selected. SQ110BSP could grow on the uninducible media. After the elimination of plasmid pBBRMCSP16, the resultant strain SQ110BSX could grow, too. This strain showed cold-sensitive phenotype for its disadvantage in ribosome assembly. Amplification of 16S rDNA and ARDRA results suggested that the 16S rRNA gene has been introduced into SQ110. The rRNA/protein ratio of of SQ110BSX was up-regulated to 148% of SQ110, while the growth rate was just the same as SQ110. This indicated that the ribosome efficiency of SQ110BSX was low.Using Red recombination system, 16S rRNA gene of SQ110 was substituted by Kan resistance gene, one recombinant SQ25 was selected. SQ25 could grow on the uninducible media MDG and MDAG After the elimination of plasmid pBBRMCSP16, the resultant strain SQ25X could grow. PCR verification and sequencing showed that the flanking regions of Kan gene just coincided with the results theoretically. Otherwise, SEFA-PCR and subcloning of Kan resistance gene have validated that some abnormal recombination has happened. Plamid replacement resulted in the lethal condition for the strain SQ171: without any 16S rRNA gene on the chromosome. Both the PCR verification and enzyme analysis showed that plasmid rearrangement happened, after the replacement of pKK3535 to pKKSma-Cm-A-1 in strain SQ171, the resultant plasmid pKKCm extracted from SQ171Cm-3 has gained the deleted sequence between two Sma I sites in 16S rRNA gene. Therefore, abnormal recombination including plasmid rearrangement may happen with high frequency during homologous recombination.