Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35‐amino‐acid cell‐killing peptide and a cis‐encoded small antisense RNA in Escherichia coli

Abstract

Summary Genome sequence analyses of Escherichia coli K‐12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR‐A, ‐B, ‐C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR‐D), 450 bp in length and nearly identical to LDR‐A, ‐B and ‐C, is located at 79.7 min, a position that is directly opposite the position of LDR‐A, ‐B and ‐C. In this study, we demonstrate that LDR‐D encodes a 35‐amino‐acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (≈ 370 nucleotides) encoding LdrD and an unstable cis‐encoded antisense RNA (≈ 60 nucleotides), which functions as a trans‐acting regulator of ldrD translation. We propose that LDR encodes a toxin–antitoxin module. LDR‐homologous sequences are not present on any known plasmids but are conserved in Salmonella and other enterobacterial species.

Small Toxic Proteins and the Antisense RNAs That Repress Them

“…The long direct repeat (LDR) sequences are approximately 530 nucleotides in length, and each encodes an Ldr toxin and an Rdl antitoxin RNA (31). The LDR sequence is repeated four times with slight variation on the E. coli K-12 chromosome; there are three tandem repeats (LDR-A, LDR-B, and LDR-C) at one locus at 27.4 min and a single repeat (LDR-D) on the opposite side of the chromosome at 79.7 min.…”

“…This close proximity may increase the efficiency of regulation, though the advantages of cis-versus trans-encoded sRNAs have not been examined. For the chromosomal pairs described here, the sRNAs encoded in trans on plasmids still act as repressors, although in these cases the sRNAs are highly overexpressed (12,30,31,49,53). It is possible that cis-encoded RNAs might pair with their targets more rapidly, which could be an advantage when the sRNA is unstable.…”

“…Overexpression of the LdrD protein leads to nucleoid condensation in E. coli (31), and Fst overexpression disrupts nucleoid structure, chromosome segregation, and cell division in E. faecalis (41). For Fst, these physiological effects have been observed at lower levels of expression than those required for membrane disruption (41).…”

“…High levels of TisB lead to decreased DNA, RNA, and protein synthesis in vivo, but these effects are likely to be indirect consequences of membrane damage, since TisB had no effects on transcription and translation in vitro (52). Whole-genome expression analysis has shown that overexpression of LdrD, IbsC, ShoB, or TisB results in the induction and repression of specific genes (12,31). While some genes, such as soxS, which encodes a regulator of the superoxide stress response, are induced by overexpression of each of the four toxins, other genes are affected by only a subset of the toxins, suggesting that these small hydrophobic proteins do not all act on the same targets.…”

The toxin-antitoxin systemtisB-istR1

“…For instance, E. coli K12 encodes four ldr/rdl loci, five ibs/sib loci, and a locus denoted shoB/ ohsC. [40][41][42] Bioinformatics suggests that they, and some additional type I systems, are abundantly present in other bacteria. 10 Their toxins, in most cases, are small hydrophobic proteins …”