Conditional Silencing of the <i>Escherichia coli pykF</i> Gene Results from Artificial Convergent Transcription Protected from Rho-Dependent Termination

Abstract

PykF is one of two pyruvate kinases in Escherichia coli K-12. λP_L was convergently integrated into the chromosome of the MG1655 strain, downstream of pykF, face-to-face with its native promoter. In the presence of λcIts857, efficient pykF ts-silencing was achieved when the 5′-terminus of the P_L-originated antisense RNA (asRNA), consisting of the rrnG-AT sequence, converted elongation complexes of RNA polymerase to a form resistant to Rho-dependent transcription termination. pykF silencing was detected by the following features: (a) impaired growth of the strain when pykA was also disrupted and when using ribose as a non-phosphotransferase system-transporting carbon source; (b) a pattern of reduced synthesis of the full-sized pykF mRNA, mediated by reverse transcription PCR, and (c) a significant decrease in PykF activity. The advantages of anti-terminated convergent transcription were clearly manifested in the strains where the rho_a-terminator was inserted specifically to interrupt asRNA synthesis. Most likely, the target gene was silenced by transcriptional interference due to collisions between converging RNA polymerases, although, strictly, the role of cis-asRNA effects could not be excluded. While details of the mechanisms have yet to be determined, anti-terminated convergent transcription is a promising new technique for silencing other target genes.

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

“…The increase in genomic copy number of native or previously modified target genes is an important tool for chromosomal editing and the construction of stably maintained bacterial genomes. The development of new approaches for the integration (Minaeva et al 2008; Rivero-Müller et al 2007) and the increase in genomic copy number (Tyo et al 2009) of recombinant DNA fragments remains relevant even for Escherichia coli and closely related Gram-negative bacteria for which recombineering-based technologies (Court et al 2002; Sawitzke et al 2007; Sharan et al 2009) have already been developed to finely modulate the expression of chromosomal genes (De Mey et al 2010; Katashkina et al 2005; Meynial-Salles et al 2005) and sometimes conditionally increase (Doroshenko et al 2010b) or silence (Krylov et al 2010) the level of their transcription. The methods of integration/amplification are especially important for bacteria for which the genetic tools for the chromosomal editing are not as comprehensive and diversified as those available for E. coli .…”

Universal Actuator for Efficient Silencing of Escherichia coli Genes Based on Convergent Transcription Resistant to Rho-Dependent Termination

“…To our knowledge, the present study, as a continuation of a previously published one, 25 is the first demonstration of the advantages of applying a CT-based strategy for silencing genes with r-transcription protected from RhoTT; this strategy could be interesting from a metabolic engineering perspective.…”

“…Thus, the approach is robust, and the efficiency of the target gene actuator-based silencing practically does not decrease, even when several strong actuators were added for silencing other genes. The unique feature of the CT-based silencing strategy implemented previously 25 and repeated again in the present study was the exploitation of the r-RNAP-mediated elongation complex that is resistant to RhoTT for the prevention of the untimely termination of nascent nontranslated asRNA. Recently, the negative effect of such termination was experimentally demonstrated for silencing by CT. 19 It was suggested 25 that after the generation of the rrnG-AT sequence in the 5′-terminus of the nascent RNA by r-RNAP, the elongation complex became resistant to RhoTT and continued transcription at a rate ≈85 nt/s, which is typical not for mRNA but for the synthesis of E. coli rRNA.…”

“…The unique feature of the CT-based silencing strategy implemented previously 25 and repeated again in the present study was the exploitation of the r-RNAP-mediated elongation complex that is resistant to RhoTT for the prevention of the untimely termination of nascent nontranslated asRNA. Recently, the negative effect of such termination was experimentally demonstrated for silencing by CT. 19 It was suggested 25 that after the generation of the rrnG-AT sequence in the 5′-terminus of the nascent RNA by r-RNAP, the elongation complex became resistant to RhoTT and continued transcription at a rate ≈85 nt/s, which is typical not for mRNA but for the synthesis of E. coli rRNA. 39 According to our knowledge, other authors who also applied CT-based silencing did not use the conversion of r-RNAP to the RhoTT resistant state.…”

Bacterial Cellular Engineering by Genome Editing and Gene Silencing

“…However, the factors that are most affected by knocking down the targeted mRNAs is still unclear, although some mechanisms have been proposed. In E. coli , the level of target mRNAs decrease following silencing as well as protein level [93–95,101,102]. It is, thus, likely that target mRNAs that are masked with asRNAs tend to be rapidly degraded in the cell.…”