A race against time: Clinically relevant antimicrobial resistance is largely spread via plasmids that disperse among bacteria during conjugation. How quickly can a resistance gene be expressed after transfer? In susceptible bacterial cells, tetracycline should inhibit protein synthesis, including from the plasmid-transferred resistance gene tetA . Unexpectedly, Nolivos et al. found that TetA can be expressed despite the presence of tetracycline (see the Perspective by Povolo and Ackermann). Immediately after plasmid transfer into a cell, TetA synthesis starts because its repressor is slow to be expressed. In addition, the ubiquitous xenobiotic efflux pump AcrAB-TolC buys time for TetA translation by keeping tetracycline concentration below toxic levels. Science , this issue p. 778 ; see also p. 737

By reviewing the properties of SMC complexes in all three domains of life, we assess their likely common biochemical mechanism of action and propose how this mi

The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter. MatP displaces wild-type MukBEF complexes from ter, thereby facilitating their association with ori, and limiting the availability of topoisomerase IV (TopoIV) at ter. Displacement of MukBEF is impaired when MukB ATP hydrolysis is compromised and when MatP is absent, leading to a stable association of ter and MukBEF. Impairing the TopoIV-MukBEF interaction delays sister ter segregation in cells lacking MatP. We propose that the interplay between MukBEF and MatP directs chromosome organization in relation to MukBEF clusters and associated topisomerase IV, thereby ensuring timely chromosome unlinking and segregation.

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.

Bacteria harbouring circular chromosomes have a Xer site-specific recombination system that resolves chromosome dimers at division. In Escherichia coli , the activity of the XerCD/ dif system is controlled and coupled with cell division by the FtsK DNA translocase. Most Xer systems, as XerCD/ dif , include two different recombinases. However, some, as the Lactococcus lactis XerS/ dif _SL system, include only one recombinase. We investigated the functional effects of this difference by studying the XerS/ dif _SL system. XerS bound and recombined dif _SL sites in vitro , both activities displaying asymmetric characteristics. Resolution of chromosome dimers by XerS/ dif _SL required translocation by division septum-borne FtsK. The translocase domain of L. lactis FtsK supported recombination by XerCD/ dif , just as E. coli FtsK supports recombination by XerS/ dif _SL . Thus, the FtsK-dependent coupling of chromosome segregation with cell division extends to non-rod-shaped bacteria and outside the phylum Proteobacteria. Both the XerCD/ dif and XerS/ dif _SL recombination systems require the control activities of the FtsKγ subdomain. However, FtsKγ activates recombination through different mechanisms in these two Xer systems. We show that FtsKγ alone activates XerCD/ dif recombination. In contrast, both FtsKγ and the translocation motor are required to activate XerS/ dif _SL recombination. These findings have implications for the mechanisms by which FtsK activates recombination.