Thomas Franch

Small regulatory RNAs control the multi‐cellular adhesive lifestyle of Escherichia coli

Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single‐cell motile lifestyle and a multi‐cellular, sessile and adhesive state that enables biofilm formation on surfaces. For this, the bacterium needs to reprogramme its gene expression, and in many E.u2003coli and Salmonella strains the lifestyle shift relies on control cascades that inhibit flagellar expression and activate the synthesis of curli, extracellular adhesive fibres important for co‐aggregation of cells and adhesion to biotic and abiotic surfaces. By combining bioinformatics, genetic and biochemical analysis we identified three small RNAs that act by an antisense mechanism to downregulate translation of CsgD, the master regulator of curli synthesis. Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five.

The antisense RNA of the par locus of pAD1 regulates the expression of a 33‐amino‐acid toxic peptide by an unusual mechanism

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA‐regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst‐encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem–loop structure at the 5′ end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300‐fold increase in production of the fst‐encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid‐free cells.

Programmed cell death in bacteria: translational repression by mRNA end‐pairing

The hok/sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid‐free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid‐free cells. The full‐length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti‐Shine–Dalgarno elements that repress translation by shielding the Shine–Dalgarno ele‐ments. The mRNAs also contain fold‐back‐inhibition elements (fbiu2003) at their 3′ ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3′fbi elements pair with the very 5′ ends of the mRNAs. This pairing sets the low rate of 3′ exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5′ ends (termed tacu2003). Thus, the fbi elements inhibit translation of the full‐length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3′ exonucleolytical processing. Mutational ana‐lyses indicate that the 3′ processing triggers refolding of the mRNA 5′ ends into translatable configurations in which the 5′tac elements base pair with the anti‐Shine–Dalgarno sequences.

Antisense RNA regulation of the par post‐segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, which are the toxin and antitoxin of the par PSK system respectively. RNAI encodes an open reading frame for a 33 amino acid toxin called Fst. Expression of fst is regulated post‐transcriptionally by RNAII. RNAII interacts with RNAI by a unique antisense RNA mechanism involving binding at the 5′ and 3′ ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem‐loop and a set of direct repeat sequences, DRa and DRb, located at the 5′ end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI–RNAII complex were analysed by partial digestion with Pb(II) and ribonucleases. Probing data for RNAI and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and terminator sequences are involved in the formation of the RNAI–RNAII complex. Mutant par RNAs were used to show that the binding reaction occurs in at least two steps. The first step is the formation of an initial kissing interaction between the transcriptional terminator stem‐loops of both RNAs. The subsequent step(s) involves an initial pairing of the complementary direct repeat sequences followed by complete hybridization of the 5′ nucleotides to stabilize the RNAI–RNAII complex.

Plasmid Stabilization by Post-Segregational Killing

Bacterial plasmids still offer some of the most versatile systems for the manipulation of genes. Bacterial plasmid-vectors have been developed for many different purposes of gene cloning. Such artificial plasmids usually consist of a replication origin, a resistance cassette and genes or expression signals for specific purposes. However, many such vectors suffer from the disadvantage of being unstably maintained. The instability of a plasmid-encoded gene will in some cases be caused by structural instability of the DNA. However, most often gene instability is caused by loss of the entire plasmid genome. In some cases, such segregational plasmid loss presents a serious practical problem either when cells are propagated in the laboratory or during optimization of gene expression.

Coupled nucleotide covariations reveal dynamic RNA interaction patterns.

Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such coupled covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts.