Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer.

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.

AES and ToF-SIMS combination for single cell chemical imaging of gold nanoparticle-labeled Escherichia coli.

A chemical fingerprint of the Escherichia coli cell surface labeled by gelatin coated gold nanoparticles was obtained by combining Auger Electron Spectroscopy (AES) for single cell level chemical images, and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) Tandem MS for unambiguous molecular identification of co-localized species.

Direct visualisation of drug-efflux in live Escherichia coli cells.

Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after ∼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells' initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer.

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.

MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation.

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.

The bacterial chromosome: architecture and action of bacterial SMC and SMC-like complexes.

Structural Maintenance of Chromosomes (SMC) protein complexes are found in all three domains of life. They are characterized by a distinctive and conserved architecture in which a globular ATPase 'head' domain is formed by the N- and C-terminal regions of the SMC protein coming together, with a c. 50-nm-long antiparallel coiled-coil separating the head from a dimerization 'hinge'. Dimerization gives both V- and O-shaped SMC dimers. The distinctive architecture points to a conserved biochemical mechanism of action. However, the details of this mechanism are incomplete, and the precise ways in which this mechanism leads to the biological functions of these complexes in chromosome organization and processing remain unclear. In this review, we introduce the properties of bacterial SMC complexes, compare them with eukaryotic complexes and discuss how their likely biochemical action relates to their roles in chromosome organization and segregation.

Co-evolution of segregation guide DNA motifs and the FtsK translocase in bacteria: identification of the atypical Lactococcus lactis KOPS motif.

Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in γ-Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis, a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5'-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo. L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity.

Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase.

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.

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn.

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.