SepF supports the recruitment of the DNA translocase SftA to the Z-ring.

In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA. SftA is active during vegetative growth whereas SpoIIIE is primarily active during sporulation and pumps the chromosome into the spore compartment. FtsK and SpoIIIE contain several transmembrane helices, however, SftA is assumed to be a cytoplasmic protein. It is unknown how SftA is recruited to the cell division site. Here we show that SftA is a peripheral membrane protein, containing an N-terminal amphipathic helix that reversibly anchors the protein to the cell membrane. Using a yeast two-hybrid screen we found that SftA interacts with the conserved cell division protein SepF. Based on extensive genetic analyses and previous data we propose that the septal localization of SftA depends on either SepF or the cell division protein FtsA. Since SftA seems to interfere with the activity of SepF, and since inactivation of SepF mitigates the sensitivity of a ∆sftA mutant for ciprofloxacin, we speculate that SftA might delay septum synthesis when chromosomal DNA is in the vicinity.

Chromosome choreography during the non-binary cell cycle of a predatory bacterium.

In bacteria, the dynamics of chromosome replication and segregation are tightly coordinated with cell-cycle progression and largely rely on specific spatiotemporal arrangement of the chromosome. Whereas these key processes are mostly investigated in species that divide by binary fission, they remain mysterious in bacteria producing larger number of descendants. Here, we establish the predatory bacterium Bdellovibrio bacteriovorus as a model to investigate the non-binary processing of a circular chromosome. We found that its single chromosome is highly compacted in a polarized nucleoid that excludes freely diffusing proteins during the non-proliferative stage of the cell cycle. A binary-like cycle of DNA replication and asymmetric segregation is followed by multiple asynchronous rounds of replication and progressive ParABS-dependent partitioning, uncoupled from cell division. Finally, we provide the first evidence for an on-off behavior of the ParB protein, which localizes at the centromere in a cell-cycle-regulated manner. Altogether, our findings support a model of complex chromosome choreography leading to the generation of variable, odd, or even numbers of offspring and highlight the adaptation of conserved mechanisms to achieve non-binary reproduction.

Membrane Curvature and the Tol-Pal Complex Determine Polar Localization of the Chemoreceptor Tar in Escherichia coli.

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years, different mechanisms have been put forward to explain this polar localization, including stochastic clustering, membrane curvature-driven localization, interactions with the Tol-Pal complex, and nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at the cell poles. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterized by strong membrane curvature. Chemoreceptors, like Tar, form trimers of dimers that bend the cell membrane due to a rigid tripod structure. The curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favorable, as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or the cell poles. These findings favor a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.IMPORTANCE Bacteria have exquisite mechanisms to sense and adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli and found that membrane curvature at cell division sites and the Tol-Pal protein complex localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains.

Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.

Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors.

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.